Data concerning their adverse effects has been lately describing central nervous system (CNS) demyelination process at escalating basis. Case Presentation. A 23-year-old male with autoimmune uveitis and a family history of multiple sclerosis (MS) developed two neurological attacks, after Adalimumab infusion, simultaneously with several cerebral lesions on magnetic resonance imaging (MRI). Hence the diagnosis of Adalimumab induced MS was suspected. Conclusion. This case is reported to tell physicians to be cautious when using anti-TNF-α in patients with family history of MS and to reconsider the risk of MS in patients with autoimmune diseases.Anti-tumor necrosis factor  It mediates inflammatory cell recruitment to the site of the infection by activating endothelial cells. Elevated levels of this cytokine, however, were detected both locally and systemically in patients with inflammatory disorders like arthritis [α have shown beyond dispute role in modulating the activity of many autoimmune diseases. Depending on their mechanism of action, anti-TNF-α drugs are divided into monoclonal antibodies  and soluble TNF-α receptors (Etanercept). Many side effects have been recorded during the relatively short history of utilizing anti-TNF-α in the field of autoimmune diseases such as infectious disease [TNF-rthritis . Drugs t disease , autoimm disease , 4, bloo disease , 6, pulm disease , congest disease , and mal disease  among otα.CNS demyelination was conducted by several reports suggesting triggering or exacerbating demyelination diseases induced by treatment with anti-TNF-In A 23-year-old Arabic gentleman from Syria had a positive family history of MS affecting two of his uncles  and idiopathic epilepsy affecting his sister. His past medical history was positive for chronic autoimmune uveitis since the age of 13. It was then treated with Methotrexate with poor response, and thus it was substituted for combination of Cyclosporine and Azathioprine which achieved a better control. A year ago, Azathioprine was suspended for a while because of the political situation and Adalimumab was initiated. Few days following the second dose of Adalimumab, the patient developed right hemibody anesthesia which lasted two weeks and was successfully treated with intravenous methylprednisolone. Adalimumab was then stopped and both of Azathioprine at a dose of 3 mg/kg and oral prednisolone were initiated. Five months later, brain magnetic resonance imaging (MRI) was requested and revealed multiple periventricular and juxtacortical lesions with high T2 signal intensity along with T2 hypersignal lesion in the spinal cord at the level of the fourth cervical vertebra C4; none of these lesions were contrast enhancing .Visually evoked potentials (VEP) were also performed and were normal. Cerebrospinal fluid (CSF) analysis was normal and negative for oligoclonal bands (OCBs).Eight months following Adalimumab infusion, the patient had right lower limb paresis which was also successfully treated with methylprednisolone, and normal strength was restored within three weeks. Six months after then, the patient was referred by a rheumatologist to a neurology clinic for consultation and followup though there has been no new attack since then.Neurological examination was normal except for anisocoria with slightly irregular pupils and bilaterally sluggish reaction to light  with normal fundoscopy and visual acuity. Mild right facial and body hemihypoesthesia, absent abdominal reflexes over the right side, generalized hyperreflexia (+3), and mild right lower limb spasticity were also noticed. To find out the relationship between the uveitis and the neurological attacks, the type of uveitis was reassessed and revealed hints of anterior and intermediate uveitis with no signs of active inflammation. An X-ray of sacroiliac joints and chest X-ray were then requested to rule out both subclinical ankylosing spondylitis and sarcoidosis and were both normal. Brain MRI with contrast was repeated and revealed no new lesions comparing with the previous one .The previous laboratory study was reviewed and showed normal ordinary labs including erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP). Antinuclear antibodies (ANA), rheumatic factor (RF), and antineutrophil cytoplasmic antibodies C-ANCA and P-ANCA were all negative. Human leukocyte antigen (HLA) analysis revealed HLA-B27. CSF analysis showed acellular fluid with immunoglobulin G (IgG) index of 0.53, normal protein, lactate dehydrogenase (LDH), and glucose levels. Despite the lack of enhancement, diagnosis of Adalimumab induced MS was suspected in this patient with HLAB-27 associated uveitis depending on Mcdonald's criteria for space and time distribution and the temporal relationship between the first neurological attack and drug initiation besides the positive family history of MS. Since there were no active lesions on MRI, treatment plan was continued with no modification and regular clinical and radiological followup was suggested.This case describes two separate neurological attacks in a young patient with HLA-B27 associated uveitis and a family history of multiple sclerosis. Although neurological attacks meet Mcdonald's criteria for time and space distribution, many differential diagnoses are plausible for this presentation, MS with associated uveitis, HLA-B27 associated uveitis and a newly diagnosed MS as two separate entities, and systemic vasculitis induced uveitis and cerebral lesions. Infectious causes and lymphoma, however, are improbable due to the prolonged history and negative laboratory investigations.A certain type of uveitis, uveitis pars planitis, is considered a manifestation of MS that often precedes or, less often, accompanies the first attacks . The typDespite its obscurity, a relationship between MS and autoimmune diseases was deduced because of the high prevalence of MS in patients with autoimmune diseases as suggested by many reports –20. Thisα treatment in patients with family history of MS was reported in many cases [α should be avoided in such patients and advocated requesting cerebral MRI before treatment initiation in patients with high risk of MS [Suspected MS following anti-TNF-ny cases , 18 whicsk of MS , 22.α infusion such as the presence of blood brain barrier (BBB). This barrier halts the entry of anti-TNF-α drugs to CNS. Meanwhile, these drugs enhance T cell receptor signaling and the functionality of antigen presenting cells and, consequently, they activate CNS demyelination. This is the essence of lack of entry theory. Presence of BBB was also the culprit in sponge theory wherein TNF-α is supposed to be drawn out of joint synovial fluid following TNF-α antagonism whereas BBB prevents this process in CNS. Thus TNF-α gradient develops across BBB and results in upregulation of TNF-α receptor expression in CNS. Moreover, severely destructed BBB in experimental autoimmune encephalopathy mice models besides the high dose used have been supposed to facilitate anti-TNF-α entry to CNS and permit the subsequent amelioration [α may prevent this natural mechanism and suppress remyelination [α drugs promote interferon γ (INF-γ) expression and this in turn disturbs the balance between INF-β and INF-γ levels which triggers demyelination process [Several mechanisms have been proposed to interpret the paradox of MS activation in spite of amelioration of systemic inflammation following anti-TNF-ioration . Other tlination . Another process . Whetherα induced MS since most of the reports of similar cases did not mention the results of OCBs analysis. The lack of enhancement, however, may be explained by the time window between performing MRI and the clinical attacks; such window would have guaranteed the resolution of enhancement if it existed in addition to treatment with Azathioprine, a drug that has been accepted as a disease modifying treatment of MS [Although some points argue against MS such as OCBs negativity and the lack of contrast enhancement, MS negative OCBs are a well-established entity that represents 20–30% of MS patients . Moreovent of MS .α agents preceding the development of clinical attacks [Asymptomatic cerebral lesions highly suggestive of MS are widely described in patients with family history of MS under the title of radiographically isolated syndrome (RIS) , 28, but attacks , 16, whiMS is highly expected in patients with family history of MS and, to a lesser degree, in patients with autoimmune diseases, and physician should consider such risks in patients who are eligible for anti-TNF treatment by detailed history, examination, and close followup as well as performing MRI prior to therapy initiation in highly suspected individuals.


Despite improvements in pre-clinical drug testing models, predictability of clinical outcomes continues to be inadequate and costly due to poor evidence of drug metabolism. Humanized miniature organs integrating decellularized rodent organs with tissue specific cells are translational models that can provide further physiological understanding and evidence. Here, we evaluated 4-Flow cannulated rat hearts as the fundamental humanized organ model for cardiovascular drug validation. Results show clearance of cellular components in all chambers in 4-Flow hearts with efficient perfusion into both coronary arteries and cardiac veins. Furthermore, material characterization depicts preserved organization and content of important matrix proteins such as collagens, laminin, and elastin. With access to the complete vascular network, different human cell types were delivered to show spatial distribution and integration into the matrix under perfusion for up to three weeks. The feature of 4-Flow cannulation is the preservation of whole heart conformity enabling ventricular pacing via the pulmonary vein as demonstrated by noninvasive monitoring with fluid pressure and ultrasound imaging. Consequently, 4-Flow hearts surmounting organ mimicry challenges with intact complexity in vasculature and mechanical compliance of the whole organ providing an ideal platform for improving pre-clinical drug validation in addition to understanding cardiovascular diseases. Hence, better predictability of molecular mechanism, specificity, and potency at the early stages of drug validation will increasing efficiency and alleviate economic strain in later stages. Translational science research strategies strive to establish highly relevant human based in vivo and in vitro models to improve prediction of targeted metabolism with minimal side effects3.Even with advancements in technology, the drug discovery pipeline stagnates at a 5% success rate and an average 13-year timeline5. The ideal model can predict at the cellular and tissue level the desired therapeutic effects on the disease in addition to the adverse effects imposed on healthy tissues. Engineered heart tissues constructed from synthetic and natural biomaterials are the most interesting modalities; however, all materials not derived from animal tissues requires fine tuning of composition, architecture, mechanical properties, and compatibility for partial mimickry of a complex organ7. Utilizing decellularized animal hearts will circumvent many engineering limitations and further reform the current translational strategy11. Elaborating from previous studies, three-dimensional platforms progress differentiation of human embryonic stem cells and human induced pluripotent stem iPS) cells towards cardiac myocytes exhibiting cell-to-cell interaction, matrix development and organization, and heterogeneous metabolism at the tissue level15. Others have shown the acellular heart can support the attachment of human iPS cells, cardiac fibroblasts, human embryonic stem cells, and iPS derived cardiac progenitor cells (iPS-CPCs)14. Consequently, a humanized heart as an in vitro model will elevate the quality and credibility of evidence during drug testing by understanding drug metabolism and interactions within a complex tissue11.With cardiovascular diseases as the leading cause of death worldwide, the demand for better cardiovascular treatments and predictions of therapeutic outcomes is a substantial challenge for translational research. Therefore, models that can simulate the heart’s physiology and functionality during normal and disease conditions will provide a comprehensive understanding on drug metabolismex vivo experiments, by cannulating the ascending aorta and preserving aortic valve function to perfuse only through the coronary arteries17. The modified Langendorff method includes an invasive insertion of a balloon through the left atrium into the left ventricle for pacing with a consequence of removing the left atrium and inducing non-physiological exertion to the ventricle wall4. For ex vivo experiments, the Langendorff method is important to evaluate cardiac functionality in response to pharmacological treatments. However, for decellularization and recellularization purposes, the Langendorff cannulation has limitations hindering the development of the whole heart model19. Additional challenges arise during the recellularization process such as complete retention of total cell number, homogenous distribution of cells within the matrix, and clearance of the vascular network for continuous nutrient perfusion. Previously, repopulation via localized manual injections showed low cell retention, low reproducibility, additional matrix damage, and clustered distribution of the cells at unspecific locations12. Others have performed perfusion delivery of cells through the aorta cannulation without confirming aortic valve competence which can lead to cells being washed out easily, or accumulating in the ventricle chamber, or precluding the vascular network19. Hence, utilizing a perfusion system with integrated pressure monitoring and feedback will streamline the decellularization process and prevent high mechanical stress on the cells during perfusion recellularization18. Even when the cells are homogenously flowing through the vascular network during recellularization, dissipation of the cells into the tissue is an important aspect to optimize for vascular clearance and cell survival. However, in utilizing the vascular network to repopulate the vascular basement membrane will retain the cells within the vessels which is optimal for endothelial cell repopulation but not all other cardiac cells20.Current methods for harvesting rodent hearts for perfusion decellularization follow the Langendorff isolation method, intended for ex vivo applications with only the aorta cannulation. Techniques such as resin casting will be used to map the perfusion network of the whole organ and multiphoton imaging will illustrate three dimensional architecture of the preserved matrix. In addition, various recellularization parameters such as cell types and culture conditions will be evaluated to demonstrate improved spatial distribution, survival, and integration within the acellular matrix. The key attribute is the capability to induce ventricle pacing emulating natural expansion through the left atrium and dynamic aortic valve. Hence, the objective is to highlight 4-Flow cannulated hearts as the humanized predictive model for translational research in cardiac drug development.Here, we have developed a method - termed 4-Flow cannulation - optimized for isolating hearts for decellularization and recellularization to maintain heart physiology with a complete circulation and organ conformity. With four different cannulations as perfusion inlets and outlets, decellularization reagents and recellularization solutions are delivered through the coronary arteries and cardiac veins. The versatility of the 4-Flow cannulation will be validated when compared to the current isolation method - Langendorff - which was adopted from The vascular network and the decellularized matrix were characterized for both cannulation methods to demonstrate that 4-Flow hearts have the proper cannulations for producing a humanized miniature organ model as an essential pre-clinical model. The 4-Flow hearts were cannulated at the superior vena cava (SV), ascending aorta (AA), pulmonary artery (PA), and pulmonary vein (PV) while Langendorff hearts only have the AA cannulation. The time lapse images of the decellularization process after a heparin rinse illustrates the differences in heart shape and time of cell clearance of the two cannulation methods under constant pressure perfusion of three liters sodium dodecyl sulfate (SDS) Fig. . Under pDNA content of the decellularized tissues was characterized quantitatively and qualitatively demonstrating that perfusion with only SDS can remove all cellular components for both 4-Flow and Langendorff cannulated hearts Fig. . In addiThe advantage of the 4-Flow cannulation is highlighted with cell repopulation by perfusion through both SV and AA Fig. . HEK293 Mechanical stimulation or ventricle pacing is the key feature of the 4-Flow cannulation achievable by preserving the left atrium via PV cannulation and the competence of the aortic valve after decellularization. Here, a decellularized 4-Flow heart underwent cyclic injections of medium into the PV to conduct ventricle pacing. The medium flows from the left atrium into the left ventricle chamber increasing fluid pressure which causes the aortic valve to open dissipate medium into the coronary circulation Fig. . When th20. An important aspect that has been previously overlooked is the retention of the full circulation network of the heart with the proper cannulation for both decellularization and recellularization. Here, we showed that 4-Flow cannulated hearts are optimized for accessibility of the complete vascular network and conservation of whole heart mechanical conformity.With complex features such as a complete vascular network and a noninvasive route for ventricle pacing, 4-Flow hearts are ideal for decellularizing and repopulating with human cells as the physiological organ model to further understanding of cardiovascular diseases and related therapeutics. Previous investigations of whole heart models focus on optimizing decellularization protocols using different detergents and reagents, and improving repopulation via injections or perfusion with different cells types17. Perfusion into both AA and SV directs flow inlet to coronary arteries and cardiac veins and flow outlets through PA and PV during decellularization and recellularization. Furthermore, utilizing perfusion at constant physiological pressure of 75 mmHg, the flow rate fluctuates to accommodate differences between each heart during decellularization4. Consequently, effective and proper decellularization can be completed by using SDS alone without the need for additional detergents11. Decellularized tissues were cleared of cellular components in addition to retaining important extracellular matrix proteins such as different collagens, elastin, and laminin for cell attachment and agrin a specific vascular protein.By accessing and controlling the perfusion into a heart via 4-Flow, decellularization is more efficient as compared to the Langendorff hearts with only access through the aorta. Furthermore, monitoring and controlling the fluid pressure right before the aortic valve ensures efficient and proper distribution of perfusates. The 4-flow cannulations has a perfusion pathway into the coronary arteries and cardiac veins as demonstrated by resin casting. The flow of the resin cast confirms the functionality of the aortic valve which is important to effectively deliver the cells through the coronary arteries and into the tissues rather than into the left ventricle chamber. In addition, utilizing the resin to visualize the perfusion pathway rather than dyes distinguishes fluid perfusion from diffusion12. Others have perfused cells through the AA to deliver cells into the coronary arteries; however, without monitoring the fluid pressure it is difficult to confirm the closure of the aortic valve and consequent flow into the arteries20. With 4-Flow under constant pressure monitoring, perfused cells are delivered with increased vascular coverage through the cardiac veins and coronary arteries with high confidence. Furthermore, distribution of the cells from the vascular network into the tissue is enhanced by increased vascular permeability. The short-term experiments with HEK293 cells demonstrated cell delivery through the SV and AA in which cells distribute throughout the matrix tissue in all chamber walls leaving the vascular lumen clear for undisturbed perfusion flow. Confirming clearance of vasculature ensures transport of medium to all populated areas within the heart. The repopulation with human primary cardiac fibroblasts provides evidence that primary cells have an affinity to the matrix to distribute, proliferate, and migrate under continuous dynamic perfusion. In h-iPS-CPC recellularization, 3 weeks of perfusion culture showed cell dispersion into the matrix and integration aligning with the orientation of the matrix fibers.One recellularization method utilizes injections of cells throughout the tissue which results in high loss of cells, localized distribution at unknown depths, and damage to the delicate tissue matrixex vivo experimentation; however, in decellularization and recellularization applications a more optimized cannulation method such as 4-Flow will improve perfusion of detergents and cells. By producing a miniature humanized heart model such as a 4-Flow heart, preservation of the physical and biological stimuli will promote progenitor cells towards mature and functional cells23. In addition, primary isolated patient cells and genetically modified progenitor cells can be used to generate patient centric models and disease models in which no translatable animal models exist. In conclusion, humanized miniature hearts can be an essential translational strategy to facilitate effective drug discovery in addition to understanding cardiac biology and diseases.To summarize, this work has demonstrated that preserving the vascular accessibility with the 4-Flow cannulation improves whole organ perfusion reducing the amount of time needed for decellularization. Furthermore, the importance of maintaining and monitoring the competence of the aortic valve was established as a critical parameter for perfusion decellularization and recellularization. The Langendorff method is important for Animal care and experiments were carried out in compliance with Swedish national legislation and AstraZeneca Bioethics policies in accredited facilities . The project protocol was approved by the institutional oversight body, Djurskyddsorgan, as well as by regional Laboratory Animal Ethics Committee of Gothenburg as required by Swedish regulations.4. Sequentially, under sterile conditions and constant pressure monitoring, the heart was perfused with heparin (150 U/mL) PBS solution (up to 50 mL), 1% sodium dodecyl sulfate solution , PBS with 1% antimycotic and antibiotic (up to 3 L), endonuclease in Hank’s balanced salt solution , ultra-pure water wash , collagenase IV solution , ultra-pure water wash , PBS with 1% antimycotic and antibiotic . To prepare for recellularization, the heart was rinsed and primed with cell specific medium into custom made glass bottles (500 mL Duran Schott) with GL45 caps or modified GL45 lids with 3 outlets for perfusion tubings.Hearts were explanted from male Sprague-Dawley rats (250–300 g) under deep anesthesia with systemic heparin (2500 U/mL). The heart was explanted by cannulating the ascending aorta (AA), superior vena cava (SV), pulmonary artery (PA), and pulmonary vein (PV) while ligating all other inlets and outlets - termed 4-Flow cannulation - to maintain the complete and enclosed circulation. Polyethylene tubings (Intramedic Clay Adams Brand) were used to cannulate and secured with sutures. The AA and SV are perfusion inlets for flow through the coronary artery and cardiac vein, respectively, of decellularization reagents and recellularization solutions. The PA and PV are outlets for used reagents to exit. Hearts were stored in a 150 U/mL heparin phosphate buffered saline (PBS) solution in −80 °C until decellularization. All decellularization and recellularization were performed using a perfusion system  controlled by constant flow or constant pressure in which fluid pressure right before the aortic valve is continuously monitored to ensure the closure of the valve for efficient delivery of all solutions through the coronary arteries. Hearts were allowed to equilibrate to room temperature before perfusion decellularization through the AA and SV at constant 75 mmHg of pressure (unless otherwise indicated) within the normal 70–130 mmHg pressure in a rat heartμL/min into the suspended heart. The resin casted heart was allowed to cure overnight submerged in water. The 4-Flow casted heart was further treated to remove the tissue by submerging the heart in alternating 5% (w/v) potassium hydroxide and water in a 50 °C water bath. The decellularized tissue of the casted Langendorff heart was intentionally preserved in order to maintain the integrity of the detailed branching of the coronary arteries.Batson No. 17 Anatomical Corrosion Kit (PolySciences Inc.) was used to demonstrate the perfusion pathways for decellularized 4-Flow and Langendorff cannulated hearts. The manufacturer’s protocol was optimized to produce 10 mL of resin solution. Briefly, 10 mL of the base solution was mixed with a droplet of pigment (red or blue) by manual stirring. Then 1 mL of the catalyst and 2 drops of the promoter were added sequentially and mixed with manual stirring. Once the solution is homogeneous, it was transferred to a 2 mL syringe for perfusion via syringe pump at a rate of 20 Freshly decellularized hearts were cut into region specific pieces and froze −80 °C directly for DNA extraction and quantification. DNA content was analyzed following the Quant-iT PicoGreen assay (Thermo Fisher) from extracted DNA obtained with the DNeasy Blood & Tissue Kit (Qiagen). Briefly, hearts were sectioned into the right ventricle, septum, and left ventricle (atria were excluded) and wet tissue weight was obtained. The section tissues were then digested for matrix characterization and DNA content according to the manufacturers’ protocols. Significant differences in DNA content were computed with two-way ANOVA using GraphPad Prism.μM ID by 20 mm) connected to a 75 μM by 500 mm analytical Easy Spray PepMap RSLC C18 column . The MS/MS spectra from each nano-LC-MS/MS fraction were searched against a selected rat tissue database (Swissprot) using the Proteome Discoverer 2.1 software (Thermo Scientific). The search criteria were as follows: tryptic digestion, one missed cleavage, carbamidomethylation (C) and TMT plex  were fixed modifications, the oxidation (M) as a variable modification, precursor ion mass tolerances were set at 6 ppm, and the fragment ion mass tolerance at 20 mmu. From the list of matched proteins, selected extracellular matrix proteins including proteoglycans, glycoproteins, and collagens were presented. For histological analysis, decellularized hearts were perfused with 4% paraformaldehyde to fixate the tissue then the samples were sent to a core facility  for paraffin embedding, sectioning at 4–8 μm thick slices, and stained with Hemotoxylin and Eosin and Masson’s trichrome. Immunostaining specifically for collagen I , collagen IV , and laminin  were performed on sections after removing the paraffin. Sections were then incubated in a blocking solution ) for 30 minutes at room temperature. Primary antibody in the blocking solution was added and incubated for 1 hour at room temperature. Sections were washed with 0.1% Tween-20 in PBS three times for 5 minutes each time before incubating with secondary antibody . Coverslips were mounted onto the sections using ProLong Gold with DAPI (Molecular Probes).Characterization of ECM content was conducted utilizing liquid chromatography tandem mass spectrometry, ECM assays (BioColor) and immunostainig of histology sections. Decellularized tissues from 4-Flow hearts were homogenized in a lysis buffer  and protease and phosphatase inhibitor cocktail (MS-SAFE) using the Precellys (Bertin Technologies) homogenization system. Protein extractions were processed to obtain tryptic peptides by using the filter aided sample preparation (FASP) method. Subsequently, tryptic peptides were quantified, TMT labelled, and dried. The dried samples were redissolved in 0.1% TFA and was subsequently fractionated into 8 fractions using a Pierce High pH Reversed-Phase Peptide Fractionation Kit, (Thermo Scientific). Samples were analyzed by nano Liquid Chromatography-tandem mass spectrometry (LC-MS/MS)  combined with Orbitrap Q Exactive mass spectrometer (Thermo Scientific) using a 2 hour gradient. The separation was performed using an Acclaim PepMap precolumn  cultured in DMEM  were used to demonstrate matrix compatibility after 2 weeks of culture. Since the fibroblasts had minimal proliferation in 2D culture, only a small population was obtained for repopulating the heart. Human induced pluripotent stem (h-iPS) cells are produced and validated as previously described24. These iPS cells were differentiated to cardiac progenitor cells by a differentiation protocol adapted from Lian et al.25. Briefly, the h-iPS cells were seeded on 24-well plates coated with growth factor reduced matrigel at a density of 0.47 million cells per centimeter square in DEF-CS medium (Takara Bio) following the manufacturer’s protocol. The h-iPS cells were cultured for 2 additional days with DEF-CS medium before differentiation was initiated. On day 0 the medium was changed to RPMI (1649 Glutamax) supplemented with B-27 (minus insulin) and CHIR (12 μM) induction was initiated and maintained for 24 hours. On day 2 IWP-2 (5 μM) was added and maintained for 2 days. At day 5 The h-iPS-CPCs were used for recellularization experiments in RPMI (1649 Glutamax) medium with B27 (minus insulin) and on day 6 the medium was changed to RPMI (1649 Glutamax) supplemented with B-27 (with insulin). Spontaneous beating could be observed from day 10 of the differentiation in the control continuously cultured on plates, confirming presence of cardiomyocytes in the culture.Three different cell types were used in this study to demonstrate different properties of recellularization. For short 24 hour studies, mammalian stable cells, HEK293 (ATCC) were used and cultured in DMEM . HEK293 are a stable cell line that is commonly used in high-throughput screening of during initial drug screeningBefore recellularization, the heart was rinsed with basal medium with recirculating perfusion of 20–30 mL of medium overnight in the incubator at 37 °C and 5% carbon dioxide with continuous monitoring of fluid pressure at the ascending aorta to ensure the valve is functional and closed. Afterwards, the heart was primed with 20–30 mL of complete medium with recirculating perfusion overnight. All recellularized hearts were cultured at 37 °C with 5% carbon dioxide and medium was changed 3 times a week. The following recellularization experiments were performed with a focus on cell distribution, attachment, and migration. Perfusion of 30 million HEK293 cells into each inlet  was conducted at a rate of 0.5 mL/min to recirculate a cell suspension volume of 10 mL with DMEM. After 24 hours, perfusion of 4% para-formaldehyde was conducted to fix the hearts. Perfusion of cardiac fibroblasts at 0.3 million cells through each inlet (SV and AA) by recirculating a total volume of 10 mL with DMEM and constant rate 0.5 mL/min (n = 3). After 2 weeks, perfusion of 4% para-formaldehyde was conducted to fix the hearts for histology sectioning and multiphoton imaging. The h-iPS-CPCs at day 5 were used for recellularization experiments in RPMI (1649 Glutamax) medium supplemented with B27 (minus insulin) (n = 3). For h-iPS-CPC recellularization, 3 rounds of cell seeding with a two hour gap were conducted to prevent cell aggregation. The first round of cell seeding with 15 mL of cell solution containing approximately 20 million cells were recirculated at 0.5 mL/min. After two hours 10 mL of the recirculated solution was removed. The second round of cell seeding with 10 mL of cell solution containing approximately 20 million cells were recirculated at 0.5 mL/min. The third round of cell seeding with an additional 10 mL of cell solution containing approximately 20 million cells were recirculated at 0.5 mL/min. The total amount of recirculating solution after three rounds of cell seeding was 25 mL which was allowed to recirculate until the next day. The medium was changed to RPMI Glutamax cont B27 with insulin the day after cell seeding and 3 times a week for the next 3 weeks. Samples were fixed by perfusing of 4% para-formaldehyde for histology sectioning.μL at a rate of 3.5 mL/min and 1 mL/min, respectively, was optimized for maintaining aortic valve opening and closing. Perfusion through both SV and AA were necessary to close the aortic valve. The mechanical pacing can be monitored by the pressure gauge right before the aortic valve.The opening and closing of the aortic valve was visualized live with ultrasound imaging (video 1).Mechanical ventricle pacing was performed using a programmable syringe pump (Aladdin) in addition to the continuous perfusion of a decellularized heart (not repopulated with cells). Continuous injection and withdraw of 450 26. Multiphoton-excited autofluorescence and second harmonic generation (SHG) is advantageous for volumetric imaging to investigate cell-cell and cell-matrix interactions28. The fine structures and organization of elastin is obtained with autofluorescence while the anisotropic collagen fibers generate SHG signals. Due to the limited autofluorescence of the cardiac fibroblasts, Hoechst staining was used to highlight the cells. The success of 4-Flow hearts is the ventricle pacing and competence of the aortic valve which can be visualized noninvasively and under sterile conditions with ultrasound imaging was performed using the FujiFilm VisualSonics VEVO 2100 with the 40 MHz probe. Images were taken at 20 frames per second and compiled into a video. Still images of the ultrasound video were analyzed using ImageJ.Histology slides were imaged using Aperio ScanScope and analyzed using ImageScope software. Fluorescent labeled immunostained slides were taken with a Nikon microscope. In consideration to the 3D and clarified nature of the samples, advanced multiphoton microscopy, at a core facility , was utilized to perform volume imaging using a LSM 710 NLO Zeiss microscope coupled to a Mai Tai Deepsee or InSight laser (Spectra-Physics) with integrated pump laser tuned at 860 nm


Neoplasia and mRNA expression of LPC and Notch markers were evaluated by histology and qPCR on isolated livers. PHD2 haplodeficiency resulted in enhanced expression of HIF target genes after 17 weeks of DEN compared to wild type (WT) littermates but had no effect on the onset of neoplastic transformation. The mRNA expression of Afp and Epcam was increased at all time points following DEN whereas CK19, Prom1 and Notch3 were increased after 17 weeks of DEN, without difference between PHD2+/- and WT mice. MDR1 mRNA expression was increased in all DEN treated mice compared to saline control with increased expression in PHD2+/- compared to WT from 15 weeks. These results indicate that the effects of PHD2 haplodeficiency on the expression of LPC and Notch markers manifest during tumour nodule formation and not early on during neoplastic transformation. Activation of the hypoxia-inducible factor (HIF)-pathway in hepatocellular carcinoma (HCC) induces therapy resistant tumours, characterized by increased liver progenitor cell (LPCs) characteristics and poor prognosis. We previously reported corresponding results in mice with HCC in which hypoxia was mimicked by prolyl hydroxylase domain (PHD) inhibition. Here, we aimed at investigating whether induction of LPC characteristics occurs during the onset of hepatocarcinogenesis and if this is associated with activation of Notch signalling. Dietheylnitrosamine (DEN) was used to induce hepatic tumours in PHD2 haplodeficient (PHD2 Liver al., 2011; Heindryal., 2011). Insufficient oxygen supply results in hypoxia, a situation known to inhibit prolyl hydroxylase domain (PHD) enzyme activity, causing stabilisation of the hypoxia inducible factor (HIF). HIF stabilisation and its nuclear translocation results in the transactivation of genes involved in cell-survival by, amongst others, stimulating (neo-)angiogenesis , and boosting the anaerobe glucose metabolism  ), in murine diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCC), results in a more aggressive mixed hepato-cholangiocarcinoma (HCC-CC) phenotype high in liver progenitor cell (LPC) characteristics, coinciding with increased expression of markers for metastasis and actors of the Notch signalling pathway . In heaLPCs then proliferate and migrate to the site of injury where they differentiate to replenish the lost cell mass by a series of tightly organised interactions controlled by the Notch and Wnt signalling pathways. Activation of the Notch pathway drives LPC's towards a cholangiocytic phenotype, while Wnt induced inhibition of Notch signalisation results in hepatic differentiation .The Notch pathway not only plays a pivotal role in the cell- fate determination of LPCs, it is also shown to be an important mediator of hepatocarcinogenesis. Interestingly, activation or inhibition of the different Notch receptors can have both pro- and anti- oncogenic effects . A heterozygous couple was used for breeding and offspring was genotyped using the following primers in a concentration of 10 µM: ACCTATGATCTCAGCATTTGGGAG, TCAGGACAGTGAAGCCTAGAAACT and AAATTCTAATCGTAGCTGATGTGAGC .2+/- and wild type (WT) littermates (129S6 background) received weekly intraperitoneal DEN injections . This induces microscopic neoplastic cells after 15 weeks, macroscopic nodule formation at 20 weeks and HCC after 25 weeks, which was previously reported by our group . These al., 2012), we admMice were euthanized at indicated time-points by cervical dislocation, the liver was excised and divided for histology and qPCR analysis, respectively submerged in 4 % formaldehyde  for paraffin embedding and stored at -80 °C in RNA later  for RNA extraction.All experiments were approved by the ethical committee for animal experiments at the faculty of medicine and health sciences of Ghent University Belgium (ECD13/61).General morphology of liver tissue was assessed using Haematoxylin-Eosin, Sirius Red and Reticulin stainings on 5 µm sections of paraffin embedded tissue as routinely described. Neoplasticity was defined as enlarged cells with normal nucleus to cytoplasm ratio (n/c), small cells with increased n/c, enlarged pleomorphic nuclei, and binucleation, (pre) neoplastic hepatocytic lesions were identified by loss of reticulin staining and sirius red staining was performed to identify potential cholangiocytic lesions marked by cholangiofibrosis, as previously described  was used to visualize structures of the cholangiocytic lineage as well as LPCs. Overall CK19 immunoreactivity was measured using Cell D software  and to evaluate the LPC response, 5 portal areas were centred at a magnification of 400 and all CK19 positive single cells were counted. RNA was extracted from 20 mg of frozen liver tissue preserved in RNA-later, according to the manufacturer's guidelines .cDNA was obtained from 1 µg RNA using the iScript cDNA synthesis kit  and quantitative PCR (qPCR) analyses were performed using the Lightcycler 480 Green I master mix , using the primersets listed in Table 1All reactions were run in duplicate; the comparative Cq method was used to determine the number of transcripts which were normalised to reference genes that showed stable expression in all samples, as also previously described  and graphs were illustrated using Graphpad prism 6 software . Kolmogorov-Smirnov test was used to test for normality. Student's T- test was then performed in case of normality; the Mann-Whitney-U test was used for not normally distributed data. P-values ≤ 0.05 were considered significant. All data are presented as mean ±SEM.2 haplodeficiency on HIF stabilisation by assessing the activation of HIF target genes. In early hepatocarcinogenesis, we did not observe a significant activation of the hypoxic pathway compared to saline control mice, in either genotype. However, HIF downstream targets showed a peak RNA expression after 17 weeks of DEN compared to other time points  and neoplastic nodules were observed from 15 weeks onwards . Sirius red staining was evaluated as previously described , Prominin 1 (Prom1), Epithelial cell adhesion molecule (Epcam), Alpha fetoprotein (Afp) and multi drug resistance protein 1 (MDR1). 2 haplodefiency- relate effect could be observed concerning Epcam mRNA expression  as well as the main Notch effector gene Hairy enhancer of split 1 (Hes1). 2+/- livers which coincides with increased expression of markers for hypoxia and HIF stabilisation  proteins, which are inherently expressed by stem -and progenitor cells  are driActivation of the Notch signalling pathway has been shown to be involved in liver and other cancers, with contradictory proposed roles  and tum2+/- and WT mice, Notch3 expression peaked after 17 weeks of DEN, like CK19 and Prom1 mRNA expression, indicating that expression of these markers coincides with nodule formation. This is in line with previous data, were Notch3 overexpressing HCC cells were shown to have increased aldehyde dehydrogenase activity , characal., 2015). 2+/-mice to evaluate the effect of increased HIF stabilisation during early hepatocarcinogenesis. However, increased HIF signalling was only observed during nodule formation, coinciding with increased mRNA expression of LPC characteristics and Notch3. We hypothesise that previously observed effects of increased HIF signalling on tumour phenotype manifest during tumour growth rather than development and are not preceded by an early LPC or Notch signature. Further elucidating possible mechanisms involved in this process could help to develop new therapeutic strategies to improve prognosis of patients with tumours growing in a fibrotic background, or receiving hypoxia- inducing therapiesIn conclusion, we used PHDThe authors would also like to thank Femke Wulgaert for her help with experiments as well as Anja Van den Bussche and Petra Van Wassenhove for their excellent technical assistance.The authors declare there is no conflict of interest.


POLR2A, an essential gene where hemizygous deletions are known to confer elevated sensitivity to pharmacological suppression. We next used this principle to identify several genes and pathways that show patterns indicative of purifying selection to avoid deleterious mutations. These include the POLR2A interacting protein INTS10 as well as genes involved in mRNA splicing, nonsense-mediated mRNA decay and other RNA processing pathways. Many of these genes belong to large protein complexes, and strong overlaps were observed with recent functional screens for gene essentiality in human cells. Our analysis supports that purifying selection acts to preserve the remaining function of many hemizygously deleted essential genes in tumors, indicating vulnerabilities that might be exploited by future therapeutic strategies.Identification of cancer driver genes using somatic mutation patterns indicative of positive selection has become a major goal in cancer genomics. However, cancer cells additionally depend on a large number of genes involved in basic cellular processes. While such genes should in theory be subject to strong purifying (negative) selection against damaging somatic mutations, these patterns have been elusive and purifying selection remains inadequately explored in cancer. Here, we hypothesized that purifying selection should be evident in hemizygous genomic regions, where damaging mutations cannot be compensated for by healthy alleles. Using a 7,781-sample pan-cancer dataset, we first confirmed this in  Cancer is one of the leading causes of death worldwide. It is caused by the accumulation of genetic changes (i.e. somatic mutations) in cells, often taking many decades. Like the evolution of a species, the evolution of cancer cells is driven by different selection processes. Changes that are advantageous for the fitness of a cancer cell will be retained (positive selection), while disadvantageous characteristics will be removed . While positive selection is well studied and has been used to identify new genes that are responsible for the development of cancer, negative selection has remained elusive and is understudied in this context. In this study, we identify genes that show signals indicative of negative selection against damaging somatic mutations. We accomplish this by focusing on genes that have lost one of their two copies, supposedly making cells more vulnerable to damaging mutations in the remaining copy. The identified genes were found to be active in cellular processes which are essential for cancer cell survival and constitute potential therapeutic targets. Cancer cells evolve by natural selection, which favors advantageous genetic changes (positive selection) and disfavors deleterious changes (negative or purifying selection). Identification of somatic mutation patterns indicative of positive selection has become a major goal in cancer genomics ,2. This POLR2A, a subunit of RNA polymerase II, creates a therapeutic vulnerability, as cancer cells carrying only a single functional copy of the gene are more sensitive to inhibition of its protein product ,,20,POLR2 samples . Observe samples  such thaPOLR2A when subjected to single-allele copy number loss.Taken together, our results suggest that purifying selection against high impact mutations is detectable in essential genes like P-value threshold of 0.05 and false discovery rate (FDR) of 50%, respectively , hemizygously deleted in 29% of samples and, interestingly, a known interaction partner of POLR2A [POLR2A, on chromosome 17p, was found on rank 14 . Several other top-ranking genes were similarly related to transcription or RNA processing, including bromodomain proteins BRPF1 and BRD1 , and PRPF8 , a key component of the spliceosome [POLR2A, PRPF8 co-localizes with TP53 (89.3% of TP53 deletions had concomitant deletion of PRPF8). A gene set enrichment analysis (GSEA) of high-ranking genes (76 genes with unadjusted P≤0.05) confirmed strong enrichment of RNA-related pathways including RNA processing (FDR = 2.8*10−3), translation (FDR = 0.13) and RNA splicing (FDR = 0.13), supporting that the integrity of RNA processing is essential for cancer cell viability , all HeZD events were attributed to broad, rather than focal, deletions, consistent with passenger effects rather than driver roles for these deletions.Similar to  results . These s results . This wa results . AdditioP = 0; Copy number data inferred from whole genome sequencing was available for a subset of 1,065 samples (13.5%) and correlated strongly with the original SNP6-based data  genes, again predominantly RNA-related and including spliceosome, integrator, and mRNA decay complexes   . These rIt has been shown that some protein complexes are comprised mostly of essential genes  . Using tFinally, the genes we identified under purifying selection were also weakly enriched for loss-of-function intolerant genes that were recently described by the Exome Aggregation Consortium  S6 Fig)S6 Fig).In addition to somatic mutations, genes can get inactivated by other genomic events like homozygous deletions (HoZD) or promoter methylations. As genes subject to purifying selection are not expected to undergo these events, we analyzed our identified genes for such alterations.P = 0.49, two-sided Wilcoxon rank sum test; P = 0.064). As we also found a higher proportion of the much more frequently occurring hemizygous deletions for these genes , as would be expected for genes under purifying selection; Unexpectedly, we found no difference between the proportion of samples that contained HoZD in the 76 top-ranked genes from our purifying selection screen as compared to the other genes screened for . While RNA processes have been found to be essential in human cell lines before ,11,34,35 pausing  and has  pausing ,37. PRPFPrevious screens in cell lines have indeed suggested that many hemizygous deletions sensitize cancer cells to inhibition ,11, but We have provided results supporting that, with the current amount of available somatic mutation data from tumors, and by considering HeZD genomic regions, purifying selection against damaging somatic mutations is becoming discernible in individual essential genes. Since these deletions occurred in up to 29% of all tumors, our approach may reveal frequently occurring cancer vulnerabilities that can be exploited by future therapies.et al. [Mutation annotation format (maf) files from 26 different cancer types were downloaded from The Cancer Genome Atlas (TCGA) data portal. Selection and processing of data was performed as suggested by Kandoth et al. . Data fret al. . All dupANNOVAR  was usedSegmented genomic copy number alteration (CNA) data  were downloaded from the Broad Institute. GISTIC 2.0 was used to derive copy number status information for each gene and sample . GISTIC Level 3 RNASeqV2  mRNA expression data were downloaded from the TCGA data portal. Gene expression was evaluated for each cancer type. A gene was considered expressed when the median RSEM expression value in a cancer type was higher than 200. Only expressed genes were used for further analysis.Human methylation 450 platform data were downloaded from the TCGA data portal with the bioconductor package TCGAbiolinks [P≤0.01) given the total number of mutations for that gene and the gene length.To avoid any bias in the detection of purifying selection due to the clustering of (silent) mutations at similar genomic locations caused by positive selection as has been described for different cancer driver genes ,47, we rAs negative selection is only expected to occur in genes that are expressed by a tumor cell, we further restricted the analysis to mutations occurring in expressed genes (as defined above). Data from somatic mutations hitting non-expressed genes were used for negative control purposes.Genes under purifying selection were defined as those that had a lower CADD functional impact score in samples harboring a hemizygous deletion as compared to copy number neutral samples (using a one-sided Wilcoxon rank sum test). Because these differences in functional impact scores can only be determined when a minimal number of mutations are present in both groups, the analysis was limited to genes containing at least 10 mutations in both the hemizygous deletion and the copy number neutral group. These filter criteria yielded a set of 1,187 genes that could be screened for signals of purifying selection. FDR values were calculated using the Benjamini Hochberg method . Purifyiet al. [The 96 mutational substitution classes as defined by Alexandrov et al.  were detsynonymous SNV were classified as silent mutations (s). All other annotated point mutations were classified as non-silent (n). The number of non-silent and silent mutation sites was calculated by simulating all three possible substitutions for each CDS position in a specific gene . Because the probability of any somatic mutation hitting a particular site is defined by the underlying mutational processes and hence its mutational signature, we determined the proportions of each substitution class (96-class model) for each gene and copy number state (HeZD and CNN) and used these as prior probabilities to determine the theoretical number of silent (S) and non-silent (N) mutation sites. Therefore, we used a sampling approach with a mutation probability determined by its specific substitution subclass. Sampling was done 1 million times to minimize variability. dN/dS was then calculated as the ratio of n/N over s/S. Fisher’s exact test (one-sided) was used to check whether dN/dS ratios were lower than 1 and hence indicative of purifying selection.The metric dN/dS is the ratio of non-silent to silent mutations per site ,14. For To determine the expected functional impact (CADD) score in the absence of any selection pressure, we calculated the scores for each possible point mutation in a gene using ANNOVAR and used the same 96-class mutational signature-based sampling approach as described higher to get the simulated functional impact score. The normalized CADD score was then calculated by normalizing the difference between each observed CADD score and the median of the simulated score to the maximal difference for each cancer  was performed to determine the pathways and protein complexes in which the retrieved genes were active. GO biological process gene sets were downloaded from the Molecular Signatures Database v5.0  and CORUS. cerevisiae essential genes as a benchmark [The purifying selection results were compared with the results from two recent orthogonal methods to identify essential genes in human cancer cell lines: two CRISPR/Cas9 methods (referred to as CRISPR-w ) and oneenchmark . Only geThe enrichment of essential genes identified by these orthogonal methods for the purifying selections results was evaluated using Fisher’s exact test. Genes were defined essential when identified as essential in at least one cell line in the corresponding CRISPR screen. The gene-trap approach can only identify essential genes in haploid regions, and diploid genes (located on chromosome 8 and part of chromosome 15 in the KBM7 cell line) were therefore excluded.S. Cerevisiae essential gene homologs were determined using the Bioconductor package [The R statistical package was used for all data processing and statistical analysis. Details on statistical tests used are reported in the respective sections. The Manhattan plot and  package .S1 FigPOLR2A was predicted using the CADD score for both copy number groups and each cancer type. p-values are indicated above the plots for those cancers that contain mutations in both groups. Nine out of eleven analyzable cancer types (as indicated below each plot) contained a lower CADD score in HeZD samples, although this never reached statistical significance. Mutations in HeZD and CNN samples are indicated by asterisks and dots respectively. Horizontal lines on plots indicate median values.The functional impact of all observed mutations in (PDF)Click here for additional data file.S2 Fig(a) Pie charts show the proportion of the 25 different cancer types in samples that are CNN for POLR2A or contain a HeZD in this gene. (b) Mutational signature examples of metastatic melanoma (SKCM) and breast cancer (BRCA). A signature is determined by the contribution of 96 mutation classes, i.e. the combination of 6 substitutions (shown in colors), 4 up- and 4 downstream base pairs, as indicated. (c) CADD scores were simulated for POLR2A using the cancer-specific mutational signature as prior mutation probabilities (see (d) Normalization approach. Each observed CADD score  is normalized to the median simulated score . (e) CADD normalization exemplified for POLR2A.ties see . Median (PDF)Click here for additional data file.S3 Fig(a) qq-plots show the negative effect (CADD score HeZD<CNN) on the upper left side and the positive effect (CADD score HeZD>CNN) on the bottom right side. (b) Copy number-specific CADD (left) and normalized CADD (right) scores from the observed mutations in RB1. (c) Proportion of Cancer Gene Census (CGC) genes for genes with (+) or without (-) signals of positive selection in the HeZD state (defined as FDR≤0.25). (d) Proportion of CGC genes known to be dominant and recessive for genes with or without signals of positive selection in the HeZD state. As classical (two-hit) tumor suppressor genes are expected to operate in a recessive way, these differences in proportions clearly suggest an enrichment of tumor suppressor rather than oncogenes.A genome-wide screen was performed on 1,187 genes to detect differences in the CADD scores between CNN and HeZD genes using a two-sided Wilcoxon rank sum test. (PDF)Click here for additional data file.S4 Fig(a) Correlation between median CADD scores of Amp versus HeZD (left) and CNN (right) genes. Spearman correlation coefficients and p-values are indicated on top of the plots. (b) Boxplots of median CADD scores for the 3 copy number states. Results from individual gene analyses are indicated in For each gene that was identified to be under purifying selection (76 top ranked genes), the median CADD score was determined for HeZD, CNN and amplified (Amp) samples. (PDF)Click here for additional data file.S5 Fig(a) Cancer types and number of samples for which WGS segmentation data were available. (b) Correlation between gene-based segmentation values derived from SNP6 arrays as a function of values derived from WGS. Each dot represents a segmentation value of one gene in one sample. For visualization purposes the points shown are limited to a random set of 10,000 points. Spearman’s correlation coefficient is shown on top. (c) Copy number-related CADD scores from the observed mutations in the top-5 ranked genes from the main analysis. Horizontal lines on plots indicate median values. WGS-based copy number segmentation data were downloaded from TCGA and analyzed using GISTIC 2.0. Results from individual gene analyses are indicated in (PDF)Click here for additional data file.S6 FigP≤0.05 respectively) and the other screened genes (P>0.05) that were previously described as intolerant to loss-of-function variants by the EXaC consortium [Bar plot shows the proportion of genes identified to be under purifying selection (at 50% FDR and nsortium .(PDF)Click here for additional data file.S7 FigP≤0.05 respectively) and the other screened genes (P>0.05) the proportion of cell lines containing hemizygous (a) or homozygous (b) deletions. (c) Bar plot shows the proportion of genes containing at least 1 HoZD. P-values were calculated using two-sided Wilcoxon rank sum test (panels a and b) or Fisher’s exact test (panel c). Copy number data were downloaded from the Cosmic Cell Lines Project [Boxplots show for both sets of genes under purifying selection (at 50% FDR and  Project .(PDF)Click here for additional data file.S1 Tablep_CADD column in first tab). p-values were calculated using a one-sided Wilcoxon rank sum test to identify genes with lower functional impact scores in the HeZD compared to the CNN state. Different tabs show the results from the main analysis (1), normalized CADD scores (2), analysis focusing on amplified genes (3) and after classifying copy number changes using WGS data (4).Genes were ranked based on increasing p-values for the CADD scores ((XLSX)Click here for additional data file.S2 TableEnrichments are calculated using Fisher’s exact test on genes showing any sign of purifying selection (76 gene set). Pathways are ranked based in increasing enrichment p-values. Tabs show results of enrichment for genes active in GO biological processes (1), CORUM protein complexes (2) and CORUM essential protein complexes (3). Essential protein complexes were defined as protein complexes that contain at least 70% essential genes, in which gene essentiality was determined based on the CRISPR screens by Hart et al. and Wang et al. ,25.(XLSX)Click here for additional data file.S3 TableMutation data from genes active in the same GO biological process (first tab) or CORUM protein complex (second tab) were pooled and both copy number states were analyzed for differences in CADD functional impact scores, similar to the main analysis. Pathways/Complexes are ranked based on increasing p-values.(XLSX)Click here for additional data file.


Here, we show that (i) three common macroalgae are more damaging to a common coral when they compete under CO2 concentrations predicted to occur in 2050 and 2100 than under present-day conditions, (ii) that two macroalgae damage corals via allelopathy, and (iii) that one macroalga is allelopathic under conditions of elevated CO2, but not at ambient levels. Lipid-soluble, surface extracts from the macroalga Canistrocarpus (=Dictyota) cervicornis were significantly more damaging to the coral Acropora intermedia growing in the field if these extracts were from thalli grown under elevated vs ambient concentrations of CO2. Extracts from the macroalgae Chlorodesmis fastigiata and Amansia glomerata were not more potent when grown under elevated CO2. Our results demonstrate increasing OA advantages seaweeds over corals, that algal allelopathy can mediate coral-algal interactions, and that OA may enhance the allelopathy of some macroalgae. Other mechanisms also affect coral-macroalgal interactions under OA, and OA further suppresses the resilience of coral reefs suffering blooms of macroalgae.Many coral reefs have phase shifted from coral to macroalgal dominance. Ocean acidification (OA) due to elevated CO Coral reefs are one of the most diverse and complex ecosystems on the planet and provide livelihoods, food, and important ecosystem services for hundreds of millions of people24672 is absorbed by the ocean and lowers pH14152 concentrations may enhance fleshy algal growth rates151714152 concentrations show increased fleshy macroalgal abundance2 in coral-macroalgal competition are currently limited to a single pair of speciesThe drivers of coral loss and macroalgal increase are varied and include herbivore loss through overfishing or disease, decreased water quality associated with nutrient enrichment and sedimentation, coral mortality caused by predators, hurricanes and cyclones, bleaching and diseases, algal colonization associated with coral morality, and a failure of juvenile corals and fishes to recruit once reefs become algal dominated291011Some algal competitive mechanisms have been recognised. Macroalgae may directly overgrow coral tissue, reduce light levels required for photosynthesis, abrade tissue, or produce chemical compounds that directly6222325272 tends to increase the production of plant secondary metabolites, but responses are variable292 decreases phenolics in seagrasses2 could increase the production and release of macroalgal primary metabolites (dissolved organic carbon -DOC), and release of DOC has been associated with a detrimental increase in microbial activity and hypoxic conditions on coral surfaces2535In terrestrial systems, elevation of COin-situ field experiments to quantify the effects of elevated CO2 on the strength of allelopathic interactions between macroalgae and a common coral on the Great Barrier Reef (GBR), Australia. We assess the allelopathic potency, rather than the concentrations, of extracts from the surfaces of macroalgae because many allelopathic compounds are unknown, may act synergistically or additively and thus their combined effects cannot be quantified by evaluating the concentrations of a few known compounds. To examine the generality of macroalgal effects, we used three species of macroalgae (seaweeds) common in GBR reefs: Chlorodesmis fastigiata, Canistrocarpus cervicornis (synonym Dictyota cervicornis) and Amansia glomerata and tested the impact of each on the common coral Acropora intermedia.Because most macroalgal secondary metabolites are carbon-based3738A. intermedia and each macroalga when in direct contact and exposed to either present day levels of CO2 or to levels projected to occur by the mid and end of the century under the RCP 8.5 CO2 emission scenarios . Once the ability of macroalgae to damage coral tissue under elevated CO2 levels was established, we conducted a second experiment to assess the effect of algal allelochemicals alone by embedding natural concentrations of lipid-soluble extracts from algal surfaces into experimental gels and applying these, and appropriate controls, to A. intermedia growing in the field. For this experiment, the three species of macroalgae were incubated (in isolation) under either high CO2 concentrations (936 CO2 ppm) or ambient CO2 concentrations (380 CO2 ppm) and their lipid-soluble, surface chemicals extracted at the end of the 14 day incubation period. Although macroalgae may induce allelopathy when competing with corals2 alone. Surface extracts were then imbedded in gel pads that were placed in contact with un-manipulated corals in situ on the reef. We used un-manipulated corals to avoid confounding altered potency of the extracts with changed susceptibility of the corals exposed to differing levels of CO2. The chlorophyll fluorescence of the coral’s symbiotic dinoflagellates, which indicates the overall photosynthetic efficiency of corals, was assessed as an indicator of stress2225First, we conducted an experiment to assess competitive outcomes between the coral 2 treatments significantly affected coral tissue loss. Macroalgal contact with corals increased the rate of coral tissue loss in all CO2 conditions  significantly more than extracts from thalli grown in ambient CO2 ] and C. cervicornis (only under high CO2) significantly reducing effective quantum yield relative to control treatments   . ExtractCorals have declined and seaweeds have increased in abundance on reefs world-wide1237912625263436Acropora intermedia under some conditions, but C. cervicornis became allelopathic only under conditions of OA (Lobophora cf. papenfussii), but the mechanism was unknown and it was unclear if the cause was enhanced algal allelopathy, altered role of DOC, enhanced coral susceptibility, or a combination of processes2. Investigations have documented macroalgal allelopathy against corals in about 75% of the 40 + coral-macroalgal combinations assayed to date22243844C. cervicornis (L.cf. papenfussiiCanistrocarpus (=Dictyota), and likely in Lobophora346474849All three macroalgae we investigated induced considerable coral tissue loss and two were allelopathic to ns of OA , Table 2vicornis , and L.cC. cervicornis grown under CO2 enriched conditions in flow through tanks caused greater damage to in-situ corals than extracts from C. cervicornis grown under ambient CO2 in equivalent flow through tanks. Because the extract experiment was conducted using in situ corals that had not been exposed to elevated CO2, CO2 effects on the coral can be ruled out. The stronger allelopathy noted in C. cervicornis becoming more allelopathic rather than the coral becoming more susceptible. The magnitude of the stress caused by the algal extracts in our experimental coral ], or that the use of different methodologies and exposure times to algal interactions causes variable damage to the corals . Importantly, however, coral fluorescence levels were significantly lower when exposed to extracts from C. cervicornis grown under high CO2 levels, compared to extracts from algae grown under ambient CO2, indicating a reduction in the photosynthetic efficiency of the coral symbionts due to lipid-soluble surface extracts alone.Surface extracts from the brown macroalga 2 generally enhances production of secondary metabolites in terrestrial plants292 on secondary metabolites in marine plants appear variable. For example, concentrations of polyphenolics decline while concentrations of dimethylsulfoniopropionate (DMSP) appear to increase with elevated CO231322238382 in some terrestrial plants due to higher availability of carbon for plant metabolism532 might have enhanced production of allelopathic lipids in C. cervicornis, but not in the other two macroalgae we investigated  (see Lobophora)Dictyota has demonstrated increased growth under OA conditions172 modifies the potency of Canistrocarpus allelopathy, or the chemical compounds involved in these interactions require additional investigation.Increased atmospheric CO.55) see , and ourA. glomerata and Ch. fastigiata also increased under OA conditions, but surface extracts from these species grown under elevated CO2 did not show enhanced allelopathic activity on corals in situ. Thus, other competitive mechanisms or some processes affecting coral physiology may be driving interactions among these species. Corals exposed to OA but free of macroalgae experienced some tissue loss, suggesting that elevated CO2 alone is a physiological stressorin situ corals we used for testing allelopathic potency  under elevated CO2. Our results show that although all experimental macroalgae caused damage to the corals when exposed to elevated CO2 levels, there is variability in the mechanisms by which algae stress and kill corals, suggesting that the relative importance of each mechanism varies under OA. Allelopathy in C. cervicornis, and possibly an altered role of DOC in A. glomerata and Ch. fastigiata, in addition to direct impacts on coral holobiont physiology and coral microbiomes may play important roles in driving coral-macroalgal interactions as concentrations of CO2 increase in future oceans.In our outdoor mesocosm experiment, the rates of tissue loss for corals in contact with  potency 5556. In 2. This suggests that the competitive advantage of some macroalgae over corals may increase and further accelerate phase shifts as OA continues. This may be exacerbated by enhanced macroalgal growth and cover under projected near-future CO215161718202 levels. For example, the brown alga C. cervicornis and the green alga Ch. fastigiata both damage corals faster  and Lobophora on many Caribbean32349602226C. cervicornis , although this hypothesis needs testing , GBR (23°26′30″S–151°54′46″E). We used the branching coral Acropora intermedia and three fleshy macroalgae, the green alga Chlorodesmis fastigiata, the brown alga Canistrocarpus (=Dictyota) cervicornis, and the red alga Amansia glomerata. These seaweeds were common and frequently in contact with corals on the reefA. intermedia was the assay coral because it is abundant in the study area . C. cervicornis and A. glomerata are also abundant in Heron reefs, the latter particularly so at the base of coral branches in coral dominated areas. Transect surveys show C. cervicornis, A. glomerata cover in average 4% and 5% respectively in shallow reefs of Heron Island . Ch. fastigiata is common but less abundant (1% cover), and similar to C. cervicornis, it produces bioactive terpenoids that harm coralsTo evaluate the effects of elevated CO2/pH manipulation. The coral-algal competition treatment included (1) coral- Ch. fastigiata interaction pair ; (2) coral- C. cervicornis interaction pair (as for the previous treatment level), (3) coral- A. glomerata interaction pair, (4) coral control ; and (5) a coral branch in contact with an inert algal mimic (monofilament fabric) attached to the base of the coral with a plastic cable tie; this assessed the effect of an alga’s physical, but not chemical or biological, presence on the coral , medium , and high  CO2, simulating current levels and those predicted for the years 2050 and for 2100, respectively, under the RCP 8.5 model of the Intergovernmental Panel for Climate Change (IPCC)2 concentrations were achieved using established methods152 bubbled into 200 l mixing sumps. pH sensors  logged measurements every 30 minutes, and calibration was linearly performed using three NIST-certified pH buffers . Ambient and CO2 treated seawater from the mixing sumps continuously fed the experimental tanks  at a flow rate of 1.79 ± 0.08 (SEM) l/min using unfiltered seawater from the reef flat. Total alkalinity was estimated from each CO2 treatment by an open-cell potentiometric titrator (Model T50 Mettler Toledo) using triplicate water samples taken from the experimental containers every 4 hours during a 24 h-period. We also collected water samples from the sumps and did not find significant differences in carbonate chemistry between sumps and experimental tanks . Total alkalinity and pH values determined in the samples were utilised to constrain the carbonate chemistry of the experimental system68Corals and macroalgae were exposed to three CO2, and macroalgal treatment levels, each tank holding four experimental sub-samples . Seawater temperature averaged 25.87 ± 0.14 (SEM) °C and salinity was 34.46 ppm (measured using a portable refractometer) during the experiment. The experiment was carried out for 25 days during March 2013. The response variable included a measurement of coral tissue loss , which was recorded daily in the tanks as the amount of sloughed tissue using four categories: 0, 1–33, 34–66 and 67–100% per coral branch. Coral branches exhibiting partial tissue loss were examined under the stereomicroscope confirming the absence of coral tissue in the affected areas. Coral tissue loss generally progressed from the base upwards along the branch.Three replicate 20 l tanks were used for each combination of competition, COples see . Species2 enhanced the allelopathic potency of seaweeds, the macroalgae C. cervicornis, A. glomerata and Ch. fastigiata were exposed to elevated CO2 concentrations, their surface metabolites extracted, these extracts applied to corals in situ, and the damage on the corals compared to control extracts from algae grown at ambient CO2 concentrations. Macroalgal collections and CO2 manipulations were conducted in the same way as described above. Macroalgae were exposed to two levels of CO2, ambient  and high  for 14 days . Ambient and high CO2 treated seawater from the mixing 200 l sumps continuously fed the experimental tanks at a flow rate of 1.79 l/min  in situ at a depth of 6 m on the fore reef of Heron Island (Harrys Bommie). Gel pads were placed approximately 3–5 cm from the tip of the coral branch and gently secured with a plastic cable tie. 15 replicates per CO2 and algal treatment combination were used in addition to 15 control pads  to test for possible effects of the gels, solvents, and extraction protocol on the corals. Each coral branch contained only one gel and gel pads from the different algal species were not intermixed in each coral branch. In total, we used 105 gel pads that were placed on coral colonies distributed over an area of approx. 150 m2. Gel pads were removed after 24 hours and the symbiotic dinoflagellate chlorophyll fluorescence  assessed in situ beneath the area that had been covered by the gel pad using a Pulse-Amplitude-Modulation Fluorometer . EQY has been previously used to estimate photosynthetic efficiency and coral health38To test whether elevated CO2 and coral-algal competition as fixed factors, tanks as the nested component, and branches as replicates. There was no significant effect of the nested (i.e. tank) component on the rate of coral tissue loss (p = 0.584). We therefore pooled the data to increase the power of the analysespost hoc Tukey tests  . This raey tests .2  and algal treatments  as fixed factors. The analyses included 15 coral branches as replicates. Significant interactions occurred between treatments, therefore we conducted subsequent one-way ANOVAs within treatment combinations followed by post hoc Tukey tests. EQY values for each CO2 and algal treatment combination were also compared against the phytagel control using a 2-sided Dunnett test using SYSTAT software. Data were tested for normality using Shapiro-Wilk test and for homogeneity of variances using Levene-Equality of Several Variance  and Cochran tests  and data were not transformed for the analyses.EQY values of corals were compared with a two-way ANOVA with COHow to cite this article: Del Monaco, C. et al. Effects of ocean acidification on the potency of macroalgal allelopathy to a common coral. Sci. Rep.7, 41053; doi: 10.1038/srep41053 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Mechanical injury to connective tissue causes changes in collagen structure and material behaviour, but the role and mechanisms of molecular damage have not been established. In the case of mechanical subfailure damage, no apparent macroscale damage can be detected, yet this damage initiates and potentiates in pathological processes. Here, we utilize collagen hybridizing peptide (CHP), which binds unfolded collagen by triple helix formation, to detect molecular level subfailure damage to collagen in mechanically stretched rat tail tendon fascicle. Our results directly reveal that collagen triple helix unfolding occurs during tensile loading of collagenous tissues and thus is an important damage mechanism. Steered molecular dynamics simulations suggest that a likely mechanism for triple helix unfolding is intermolecular shearing of collagen α-chains. Our results elucidate a probable molecular failure mechanism associated with subfailure injuries, and demonstrate the potential of CHP targeting for diagnosis, treatment and monitoring of tissue disease and injury. Collagen denaturation is thought to occur during tissue mechanical damage, but its role in damage initiation is still unclear. Here, the authors use a collagen hybridizing peptide to provide insights into the molecular mechanisms leading to collagen unfolding during tendon mechanical stretch. Many painful and physically debilitating conditions involve subfailure tissue damage, or mechanical damage to seemingly intact connective tissues. Examples include rotator cuff disease13568910111213Several experimental15161815161722151617To date, studies that examined mechanical damage to collagen fibrils in tendon were performed on severely damaged samples 1516172021n can hybridize to unfolded collagen strands that have been denatured by heat or by protease activity, both in vitro and in vivo26A quantitative technique capable of direct detection and spatial localization of collagen molecular damage at multiple physical scales would be highly useful for understanding the relationship between tissue injury and molecular damage, while potentially providing a means for early detection and treatment of tissue injuries. This led us to consider the use of collagen hybridizing peptide (CHP), also known as collagen mimetic peptide, to detect molecular level damage to collagen252728Considering the ability of CHP to specifically hybridize with the unfolded collagen triple helix, we hypothesized that carboxyfluorescein (CF) labeled CHP (CF-CHP) could be used to detect, localize and quantitatively compare molecular level damage to collagen caused by mechanical loading. We used CF-CHP to detect damage due to collagen unfolding during subfailure loading for a series of mechanically stretched rat tail tendon (RTT) fascicles. We show that the triple helix-mediated hybridization process effectively reports the location and level of molecular damage of the collagens in tendon, which provides new insights into the mechanism of mechanical damage to fibrous collagens in load bearing tissues. We also conducted steered molecular dynamics (MD) simulations to investigate the molecular response of the collagen triple helix under loading at the atomic scale and identified a new molecular mechanism that has been previously overlooked but is capable of causing permanent mechanical damage to fibrous collagens in load bearing tissues.3–(GPO)9], washed and imaged using wide-field fluorescence microscopy to assess the CHP binding , which was previously used to detect denatured collagen for only highly damaged tendon samples15CF-CHP fluorescence within fascicles was imaged using multiphoton confocal microscopy  to corre826SCHP, SCHP, confirming our observation that CHP binding in damaged fascicles is specifically via the triple-helical hybridization to unfolded collagen. The mean areal density of NP-CHP in the 12% strain group was significantly higher than the undamaged group (P<0.05). The mean areal density of NP-CHP binding in regions with visible collagen fibril disruption was higher than the mean areal density in regions with retained d-banding in the 12% strain samples, although the difference was not statistically significant  under transmission electron microscopy (TEM). TEM has long been used to interrogate the integrity of collagen fibrils through visualization of the characteristic 67 nm periodic d-banding pattern P=0.10,  inset.To investigate the effects of repeated subfailure loading on collagen molecular damage, we tested fascicles in cyclic fatigue at physiological and quasi-static loading frequenciesThe two different loading frequencies produced different CF-CHP staining patterns . While s41We performed computational simulations to gain insights into the potential molecular mechanism of triple helix unfolding under tension that permits CHP binding. Although there are a variety of ways that the collagen molecule could unfold1943444546196(GPA)2(GPO)2] also indicated that triple helix unfolding by strand sliding as a result of shear-dominant loading takes place long before the tension-dominant loading can cause any type of irreversible damage to the collagen model peptide . Moreover, at a physiologically relevant loading rate (dɛ/dt<1 s−1), the peak force, which is the force needed to fail full-length collagen molecule, was much lower than the mechanical strength of a covalent crosslink (6–8 nN). Therefore, the shear-dominant mode of collagen molecular unfolding is a realistic failure mechanism for a full-collagen molecule inside a fibril under tensile loading conditions.To demonstrate that the molecular mechanism for shear-induced unfolding of a model collagen triple helix is valid for a full-length collagen molecule confined inside a microfibril and interacting with its neighboring molecules, we built a full atomic scale molecular model of a collagen microfibril and repeated the cal test . This mocal test . The crical test  as this Mechanical damage of collagen fibrils and fibers in connective tissue has been extensively studied by both experiments and computational modeling1113161724Previous research on damage to collagen across its hierarchy of structure has primarily focused on the level of the collagen fibril1316321515212244By improving the extraction methods for detecting mechanically denatured collagen, we were able to correlate the amount of collagen digested by trypsin with CHP binding even for samples subjected to a single cycle of moderate subfailure loading. To our knowledge, this is the first direct and convincing evidence other than the CHP binding that collagen denaturation is associated with the onset of mechanical damage. Although the improved digestion method yielded quantitative estimation of collagen denaturation, it is a complicated multistep process requiring over 64 h of assay time. CHP-mediated imaging, on the other hand, is a simple and easy method that can reveal location and level of collagen damage from a single fluorescence photograph and the versatility of CHP functionalization allows multi-modality imaging at multiple physical scales, as demonstrated here with fluorescence and electron microscopy. In addition, CHP binding and detection is a non-destructive method with significant potential for diagnostic and therapeutic applications.Continuous research over more than two decades has focused on explaining molecular level deformation mechanisms of collagen. Based on x-ray diffraction measurements of d-band spacing and orientation, Sasaki and Odajima555657Our results provide an explanation for the progression of mechanical damage in collagen-rich tissues, as well as implications for initiation of the inflammatory response and subsequent healing. It is now widely accepted that overuse tendinopathies involve tissue microtrauma or subfailure damage, which causes measurable changes in tissue scale material properties, and often progress to observable and symptomatic damage in the form of partial and full tendon tears1235260in vivo detection of collagen damage associated with traumatic injuries, for the initial detection and monitoring of diseases such as osteoarthritis and tendinosis, as well as detection of mechanical damage to collagenous tissues caused by extreme mechanical conditions such as tendon fatigue before tissue failure under cyclic loading. Since CHP can be readily conjugated to bioactive molecules26CHP binding provides an exciting and unprecedented tool for detection and quantitative comparison of mechanical damage to collagen in connective tissues, and offers the potential for translational application to diagnosis, staging and treatment of numerous connective tissue disorders and diseases. Standard antibody staining methods that rely on epitopes of a few amino acid residues do not provide sufficient binding sensitivity to distinguish damaged collagen molecules from intact molecules see . Unlike 30−1 reactive site density. For CF-CHP synthesis, the sequence GGG(GPO)9 was completed by N,NN′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU) chemistry, followed by on-resin labeling with 6 molar equivalents of CF, activated by 6 molar equivalents of PyAOP in NMP for over 24 h. The full-length fluorescent CHP was cleaved from the resin by treating the resin with trifluoroacetic acid (TFA)/triisopropylsilane (TIS)/H2O (95:2.5:2.5) for 3 h, and the cleaved peptide was purified by reverse phase HPLC on a semi-preparative Vydac C18 column using a linear gradient mixture of water (0.1% TFA) and acetonitrile . The purified peptide was subsequently lyophilized, confirmed for purity by matrix assisted laser desorption ionization time of-flight mass spectrometry , and reconstituted in 1 × phosphate-buffered saline (PBS).All peptides were synthesized by automated solid phase peptide synthesis using TentaGel R RAM resin with 0.18 mmol g9 and the scrambled sequence (CPGOGPGPOPOGOGOPPGOOPGGOOPPG) were prepared by automated solid phase peptide synthesis, purified by reverse phase HPLC and their molecular weight confirmed using matrix assisted laser desorption ionization time of-flight mass spectrometry, as described above. Purified C(GPO)9 was mixed with 10 nm citrate-stabilized gold nanoparticle solution , giving a final concentration of 25 μM CHP and 5 nM gold nanoparticles, respectively. The CHP and gold nanoparticle reaction mixture was incubated overnight at room temperature and unreacted C(GPO)9 was removed by repeated centrifugation and washing with deionized water.For synthesizing gold nanoparticle conjugated CHP (NP-CHP), the sequence C(GPO)RTT fascicles were dissected from Sprague Dawley rat tails  by holding the proximal tail end while gripping the distal end with a hemostat. The hemostats were twisted around the axis of the tail until the skin separated, at which point the distal end was pulled away from the tail revealing tendon fascicles. The fascicles were cut away and individual fascicles were tested by one of the two protocols in a mechanical tester. All testing was performed on a custom micro-materials test system  consistin=3 samples for each strain group). Unloaded fascicles were used as a negative control (n=5). All tests were performed at room temperature with samples immersed in 1 × PBS. For stress calculations, the fascicles were assumed to have a circular cross-section and the average diameter of each fascicle was calculated from 5 measurements along the length of each fascicle.Tensile tests were performed by loading individual fascicles into the mechanical tester sample clamps and applying a 0.03 N preload to remove slack from the samples and provide a consistent point for the zero-strain length measurement9n=3 samples for each group). The control groups for the fatigue testing consisted of unloaded (n=3) and preconditioned-only (n=3) fascicles. All tests were performed at room temperature with samples immersed in 1 × PBS.Cyclic tests were performed by loading individual fascicles into the mechanical tester sample clamps followed by application of a 0.03 N preload. Fascicles were preconditioned for 10 cycles at 1 Hz from 0 to 4% strain, and 0.03 N preload was reapplied to give a new zero-strain length. Immediately following preconditioning, fascicles were loaded at 1 Hz from 1 to 5% strain for 10, 100 or 1,000 cycles or at 0.1 Hz from 1 to 5% strain for 10 or 100 cycles  channel and 488 nm excitation. Images of whole samples were captured by CCD camera at 16-bit depth using the built-in large image acquisition feature.t-tests with the Holm–Sidak correction for multiple comparisons, with a significance of α=0.05.Wide-field fluorescence images were quantified for fluorescence intensity using a thresholding segmentation method. Micrographs of tensile test samples were thresholded by Otsu's methodSamples that were subjected to tensile testing were imaged by two-photon confocal microscopy, using a custom Prairie View Ultima multiphoton microscope  to localize CF-CHP binding within the tissue. Fascicles were mounted in Fluoroshield mounting media  and imaged with 910 nm excitation and detection in 435–485 nm and 500–550 nm for collagen SHG and CF two-photon excited fluorescence, respectively.Samples were stained with NP-CHP for TEM study to understand the location and condition of CHP binding on collagen fibrils. Tissue sections of damaged and undamaged RTT fascicle were prepared as described by Starborg and coworkersSCHP (n=5 images per group). Staining density of NP-CHP was quantified by the areal density of gold nanoparticles on TEM images, defined as the number of nanoparticles per unit area (# μm−2). For all images, areal density of NP-CHP was determined as the total number of nanoparticles in the image divided by the total image area. Additionally, 12% strain images stained with NP-CHP were quantified to compare areal staining density on undamaged (regions retaining characteristic d-banding pattern) and damaged (regions missing d-banding pattern) areas. All analysis of TEM images was performed in ImageJ; nanoparticles were highlighted and counted using the ‘mark-points' tool and the size of undamaged tissue in the 12% strain images was measured by creating a selection that included all tissue with the characteristic d-banding pattern. T-tests were performed to compare unloaded and loaded tissues, damaged and undamaged regions of loaded tissue, and unloaded tissue stained with NP-CHP and loaded tissue stained with scrambled sequence NP-SCHP. The Holm–Sidak method was used to correct for multiple comparisons.Three groups of samples were stained using NP-CHP: unstretched RTT fascicle, RTT fascicle stretched to 12% strain, and RTT fascicle stretched to 12% strain and stained with a scrambled sequence NP-Mus musculus with the sequence encoded as GEV GPO GPO GPA GEK GSO GAD GPA GSO GTO, which is close to the location of the known intermolecular covalent crosslink at position 930 , constant pressure of 1 atm and constant temperature of 310 K) by using LAMMPS open-source code with interatomic interactions defined by the CHARMM22 force field for proteins and solvent. The simulation time step was 2 fs using a rigid bonds model for all of the covalent bonds between hydrogen atoms and other heavy atoms. Energy minimization and equilibrium simulation was performed for 22 ns for a fully equilibrated structure before mechanical test. A particle mesh Ewald function was used with a grid width <1 Å to calculate the electronic interactions. The steered MD method was used to deform the molecule with a constant loading speed at 0.002 Å ps−1. The triple helix collagen molecule was deformed by fixing one alpha carbon atom at the N-terminus of one chain and tensile force is applied to an alpha carbon atom at the C-terminus of another chain. After a certain amount of tensile strain, the structure was taken out and allowed to stress relax for 40 ns. SASA was computed during the last 20 ns of the relaxation simulation using the VMD Molecular Graphics Viewer6(GPA)2(GPO)2], and the results of these simulations are detailed in The computational model of the collagen triple helix molecule represented type I collagen from 930 ref. . The N-t−1 NaCl was used to fill the gap between collagen molecules, and the system was equilibrated in a NPT ensemble for 6 ns before subjecting to tensile tests. Deformation was applied to the entire system with a constant strain rate during a dynamic run while the system could simultaneously relax. The movement of the alpha carbon atom at the far end of each crosslink was remapped by the deformation of the system for each time step to ensure that the central collagen was deformed with a constant strain rate under a full control. The forces in two crosslinks were averaged and recorded during the loading test.Four collagen molecules were explicitly included in the system and full periodic boundary conditions were applied in all directions. Each collagen molecule with ∼300 nm in length was built according to the coordinates of alpha carbon atoms based on Protein Data Bank entry 3HR2. Two crosslinks, K87 and K930, were created as shown in http://lammps.sandia.gov.The data that support the findings of this study are available from the authors upon reasonable request. The LAMMPS open-source code is publicly available at How to cite this article: Zitnay, J. L. et al. Molecular level detection and localization of mechanical damage in collagen enabled by collagen hybridizing peptides. Nat. Commun.8, 14913 doi: 10.1038/ncomms14913 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Table, Supplementary Methods and Supplementary References