Four non-growing orthodontic patients (age range 14–47 years old) with Class II skeletal patterns (convex profiles with retrognathic mandibles) who sought correction of their crowded teeth and non-surgical correction of their convex profiles were included in this study. These patients were treated using Invisalign clear aligners together with high-frequency vibration (HFV) devices (120 Hz) (VPro5™) that were used by all patients for five minutes per day during active orthodontic treatment. Vertical control and forward rotation of the mandible for each patient was achieved through pre-programming the Invisalign to produce posterior teeth intrusion. Successful forward rotation of the mandibles achieved in all patients led to improvement of their facial convex profiles  improved 2.1 ± 0.5 degrees; FMA (Frankfurt mandibular plane angle) improved 1.2 + 1.1 degrees). Dental decompensation was achieved by lingual tipping of the lower incisors and palatal root torque of upper incisors. The use of HFV together with Invisalign facilitated achieving these results within a 12 ± 6 months period. In addition, more bone labial to the lower incisors after their lingual movement was noted. In conclusion, the use of HFV concurrent with SmartTrack Invisalign aligners allowed complex tooth movement and forward mandibular projection without surgery in non-growing patients with skeletal Class II relationships. The clinical impact and implications of this case series are: (1) the use of HFV facilitates complex orthodontic tooth movement including posterior teeth intrusion and incisor decompensation; (2) forward mandibular projection of the mandible and increased bone formation labial to lower incisors can be achieved in non-growing patients that may minimize the need for surgical intervention in similar cases or gum recession due to lower incisors labial inclination. This study presents a novel technique utilizing high-frequency vibration to shorten treatment time and preserve alveolar bone in challenging orthodontic cases that have been treated with Invisalign Clear aligners are chosen mainly for esthetics, better oral hygiene, and no food limitations or constriction like with fixed orthodontic appliances ,2. Tradiven days . The redven days . This prven days ,5. For taligners ,7,8. Comaligners ,11,12,13aligners . The effaligners . Comparialigners .The common biological effect of these techniques is the activation of local cytokines that accelerate osteoclastic activities that in general activate osteoblasts as well ,11,12,13In this study, four non-growing patients with hyperdivergent and convex profiles due to reterognathic mandibles were treated with Invisalign clear aligners  and HFV was utilized using the VPro5 device  that delivers a frequency of 120 Hz and was prescribed for patients to use once a day for 5 minutes. All patients utilized vertical pull chin cup  and they were instructed to change aligners whenever new aligners could fit without gaps between the teeth and aligners, and if no intolerable force/pressure was felt by use of the new aligners.Case PresentationsThese four cases had the common chief complaint of convex profiles with recessive chin. None of them were interested in any surgical option, while they were interested in improvement of their bites and profiles. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of the University of Alberta .This 28-year-old female presented with the chief complaint that she wanted to correct her crowded teeth (4 mm of crowding in the upper and lower arches) as well as her increased overjet, in addition to improving her convex profile, specifically her recessive chin. Her medical history was noncontributory, clinical records A showed ® retainers and was instructed to rotate between them on a monthly basis and to use fulltime for 6 months, in addition to the continuation of using the VPro5 every day as she did during her active treatment period. Then, she was instructed to wear the retainers at night time for the rest of her life as a night guard. After 6 months, the patient finished her active orthodontic treatment, and she was satisfied with overall results. This 47-year-old male presented with chief concerns including correcting his crowded teeth (upper arch 5 mm and lower arch 4 mm arch length deficiencies), increased overjet (7 mm), and improving his recessive chin without surgical intervention, if possible. The patient was also not interested in regular fixed appliance. Clinical records A–E revea®) included a total of 140 aligners. Interproximal reduction (IPR) was also prescribed for this patient to minimize black triangles between front teeth as well as to move the upper and lower incisors lingually . Intraoral photos and digital models revealed a Class II division 2 malocclusion with increased overjet (5.5 mm) and impinging overbite (95%), as well as moderate upper and lower dental crowding (5 mm in each arch). Treatment plan and progress: The patient and parents were made aware of the challenges of mouth breathing habit and its detrimental effects on treatment outcomes and stability. In addition, myofunctional therapy was proposed using a trainer for braces  that was prescribed to be worn one hour during the day and night time with vertical-pull chin cup . The treatment plan included sequential distalization of the upper posterior teeth utilizing light (3/16 3.5 ounce) Class II elastics for inter-maxillary anchorage and torqueing of upper incisors’ roots palatally. The vertical-pull chin cup together with occlusal coverage of the posterior teeth with the Invisalign aligners (working as posterior bite blocks) were intended to intrude posterior teeth. The first batch of aligners included 69 aligners; however, at stage 35 F–H, the When she used VPro5, her total treatment time was one year. Final records M–Q showeThis 14-year-old female presented with chief concerns that she wanted to improve her crowded teeth and deep bite as well as improve her recessive chin as much as possible without surgical intervention. The patient maturity indicator from CVM and physical/menstrual history confirmed that she already passed her growth spurt.The patient was not interested in a fixed orthodontic appliance. Clinical records A–E and TTreatment plan and progress: The minimum overjet was challenging to allow the mandible to be projected forward, so dental decompensation protocol was applied to this patient. In other words, treatment planning included advancing the upper arch forward using class III elastics (3/16-4.5 ounces full time) as well as expanding the upper arch at the same time in addition to intruding posterior teeth as moving the upper arch forward. Another challenge in this case was the excessive curve of Spee with moderate lower incisor crowding (6 mm). So in order to advance the lower arch, the excessive curve of Spee should be leveled, also due to the high angle presentation of this case (FMA = 30.4° Her treatment involved 23 aligners, and no additional aligners were needed afterwards. Treatment time was 4 months. Then she was given 4 sets of Vivera retainers. Final records were taken at one year from the original records that show better chin-forward projection I–M Tabl. SuperimGeneral results in all cases included improved facial convexity  and FigM by forwThe increased demand of non-surgical techniques to correct skeletal mal-relationship cases especially in non-growing or slow growing patients is increasing ,26. In t® Invisalign aligners and vertical-pull chin cup in this case series allowed achievement of complex tooth movement including posterior teeth intrusion and expansion simultaneously as well as forward mandibular rotation/projection in non-growing patients with a skeletal Class II relationship, which otherwise would have required surgery. These results should be confirmed with future larger samples size prospective controlled clinical trials.The use of HFV concurrent with SmartTrackAlthough the improvement of patients’ profiles was not as ideal as could possibly achieved with surgical intervention, the achieved improvement was satisfactory to all patients/parents.The clinical impact and implications of this case series are that the use of HFV facilitates complex orthodontic tooth movement including posterior teeth intrusion and incisor decompensation in addition to increasing bone formation labial to lower incisors that may minimize future gum recession due to their labial inclination. Again, the presented results should be confirmed with future larger samples size prospective controlled clinical trials.


Recent work has demonstrated that Foxk proteins predominantly act as transcriptional repressors in muscle cells and fibroblasts, where they recruit the Sin3-HDAC complex to silence targeted loci . However, Foxk proteins can also associate with the DNA demethylase TET1 in vivo and in vitro , which is suggestive of their role in transcriptional activation and formation of open chromatin. To date, the majority of Foxk studies have been carried out in cultured cells, with limited information available on in vivo developmental requirements for Foxk function .Foxk proteins are transcription factors from the forkhead box family, implicated in diverse biological processes such as glucose metabolism, inhibition of myogenic differentiation, and repression of autophagy  and found them to be on average highly expressed (10 – 125 TPM) during the first 48 hours of embryogenesis . We next sought to investigate how the depletion of Foxk proteins affects early embryonic development by employing targeted morpholino (MO) knockdown of three zebrafish Foxk orthologs: Foxk1 (forkhead box K1) , Foxk2a (forkhead box K2a) , and Foxk2b (forkhead box K2b) . Zebrafish embryos were injected at 1cell stage with MOs targeting either single foxk transcripts or all three transcripts together (foxk1/foxk2a/foxk2b – triple MO). Whereas the injections of single foxk MOs had no notable effect on embryonic development, the triple MO resulted in severe developmental phenotypes including grave nervous system defects and abnormally short bodies in 32% of injected embryos . We next attempted a rescue assay with full length, wild type human foxk1 transcript (hfoxk1) as well as with a mutant transcript (hfoxk1-mut), which contains a point mutation (H355A) that is predicted to disrupt its DNA-binding domain . The wild type transcript reduced the number of affected embryos, from 75% displaying strong or mild phenotype, to 55%. This effect was not observed when foxk1 mutant RNA was injected together with the triple MO mixture .To investigate the contribution of Foxk proteins to zebrafish embryonic development, we explored the steady state levels of Fig. 1B). The data separated by condition, as expected from the experimental design. We next employed differential gene expression analysis  to understand the impact of Foxk loss on gene expression. We detected 9,130 differentially expressed genes , suggestive of significant transcriptional changes caused by the loss of Foxk function . To validate these RNA-seq results, we searched the literature for previous examples of genes upregulated upon Foxk loss. Notable transcriptional upregulation was observed from two loci (fbxo32 and ulk1b) in the triple MO condition, which were previously reported as Foxk targets in mammalian cells  . To obtain further insight into genes that become misregulated upon Foxkknockdown, we performed gene ontology analysis  on the most highly affected upregulated and downregulated genes. Genes upregulated in the triple MO displayed significant enrichment in ontologies associated with autophagy and starvation ,whereas downregulated genes were highly enriched in cell cycle regulators. Foxk1 proteins have previously been implicated in cell cycle control . We also observed downregulation of genes associated with hydrolase activity, in line with the proposed roles of Foxk proteins in autophagy . It is worth noting, however, that the tissue complexity of 24hpf embryos as well as the strong phenotypes characterised by significant developmental delay, make it challenging to draw precise conclusions regarding the impact of Foxk proteins on transcriptional regulation.To assess how the loss of Foxk function affects the embryonic transcriptome, we extracted RNA from 24hpf embryos and subjected it to stranded RNA-seq library preparation and sequencing, generating an average of 40 million reads per sample. Upon mapping, we first generated a distance matrix to obtain insight into similarities between samples . The triple MO severely affected early embryogenesis but was nevertheless ameliorated by the injection of a full length human foxk1 transcript. The transcript coding for mutated Foxk1 that inhibits DNA binding was not able to rescue the triple MO phenotype, suggestive of a requirement for Foxk DNA binding during embryogenesis . The triple MO resulted in global transcriptional misregulation, including upregulation of autophagy-related genes and downregulation of cell cycle regulators . Overall, zebrafish recapitulates the majority of molecular phenotypes previously associated with Foxk loss in vitro and can serve as a useful model system for the further exploration of Foxk function in vivo.Our comprehensive assessment of Foxk function in zebrafish embryos demonstrates that Foxk proteins display redundant roles during embryogenesis, as evidenced by the lack of phenotype upon single Foxk morpholino knockdown and rescue experiments: Either 9 ng of a single morpholino or 3 ng each for the combination of all three morpholinos  was injected into one-cell stage zebrafish embryos. The observed phenotypes were recorded at different time points using a stereoscope . Full length and mutant (H355A) human foxk1 RNA required for rescue experiments were in vitro transcribed from pAS2255 and pAS2259 plasmids , respectively.RNA-seq library preparation and data analysis: RNA-seq libraries were prepared using the TruSeq Illumina stranded RNA-seq kit following manufacturer’s instructions, starting with 500 ng of RNA per sample. The libraries were sequenced on the Illumina HiSeq1500 sequencing platform. RNA-seq reads were trimmed to remove adapter sequences and low-quality nucleotides with Trimmomatic  and mapped using Kallisto  with the following settings: kallisto quant –single -l 100 -s 20 -t 4. The sequence required for the generation of the reference transcriptome (Danio_rerio.GRCz11) was obtained from ENSEMBL. Differential gene expression analysis was conducted in R with the DEseq2 package . Gene ontology analysis was performed with gProfiler  on 500 most upregulated and 500 most downregulated genes, sorted by adjusted P values, as per DEseq2 results. RNA sequencing data have been deposited to the ArrayExpress database (https://www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-9777.Animal handling: All animal experiments were conducted following the guidelines established and approved by the local governments and the Institutional Animal Care and Use Committee, , always in accordance with best practices outlined by the European Union.The sequences of morpholinos targeting each foxk transcript are as follows:foxk1 (Ensembl ID: ENSDARG00000037872): CGGTATCATCCCCTAAATCAGCCATfoxk2a (Ensembl ID: ENSDARG00000030583): CCATCTGTACCGCCGCTGACCGGGAfoxk2b (Ensembl ID: ENSDARG00000011609): ACGGGCCATCGCTGCCATCTTTATC


Gasterosteus aculeatus) fish are a tractable model for attacking this problem because of abundant population-level genetic and phenotypic variation in the gut inflammatory response. Previous work in our laboratory identified genetically divergent stickleback populations exhibiting differences in intestinal neutrophil activity. We took advantage of this diversity to genetically map variation in an emblematic element of gut inflammation - intestinal neutrophil recruitment - using an F2-intercross mapping framework. We identified two regions of the genome associated with increased intestinal inflammation containing several promising candidate genes. Within these regions we found candidates in the Coagulation/Complement System, NFkB and MAPK pathways along with several genes associated with intestinal diseases and neurological diseases commonly accompanying intestinal inflammation as a secondary symptom. These findings highlight the utility of using naturally genetically diverse ‘evolutionary mutant models’ such as threespine stickleback to better understand interactions among host genetic diversity and microbiome variation in health and disease states.Selection, via host immunity, is often required to foster beneficial microbial symbionts and suppress deleterious pathogens. In animals, the host immune system is at the center of this relationship. Failed host immune system-microbial interactions can result in a persistent inflammatory response in which the immune system indiscriminately attacks resident microbes, and at times the host cells themselves, leading to diseases such as Ulcerative Colitis, Crohn’s Disease, and Psoriasis. Host genetic variation has been linked to both microbiome diversity and to severity of such inflammatory disease states in humans. However, the microbiome and inflammatory states manifest as quantitative traits, which encompass many genes interacting with one another and the environment. The mechanistic relationships among all of these interacting components are still not clear. Developing natural genetic models of host-microbe interactions is therefore fundamental to understanding the complex genetics of these and other diseases. Threespine stickleback ( Animals harbor an array of microbes on and in their bodies which perform essential functions that are fundamental to host health .The relationship between host immune system and resident microbes is complex. Some microbes cause disease states only in specific host genetic backgrounds or in the presence of other microbes . For exaWhat is needed are studies that can link quantifiable microbe-induced differences in immune response to host genomic loci and genetic variants. One way to quantify the inflammatory response is through assessment of neutrophils, specialized white blood cells that are recruited during an inflammatory response . These cnatural genetic variants influencing complex phenotypes such as inflammation (Gasterosteus aculeatus) as such an outbred ‘evolutionary mutant model’  and a female from the anadromous population Rabbit Slough . An F2 family (n = 64) was produced intercrossing F1 siblings. Fertilized eggs were incubated overnight in one-micrometer filter sterilized antibiotic embryo media containing 100 mg/mL Ampicillin, 50 mg/mL Kanamycin, and 8 mg/mL Amphotericin .Whole fish were stained using the Sigma Myeloperoxidase kit , which preferentially stains neutrophils. Fish were then embedded in paraffin, and their bodies were cross-sectioned in 7-micrometer sections from just posterior to the gill rakers to the urogenital opening. Every 10P = 0.14) , as males and females exhibited similar distributions of average intestinal neutrophils. To determine if differences in neutrophil density correlated with minor changes in developmental timing and growth rate, we used standard length which is a common metric used for fish. In doing this, we found that neutrophil abundance is correlated with standard length, with larger fish exhibiting higher average neutrophil density  . This measurement of standard length was additionally correlated with gut size, with larger fish exhibiting larger guts  . However, there is no direct relationship between gut size and neutrophil density  . We therefore concluded that differences in developmental timing, measured by standard length, should be included as a covariate for subsequent analyses. To account for size as a covariate, we calculated residuals from the regression of neutrophil density and standard length, to be included as trait values in our QTL mapping.Intestinal neutrophil abundance in 14 days post fertilization (dpf) F2s was distributed roughly normally, with full siblings ranging from less than one neutrophil per section on average to over seven neutrophils per section . This vaTail samples were flash frozen in liquid nitrogen and stored at -80°. DNA was extracted using the Qiagen DNeasy Blood and Tissue Kit . DNA was then quantified using the Qubit fluorometer broad range kit. Individual sex phenotypes were determined using PCR amplification of a sex specific region of the genome using the GA1 primer pair , and malGenomic DNA from each F1 parent and F2 offspring was standardized to 10 ng/uL and digested with the endonuclease SbfI-HF (NEB), and RAD-seq libraries were generated using protocols established by the Cresko Laboratory  to the sscanone function with Haley-Knott regression  under project ID: PRJNA591028 Supplemental material available at figshare: We identified 18,394 SNPs to be used for QTL mapping. Using raw intestinal neutrophil densities (number of neutrophils per section), we detected 13 linkage groups (LG) with QTL associated with variation in neutrophil density (LOD > 3) ; Table 1In some instances, low coverage or missing genotypes can falsely inflate LOD scores of individual SNPS generating false positives. To evaluate our data for false positives, we randomized the raw phenotype data and the residuals with respect to the F2 genotypes and re-ran r/QTL. This method is similar to large scale permutation tests used to designate a significant alpha value, but can also indicate regions where low coverage in specific SNPs are generating false positives. In our original runs, high-LOD regions were concentrated in pileups , indicatP = 0.28; df = 34.7, t = -1.11; P = 0.28; df = 34.7 and t = -1.30; P = 0.20; df = 43.4). In this case, one parent was a homozygote while the other was a heterozygote so there are only two genotypes in the progeny (homo and het) .To test if low coverage in a few SNPS could be generating false positives, we assessed phenotype distributions for each genotype in the SNPs with LOD > 3, in the regions with retained signal in both the raw and residual maps. On LG12 we found evidence of false positives, with neutrophil densities spanning similar phenotypic space regardless of genotype , with heterozygous and homozygous siblings exhibiting similar average neutrophil densities . In LG3, we identified 11 SNP clusters LOD > 3, including one marker with a score below the initial threshold of LOD > 3. We chose to include this locus, however, as the LOD score of the residual was one of the highest (LOD = 4.13) and due to linkage, these pileups are used to identify fairly large genomic regions of association. In LG8 we identified 17 such regions (Table S3).To ascertain biological relevance of each of our QTL we used the threespine stickleback genome annotation to compile a complete list of candidate genes associated with each genomic interval. Within these boundaries we assembled a complete gene list and identified those with known functions and if available assigned directionality of differential expression from the parental populations .f3, c8a, and c8b on LG3 (th exon of c8a (Table S4). A second group included members of the ERK signaling and MAPK pathways. Interestingly, the MAPK Pathway was also enriched for genes sensitive to the presence of microbes in freshwater families  in addition to the number of genes in this pathway is highly suggestive of a strong association between MAPK/ERK signaling and intestinal inflammation. Another immune pathway associated with several candidate genes was the NfKB immune pathway. This included ripk2 - an activator of NFkB - on LG3  : To our knowledge this is the first study in which an F2 intercross mapping framework has been used to identify genomic loci underlying a complex immune trait. The identification of multiple genomic loci significantly associated with variation in neutrophil density suggests several genes throughout the genome contribute to this complex phenotype. Our results highlight several genomic loci with relatively large effects contributing specifically to intestinal neutrophil recruitment. The persistence of high LOD SNP pile-ups on LG3 and LG8, after accounting for standard length as a covariate and testing for false positives, argue for the biological relevance of the QTL detected in this analysis. However, the modest size of this single family of F2 progeny (n = 64) means that other genomic regions of small effect would likely have gone undetected. Although other genes of small effect with bearing on the observed variation in intestinal neutrophil activity may have been missed, we can be confident in our identification of several genomic intervals on LG3, and LG8 with strong associations to intestinal neutrophil activity. It is important to note that this study does not suggest causality of any one of these SNPs individually. Instead these data are highly suggestive of candidate genomic regions and specific genes that warrant further investigation into their ties to intestinal inflammation.c8a and c8b were both found in the same genomic interval with the associated SNP located within c8a. These proteins both function as a part of the Complement Cascade, a pathway enriched for differentially expressed genes between oceanic and freshwater stickleback (map2k2a/map2k2b and tcf3a/tcf3b are also excellent candidates for impacting the inflammatory response as tcf3 has been shown to play a role in the regulation of B cell maturation and map2k2 is an activator of MAPK. These pairs are of particular interest, however, as they were found on separate linkage groups with map2k2b/tcf3a on LG3 and map2k2a/tcf3b on LG8.More specifically, the large number of immune genes identified in the associated genomic intervals is not an unexpected result. The Coagulation/Complement Cascade, MAPK, ERK, and NfkB pathways are all essential immune pathways activated early in development and play roles in the regulation of the inflammatory response (rgs1 and scp2a, which contained three markers within intronic regions (The identification of several genes involved in human intestinal disease via the formation and regulation of tight junctions was also intriguing. Tight junctions are extremely important in regulating intestinal permeability and the intestinal immune response, and have been tied to many intestinal diseases including IBD, Celiac Disease, and Type 1 Diabetes (Lastly, the identification of variants within neurological and neuromuscular disease genes provides potential links between intestinal and neurological health. Our findings included variants associated with known disease genes impacting ALS, Autism, and Muscular Dystrophy. All of these diseases are of particular interest to those studying intestinal inflammation, as individuals who exhibit them often report symptoms consistent with colitis and other types on intestinal inflammation at greater rates; and in many cases targeted treatments of the microbiome have been successful in alleviating or slowing progression of symptoms (This study provides a strong example of the power of threespine stickleback as a model for mapping natural variants contributing to genetically complex phenotypes relevant to human disease. To expand upon these findings, we can use the stickleback system to map other related immune system phenotypes and expand studies of inflammation to other tissue types. Though this study does not include functional testing of candidate loci, these findings additionally provide potential targets for functional testing using CRISPR-Cas9 genome editing to connect systems-level genetic links between tight junction, neurological disease, and intestinal health. Because of the amenability of stickleback for gnotobiotic studies, these genetic approaches will be particularly useful to manipulate both host genes and microbiomes simultaneously to perform functional tests not possible in other organisms. Our findings have broader impacts in elucidating the complex roles of natural genetic variation in chronic intestinal inflammation and provide further evidence of a strong link between intestinal health and systemic health.


To report the observation of the choriocapillaris island (CCI) on optical coherence tomography angiography (OCTA) in eyes with active central serous chorioretinopathy (CSCR), and to investigate its associated clinical features.Retrospective observational study.en face choriocapillaris slab was examined to demonstrate CCI, defined as an area of detectable choriocapillaris flow surrounded by an area of undetectable or diminished flow. Electronic medical records (EMR) were reviewed for demographics, clinical data, other imaging modalities and any intervention, and these parameters were correlated with CCI findings.Patients diagnosed with active CSCR underwent OCTA imaging , and the software built-in en face choriocapillaris density maps. 24 eyes had focal retinal pigment epithelium (RPE) alterations overlying CCI. All 14 eyes with simultaneous fundus fluorescein angiography (FA) showed actively leaking point(s) well corresponding to the CCI location. Resolution of sub-retinal fluid in 4 eyes was associated with disappearance of CCI on follow-up OCTA scans. 1 eye showed complicating neovascularization 5 months after the initial presentation at the same location of the CCI.25 eyes of 25 patients were recruited. CCI was detected in all examined eyes and was best elucidated on the We demonstrate the observation of the “choriocapillaris island” an OCTA finding in eyes with active CSCR underneath the area of neurosensory detachment. CCI may constitute an angiographic representation of the focal area of choriocapillaris structural and functional affection, with secondary RPE alteration jeopardizing its barrier function. Larger longitudinal studies are needed to further elucidate this finding. Central serous chorioretinopathy (CSCR) is an idiopathic chorioretinal disorder typically affecting young and middle-aged adults with a higher male predilection, and represents the fourth most common non-surgical retinal disease in terms of incidence , 2. It wThe pathophysiology of CSCR is not yet fully understood, however recent studies have suggested that leaking pachy-choroidal vessels may exert back pressure changes on the overlying choriocapillaris (CC) leading to secondary CC disruption and retinal pigment epithelium (RPE) alterations –10. LocaCSCR characterization has been further validated through multiple imaging modalities including fluorescein angiography (FA) , fundus The purpose of our study was to document the “choriocapillaris island”, an OCTA sign observed in the choriocapillaris slab in eyes with active CSCR, and attempt to correlate this finding with various clinical and interventional data.This was a retrospective analysis of patients diagnosed with active CSCR recruited in the Watany Eye Hospital (WEH) in Cairo, Egypt between January 2019 and September 2019. The study was approved by the institutional review board of Watany Research and Development Center (WRDC) and followed the tenets of the Declaration of Helsinki. A written informed consent was obtained from all participants.We included eyes with active CSCR based on the clinical evaluation of a vitreoretinal consultant. The diagnosis was made by the detection of well circumscribed posterior pole neurosensory detachment on fundus examination, presence of subretinal fluid by OCT, pachy choroid or dilated choroidal vessels by enhanced depth imaging (EDI) OCT, and a leaking point (hot spot) on FA. Only eyes that had OCTA images with a quality index (Q) of 6 or more were considered eligible.Exclusion criteria were patients with history of any systemic illnesses or medication intake that may potentially cause subretinal fluid, other vitreoretinal disorders that may confound our results, and previous ocular surgery or treatment  or steroids). Electronic medical records  of recruited patients were reviewed to extract demographics and clinical information.OCT imaging was performed using a Spectralis OCT machine , and the following parameters were documented for all eyes: central sub-field thickness (CST), the presence of retinal pigment epithelium (RPE) alterations, and sub-foveal choroidal thickness (SCT) using the caliber option defined as "the distance extending from the outer portion of the hyperreflective line corresponding to the retinal pigment epithelium (RPE) to the hyporeflective line corresponding to the sclerochoroidal interface" .Patients who have received simultaneous fundus fluorescein angiography (FA) imaging using the Heidelberg Retinal Angiogram  were reviewed for the presence and location of actively leaking hot spot(s).en face image of the software built-in choriocapillaris slab (extending between the Bruch’s membrane as an upper boundary and 30 microns below the Bruch's membrane as a lower boundary) was examined by three graders , to determine the presence of the choriocapillaris island (CCI). No subsequent image modification or processing was performed.Patients underwent imaging using RTVue-XR Avanti device , 3D Projection artifact removal (3D-PAR) technology was used to obtain 3 × 3 and 6 × 6 mm macular scans. The en face OCTA slab and structural appearance on the corresponding cross-sectional B scan, was documented for each eye.CCI was defined as an island of detectable choriocapillaris flow surrounded by an area of undetectable or diminished flow underneath the area of neuro-sensory detachment Fig. . CharactAdobe Photoshop CS2  was used to superimpose images obtained from different imaging modalities  by aligning the retinal blood vessels for each patient, to display CCI location in relation to leaking point(s) on FA, and chorioretinal structural changes on corresponding structural OCT  met the inclusion criteria and were included in the study. Mean patient age was 40.6 ± 9.7 years. Mean onset of complaint was 5.2 ± 5.1 month. The Mean BCVA was 20/28 ± 20/95. The mean central subfield thickness was 417.7 ± 106.1 microns, and the sub-foveal choroidal thickness was 433.3 ± 94.6 microns. 24 eyes had retinal pigment epithelium (RPE) alterations overlying CCI.CCI was best visualized on the OCTA software-generated choriocapillaris density map, defined as the ratio of area occupied by vessels divided by the total area of the region of interest Figs. b, 3b, 4denface CC slab  on FA and chorioretinal structural changes on OCT.en face slabs of OCTA, the blood flow in the choriocapillaris layer gives off a homogenous granular appearance in normal eyes [The choriocapillaris represents the innermost layer of the choroid underlying Bruch’s membrane. Histologically, it is composed of compacted fenestrated anastomotic capillaries, and is responsible for providing RPE and photoreceptors nourishment and removing their metabolic waste . In thatmal eyes , 25.In eyes with active CSCR, our data demonstrates diminished or absent CC signal underneath the area of neurosensory detachment. Although a clear explanation for this finding cannot be determined, several theories exist. One assumption is that choroidal congestion characterizing CSCR may be accompanied with sluggish choriocapillaris flow caused by mechanical back pressure changes by the dilated large choroidal vessels, thus falling below OCTA detectable threshold . Anotheren face CC slab, whereas it is best visualized on the OCTA density map because of the color coding which enables a more vivid appreciation of differences in flow volumes  . In 24 eFluorescein angiography (FA) is still considered the gold standard for localization of actively leaking point(s) in CSCR . In all Spontaneous resolution of sub-retinal fluid has been reported in eyes with CSCR eyes , 4, 5, wenface CC slab, which corresponded well to the previously observed CCI location  complicating CSCR , 35. A sion Fig. .It is poFrom the data provided, it cannot be ascertained whether the presence of CCI should be ascribed to focal choriocapillaris affection or to a newly forming abnormal choroidal neovascularization. However, spontaneous resolution of subretinal fluid was documented in 2 eyes, which was not previously reported in pachychoroid Neovasculopathy. When taking into consideration the unique choriocapillaris histological architecture of multiple, well separated vascular lobular units, filled and drained independently from each other, one can determine that the choriocapillaris is actually divided into multiple functional units , 37. BasThe limitations of our study include its retrospective nature with lack of FA in some patients and its short-term follow-up visits, denying the opportunity to achieve more insight into the natural and treatment course of our observation in the context of evolution of CSCR. Moreover, the lack of access to ICGA did not allow a correlation of this important imaging tool in choroidal disorders with our findings. Another possible limitation is the use of SD-OCTA system that employed a shorter wavelength with a rather limited light penetration compared to SS-OCTA which utilizes a longer wave length and provides greater penetration power. However, we believe that reporting this OCTA sign would encourage further characterization and validation though larger prospective controlled studies.We demonstrated the CCI as a finding in the choriocapillaris OCTA slab in eyes with CSCR, characterized by the presence of a detectable choriocapillaris island of flow within an area of presumedly artifactual choriocapillaris flow impairment underneath serous neurosensory retinal detachment. This finding could be explored as a dye-less tool for the identification of the focal area of RPE/CC structural and functional affection in eyes with CSCR. Larger longitudinal studies are needed to provide more insight into the natural course of this finding, its prognostic implications, and its impact on various intervention modalities.


Uncovering cellular responses from heterogeneous genomic data is crucial for molecular medicine in particular for drug safety. This can be realized by integrating the molecular activities in networks of interacting proteins. As proof-of-concept we challenge network modeling with time-resolved proteome, transcriptome and methylome measurements in iPSC-derived human 3D cardiac microtissues to elucidate adverse mechanisms of anthracycline cardiotoxicity measured with four different drugs . Dynamic molecular analysis at in vivo drug exposure levels reveal a network of 175 disease-associated proteins and identify common modules of anthracycline cardiotoxicity in vitro, related to mitochondrial and sarcomere function as well as remodeling of extracellular matrix. These in vitro-identified modules are transferable and are evaluated with biopsies of cardiomyopathy patients. This to our knowledge most comprehensive study on anthracycline cardiotoxicity demonstrates a reproducible workflow for molecular medicine and serves as a template for detecting adverse drug responses from complex omics data. Using a network propagation approach with integrated multi-omic data, Selevsek et al. develop a reproducible workflow for identifying drug toxicity effects in cellular systems. This is demonstrated with the analysis of anthracycline cardiotoxicity in cardiac microtissues under the effect of multiple drugs. Crucial for realizing this goal is a comprehensive characterization of adverse pathways across multiple regulatory systems. In this context the identification of mechanisms through application of multiple-omics technologies that enable capturing the full width of molecular responses upon drug treatment combined with integrative data analysis approaches has been defined as the key strategy for solving the task2. Moreover, unforeseen cardiovascular toxicity is among the most important reason for drug candidate failure4 and accounts for 14% of drug withdrawals upon market introduction due to adverse reactions8. This is mainly due to preclinical non-human test models for drug toxicity that translate only poorly to the human conditions; in fact, across a range of pharmaceuticals it has been demonstrated that to the best, only 63% of compounds show concordance of toxicity between animal test results and human responses10. Overall, an understanding of underlying mechanisms of toxicity has been recognized as one of the most effective ways to improve drug safety. Against this background, Collins et al.11 proposed to bypass animal-based drug toxicity testing, by developing molecular response patterns generated from human cell-based in vitro systems that are indicative of human disease phenotypes, and by identifying pivotal signaling pathways leading to toxicity. Here, the toxicogenomics approach has been claimed to be capable of identifying new genomic entities yielding innovative prediction models for adverse drug reactions thereby improving risk assessment12. An exemplar study already demonstrated the usefulness of global gene expression-based in vitro modeling for predicting drug-induced hepatic cytotoxicity13. But a true integrative understanding of molecular mechanisms of adverse drug reactions can only be accomplished by interrogating different levels of molecular activities in a dynamic fashion, and this approach requires (i) capturing of the dynamic responses across time and dose in the system under study enabling dynamic network inference and quantitative modeling15, (ii) identification of mechanistic networks of interacting proteins that are responsible for the systemic drug response17, and (iii) validation of these networks in human patients.Such integrated approaches have impact on standard-of-care therapies as well as on the development of novel drugs. In particular, adverse effects of anti-cancer drugs on the heart are a growing clinical problem with the ever increasing number of cancer patients18. ACs are widely used chemotherapies despite the fact that they induce cardiotoxicities in up to 23% of the patients. AC-induced cardiotoxicity (ACT) represents a cumulative systemic effect over time and refers to changes in myocardial functions for example in left-ventricular ejection fraction (LVEF), diastolic functions, arrhythmias as well as cardiac stress responses. The major molecular hallmarks of ACT include the generation of reactive oxygen species (ROS) and changes in mitochondrial response pathways that ultimately lead to mitochondrial damage, the interference of ACs with topoisomerase 2 (TOP2B) and thus the disruption of gene regulation and DNA damage repair mechanisms as well as changes in sarcomere function20.Consequently, in a yet unmet endeavor, we obtained proof-of-principle for such an integrated approach with respect to cardiovascular drug toxicity. We generated dose-over-time cross-omics data from an advanced iPSC-derived human cardiac 3D microtissue model exposed to physiologically relevant doses of anthracyclines (ACs) over a time period of 14 days21. We identified a network of 175 proteins representing the common signature of ACT. We then undertook the challenge of acquiring cardiac biopsies taken from patients with and without historic AC treatment and now suffering from chronic cardiac failure and in a yet untried approach, compared the in vitro results with proteome analysis of these target tissue samples. We demonstrate that proteins of the ACT network were (i) clinically transferable to patients suffering from drug-induced cardiomyopathies and (ii) physiologically relevant as tested with a previously defined model for predicting ACT of the cardiomyocyte mitochondrion22 and thus that network-based data integration has major potential for advancing the field of molecular medicine.We have generated dynamic quantitative proteomics (LC–MS), transcriptomics (RNA-seq) and methylation (MeDIP-seq) landscapes from the cardiac cell model for four widely used anthracyclines (doxorubicin (DOX), epirubicin (EPI), idarubicin (IDA), and daunorubicin (DAU)) and conducted an integrated computational approach using the results of longitudinal expression analyses and network propagation based on insulated heat diffusionA workflow of the analyses is shown in Supplementary Fig. 24. Microtissues are composed of iPSC-derived human cardiomyocyte and cardiac fibroblast cells (proportion 4:1). They show spontaneous contractile activity, Ca2+ responses, homogenous tissue without central necrosis, a spherical shape and were filled with myofibrils, a tissue structure characteristic for the mammalian heart. Essential functions such as contractility, microtissue size, and ATP generation and consumption are observable over the entire time period . We examined whether the microtissues express previously found in vivo epigenetic characteristics of adult cardiomyocytes. In mouse as well as in human adult cardiomyocytes it has been observed that gene body methylation is inversely correlated with gene expression27. This overall trend in vivo indeed appears to be preserved in the iPSC-derived cardiac microtissues: Lowly expressed genes show a higher level of promoter and gene body methylation than highly expressed genes  and found good agreement of methylation levels between 3D cardiac microtissues and human heart-specific cell lines  and intronic (41–43%) regions with only a small fraction (5–8%) in promoter regions  were found consistently differentially methylated at therapeutic and toxic doses, respectively, with all four ACs. At therapeutic dose 984 DMRs were located in gene bodies  and 151 DMRs were found in promoter sequences, corresponding to 758 different genes. At toxic dose 322 DMRs were located in gene bodies  and 30 DMRs in promoter sequences, corresponding to 250 different genes. These genes enrich important pathways such as adrenergic signaling in cardiomyocytes , ETS1  and SRF  binding sites showed strong enrichment of TFBSs in hypermethylated DMRs, which indicates that gene regulation of these factors is impaired by AC-induced methylation demonstrating profound disturbance of regulatory pathways crucial for cardiac physiology (cf. “Discussion” section).To examine the association of AC-induced methylation patterns with transcriptional regulation, we contrasted DMRs against transcription factor-binding sites (TFBSs) as measured by ENCODEy32 Fig. . In part33. This yielded for each protein an estimation of its temporal response as well as the significance of the deviation of the treatment temporal profile from the respective control profile , which is dynamically altered with respect to EPI, IDA, and DAU at therapeutic doses and with DOX, EPI, and DAU at toxic doses. Time-point specific analysis , myosins , tropomyosins  among others. In total, 47 out of 199 sarcomere-related proteins  showed a significantly altered time profile upon AC treatment when compared to control microtissues. Further largely enriched functional groups relate to mitochondrial dysfunction and response pathways34 in particular electron transport chain , respiratory chain complex  and response to oxidative stress .ACs induced heterogeneous dynamic responses in terms of protein content with DOX (359 proteins at both doses) and EPI (313) affecting higher numbers of proteins followed by IDA 230) and DAU , mitochondrial membrane proteins  and electron transport chain components . A summary of these transcriptome responses for all ACs is shown in Fig. On the transcriptome level, for each treatment and dose time-resolved RNA-seq analysis was performed and analyzed with longitudinal data analysis  using a adrenergic signaling pathway  the cardiomyopathies were a consequence of prior anti-cancer therapy. The remaining patients (n = 8) had developed cardiomyopathies because of other reasons not related to drug toxicity. The major clinical indication of cardiotoxicity is decrease of the LVEF. We observed that on average the LVEF is not different between the two patient groups , MYOM1 (c = 0.78), TNNI1 (c = −0.59), members of the electron transport chain COX4I1 (c = −0.84), NDUFV1 (c = 0.61) and mitochondrial-related proteins SUCLA2 (c = 0.72), DECR1 (c = 0.71), MDH2 (c = 0.57) , a protein not predominantly known in the context of cardiotoxicity and thus representing a promising candidate for diagnosis. Laminin Subunit Gamma 1 (LAMC1) is a member of the ECM-receptor interactions and Focal adhesion pathways that have been associated functionally with cardiomyopathy previously (cf. “Discussion” section). Interestingly, the high correlation of the protein expression of these genes with the LVEFs of the chronic cardiotoxic cancer patients is in most cases accompanied by a low or even opposite correlation with the LVEFs of the control group of cardiomyopathy patients  proteins from the in vitro-derived ACT response network Fig.  were ide57) Fig. . Among tnts Fig. . This sut-test and observed that rather the intermediate and late time points contribute to the changes in protein expression of those proteins in vitro which translate to protein markers of AC-induced cardiotoxicity in cardiomyopathy patients. For example, upon DOX treatment at therapeutic dose we observe, among others, significant late responses (Q < 0.05) of ACTN2 (240 and 336 h) and LAMC1  in contrast to TNNI1 which has an intermediate peak response at 72 h  and electron transport chain proteins . For the other ACs, we found six out of seven significant model components being covered by network module proteins for EPI, seven out of nine for IDA and three out of four for DAU. These covered components relate mostly to the TCA cycle and the electron transport chain , MHC genes, TTN (titin), TNNC1, among others54 which has a high level of overlap with the inferred proteome and transcriptome changes. Changes in extracellular matrix/structure organization protein regulation are consistent with observations in patients55 and mice56 that find increased fibrosis following anthracycline exposure. The fibrosis may be reparative in response to apoptosis or interstitial in response to changes in loading. Changes in extracellular matrix/structure organization proteins may be due to direct effects of anthracyclines or secondary due to increased ROS57 or changes in mechanical loading58. Of the proteins associated with LVEF in biopsies we identified the extracellular matrix protein LAMC1, consistent with either a dominant or integrating effect of the extracellular matrix in determining LVEF in cardiotoxic patients.The dominant pathway changes may be attributable to direct action of anthracyclines or may be a secondary change due to anthracycline action on one or more of the other pathways. Specifically, anthracyclines are known to act on the mitochondria increasing ROS production, which can modulate downstream protein expression. Increased ROS production alters the beta-adrenergic signaling pathways, sarcomere proteins, and extracellular matrix synthesis59 showed that, in rat at maximum tolerated doses, IDA showed significantly lower cardiotoxicity than DOX. Furthermore, the computer simulations predicted differences in protein changes between different ACs. Despite similar predicted changes in membrane potential and ATP for DOX, EPI, and IDA, the cause was distinct between compounds. DOX increases ETC and TCA fluxes; conversely IDA and EPI cause a decrease in TCA and ETC fluxes  that the most plausible order of toxicity is DOX > EPI > IDA > DAU. This is in line with in vivo-derived phenotypic observations: for example, Platel et al. This study has generated several novelties. Firstly, using integrated network modeling we were able to identify potential biomarkers that translate from in vitro microtissues to patient biopsies and that are indicative of clinical parameters Fig. . This is27. Furthermore, it has been observed in murine cardiomyocyctes that this gene body methylation of key sarcomere genes, such as troponin I isoforms (Tnni1 and Tnni3) is highly dynamic26: While Tnni1 gene body was de novo methylated in adult murine cardiomyocytes, Tnni3 was demethylated. In our microtissues we observed that TNNI1 is methylated in cardiac microtissues throughout the gene body while TNNI3 has a significant reduction in methylation at exons 4 and 5  that belong to the superfamily of TRIM proteins that function as regulators for a variety of human diseases65. TRIM55 is also known as Muscle-specific Ring Finger protein 2 and has been shown to localize to the sarcomere and to regulate muscle protein turnover66. TRIM55 has several connections to sarcomere genes .Thirdly, dynamic network modeling identified proteins that are typically not easy to identify with MS-approaches such as transcription factors. Here, we showed evidence for a role of TRIM proteins in AC cardiotoxicity, in particular nes Fig.  and thusDAU Fig. , and IDAp-value scoring and to perform network propagation analysis in order to compute for each drug a subnetwork accumulating the major dynamic responses from the integrated data sets. This approach is rather generic and can be applied to various other data  as long as the results can be quantified by p-values. It thus might serve as a template for extracting common information from complex multi-omics data sets, likely to be massively generated in future precision cardio-oncology67. We combined the four individual drug subnetworks at therapeutic doses to an ACT response network consisting of 175 proteins and showed that 70% of these proteins were in fact expressed in cardiac biopsies of patients and that potential biomarkers inferred from the network analysis correlate with pathological parameters and mechanisms.In conclusion, our study proofs iPSC-derived 3D cardiac microtissues as a suitable in vitro cell model for screening dynamic drug responses at multiple molecular layers. We have identified a set of 641 proteins and 904 transcripts that are dynamically changed over time when compared to DMSO control experiments. Although the overlap across these data sets is fairly low, the biological pathways and functions that are enriched by the different molecular layers are similar. This prompted us to integrate molecular data in a PPI network with 68.A previously established workflow for model-based assay design was used to reproduce in vivo patterns of drug-induced toxicity in spheroids. For each of the four anthracyclines physiologically based pharmacokinetic (PBPK) models were developed. The models were built with the open source PBPK modeling software PK-Sim and validated according to best practice guidelines for PBPK model qualificationEach PBPK model was used to simulate in vivo drug exposure in the interstitial space of the heart following administration of either a therapeutic or toxic drug dose over 2 weeks of a once daily administration schedule. Here, the therapeutic dose was based on a standard clinical dosing regimen according to the specific drug label. Toxic doses were estimated based on in vitro viability experiments in 3D cardiac microtissues (IC20). A toxic dose for each drug was then calculated from the IC20 values by reverse dosimetry. To this end it was assumed that the unbound drug exposure in the interstitial heart compartment of the PBPK model was equal to the in vitro drug. Finally, the continuous exposure profiles of the PBPK models where translated into an experimental setting with three daily media changes mimicking the estimated in vivo PK exposure profile  were used, containing ~4000 iPSC-derived human cardiomyocytes  and 1000 cardiac fibroblasts  per microtissue. The microtissues were cultured in 50 μl per well 3D Insight™ Human Cardiac Microtissue Maintenance Medium .Commercially available human iPSC-derived cardiomyocytes were obtained from Cellular Dynamics International Inc. . Shortly, these iCell cardiomyocytes were derived from an engineered hiPSC clonal line reprogramming human fibroblasts from a female Caucasian donor as described in Ma et al. To accomplish the PBPK-based repetitive dosing profile, the medium of the microtissues was changed three times daily on working days at intervals of 2, 6, and 16 h with the PBPK-determined concentrations , first had a physical examination, blood sampling, 12-lead electrocardiogram, 24-h Holter monitoring on indication, and a complete echocardiographic and Doppler evaluation. Significant coronary artery disease as a cause of the decreased ejection fraction was excluded by a coronary angiography (CAG) or a CT-angiography at baseline. EMB were performed as part of routine diagnostic work-up in non-ischemic, non-valvular cardiomyopathy, upon consent of the patient, as part of the Maastricht Cardiomyopathy Registry with inclusion and exclusion criteria as described previously2 (men) or >32 mm/m2 (women) measured by echocardiography; and HNDC defined as LVEF < 50% with an LVEDDi ≤ 33 mm/m2 (men) or ≤32 mm/m2 (women) measured by echocardiography in the absence of a (i) myocardial infarction and/or significant coronary artery disease; (ii) primary valvular disease; (iii) hypertensive or congenital heart disease; (iv) acute myocarditis; (v) arrhythmogenic right ventricular dysplasia; and (vi) hypertrophic, restrictive or peripartum cardiomyopathy. For the present study, we included cases with a previous history of cardiotoxic-anthracycline chemotherapy, and control DCM/HNDC without. The study was performed according to the declaration of Helsinki and was approved by the Medical Ethics Committee of Maastricht University Medical Centre. All patients gave written informed consent.In short, both DCM and hypokinetic non-dilated cardiomyopathy  according to the latest ESC proposal were included (DCM defined as LVEF < 50% with an indexed left ventricular end diastolic diameter (LVEDDi) >33 mm/mg for 15 min at 4 °C and protein concentrations were assessed with the Qubit™ Protein Assay Kit . Protein isolates were then submitted to in-solution digestion71 or filter aided sample preparation (FASP)72. Protein digestions were stopped by adding formic acid to a final concentration of 1%. The peptides were cleaned up using Sep-Pak tC18 cartridges (Waters) according to the manufacturer’s instructions, and eluted with 60% ACN and 0.1% formic acid . Patient biopsies were prepared as described in Guo et al. 73.Cardiac spheroids and cardiomyocytes were resuspended in 100 μl lysis buffer containing 8 M urea, 1 mM dithiothreitol, 0.1 M ammonium bicarbonate, pH 7.8. After four freeze–thaw cycles, the samples were centrifuged at 16,000×m/z) at 120,000 resolution at 200m/z; precursor automated gain control (AGC) target was set to 400,000. Charge-state screening was enabled, and precursors with +2 to +7 charge states and intensities >5000 were selected for tandem mass spectrometry (MS/MS). Ions were isolated by use of the quadrupole mass filter with a 1.6m/z isolation window. Wide quadrupole isolation was used, and injection time was set to 50 ms. The AGC values for MS/MS analysis were set to 5000 and the maximum injection time was 300 ms. HCD fragmentations were performed at a normalized collision energy (NCE) of 30%. MS/MS were detected in the ion trap in centroid mode. Precursor masses previously selected for MS/MS measurement were excluded from further selection for 25 s, and the exclusion window was set at 10 ppm.Samples were submitted to an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific) coupled to a NanoLC-2D HPLC system  or EASY-nLC 1000 system . Samples were loaded onto a self-made column (75 μm × 150 mm) packed with reverse-phase C18 material  when coupled with the EASY-nLC 1000 system and onto an Easy-Spray Column (75 μm × 500 mm) packed with reverse-phase C18 material  when coupled with the NanoLC-2D HPLC system. Peptides were separated with a linear gradient of acetonitrile/water, containing 0.1% formic acid, at a flow rate of 300 nl/min. A gradient from 5% to 30% acetonitrile in 60 min was used. The mass spectrometer was set to acquire full-scan MS spectra  and Analyst . In short, after noise reduction and normalization, LC–MS peaks were detected and their properties calculated . Individual peaks where grouped into clusters and MS/MS data associated to these clusters were annotated with MS/MS Ions Search (Mascot 2.6) using peptide tolerance: 10.0 ppm, MS/MS tolerance: 0.50 Da, max missed cleavages: 2 and database: Uniprot Swiss-Prot 29062016, Taxonomy Homo sapiens (human). Results are validated by applying a threshold of 5% normalized false discovery rate (FDR). Protein interference was done based on peptide and protein annotations. Redundant proteins were ignored according to the Occam’s razor principle, and at least two peptides were required for a positive protein identification (shared peptides were ignored). Protein intensities were computed using the Hi3 method. A maximum of the top 3 peptides per protein (based on the average intensity across samples) was used in the calculation. If a peptide was identified in multiple charges  and modification states (carbamidomethyl (C), deamidated (NQ) or oxidation (M)), values were consolidated into a single peptide intensity. The volume of a peak is computed as the area under the intensity curve inside the peak region. The area under the intensity curve is subdivided into trapezoids at the data points according to the trapezoidal rule. After the data pre-processing, the intensities were log2 transformed. Normalization was performed as follows: (i) the transformed data of the DMSO (control) samples were shifted to the median of the medians determined by a reference group consisting of the proteins found in all these control samples, (ii) for every treatment/dose combination and for each time-point the common protein set between the controls and the treatment samples was determined, (iii) the median of the medians of the  normalized control samples was determined using this common protein set between the controls and the treatment samples, and (iv) the data from the samples of the treatments were shifted to these medians. Two-sided Student’s t-tests were then used for the determination of differentially expressed proteins (DEPs) comparing the proteins of each time-point and dose against the corresponding time-matched control. These normalized data sets were also used for the two-step regression model in order to identify temporal protein expression changes.Raw MS data were processed using Genedata ExpressionistTotal RNA was isolated for each exposed microtissues using Qiagen AllPrep DNA/RNA/miRNA Universal Kit (Cat #80224). Sample were depleted of ribosomal RNA using the Illumina RiboZero Gold kit (Cat #MRZG12324) and libraries were prepared for sequencing using Lexogen SENSE total RNA library preparation kit (Cat #009.96). The samples were sequenced on the HiSeq2500 (100 bp paired-end).® software v.11.0. The first 12bases of the 5′end of all reads and adapter sequences were removed using Trimmomatic version 0.3274. Data quality was checked using FastQC before and after trimming. Sequencing reads were mapped to the human genome version hg38 with the splice junction mapper STAR (version 2.5.3a)75 using as annotation the reference genome gencode version 26 —Ensembl 88. Quantification of genes or transcripts, respectively, was performed with an algorithm based on Cufflinks76. Features used for quantification were protein coding and non-protein coding sequences (e.g. pseudo-genes missing a CDS of the transcripts). Differential expression analysis of the RNA-seq experiments was performed with DESeq2 (version 1.14.1)77. The quality of the samples was assessed according to the amount of (mapped) reads, Cook’s distance, hierarchical clustering, principal component analysis, and sample dispersion. Outliers were excluded from further analyses  q-value of ≤0.05, an R-square of the regression model of ≥0.7, and a minimal observation number of 10. For the two-step regression model searching for significant different time-dependent gene expression profiles across the analytical groups, the input matrices for DESeq2 consisted of all samples from the controls and the treatment/condition groups (therapeutic and toxic) and the DESeq2-derived rlog-transformed data were then used for the analysis of the time-dependent gene expression profiles. For the analysis of the time-dependent protein expression profiles the previously described normalized data sets were used as input. For each treatment and dose the analysis was carried out separately, the resulting dynamic response genes/proteins are summarized in Supplementary Data We have used MaSigPro (version 1.46.0) to calculate for each gene or protein, the fit of the quadratic regression model degree = 2)::1\docume78 was modified. DNA was fragmented to 100–200 bp using the Covaris S2 system. Because of low DNA yield for DOX and IDA samples, the triplicate samples were pooled before fragmentation. End repair and A tailing was performed using the NEBNext® Ultra™ library prep kit for Illumina® (NEB), adapters were ligated with NEBNext® Ultra™ Ligation Module (NEB) and samples were purified using Agencourt® AMPure® XP beads (Beckman Coulter). Methylated fragments were captured using the MagMeDIP kit (Diagenode). In short, denatured DNA was mixed with anti-5-meC-antibody and captured using magnetic beads. Capture efficiency was determined by qPCR against spiked-in Lambda-DNA fragments in precapture and postcapture library samples. Libraries were amplified in a final PCR step using barcoded TruSeq primers. Quality was assessed on Agilent Bioanalyzer 2100 and library concentration was determined by Qubit™ and qPCR.For preparation of microtissue MeDIP-Seq libraries, a previously published low input MeDIP protocol79, and analyzed in 250 bp windows using the R/bioconductor package QSEA25 with standard parameters. Within QSEA, the MeDIP enrichment was calibrated with 450k methylation array measurements of primary hepatocytes (GSM999339) and cardiac myocytes  from ENCODE80, for the hepatic and cardiac micro-tissues, respectively. To this end, beta values of the calibration samples were computed with the R/Bioconductor package Minfi81, genomic locations of the array probes were mapped from GRCh37 to GRCh38 using the UCSC liftOver command line tool82, and probes within 250 base windows were averaged. DMRs obtained from QSEA were annotated with gene, exon, and promoter (transcription start site ±2 kilobases) information from RefSeq, ENCODE TFBS and model-based CpG islands, all obtained via the UCSC table browser. Since ENCODE TFBS were not available for GRCh38, genomic locations were mapped from GRCh37 using the liftOver tool.In order to gain exhaustive genome-wide coverage the triplicate samples that have been sequenced individually were merged before alignment. MeDIP sequencing reads were aligned to the GRCh38 reference genome using bwa Version 0.7.15-r1140Interaction network generation: We agglomerated a large PPI from 19 different data resources83 which has been proven to be one of the most effective networks for identifying disease genes in a recent comparison84. We have performed quality assessment of the individual interactions using different supervised and unsupervised methods85 in order to generate a high-quality PPI network consisting of 10,707 proteins and 114,516 interactions.Node scoring: In order to weight the proteins/genes according to their information content with respect to time-sensitive AC treatment responses we used scores that reflect the dynamic changes of the proteins after AC treatment: For each protein i, the fit of the quadratic regression model (above section) is described by the estimated regression parameters p-values, Network propagation: Network propagation was done with Hotnet221 based on a version of the random walk with restart method (insulated heat diffusion). This method was developed initially for tracking the effect of mutations across a network. We adapted this approach to analyze the effects of proteome/gene expression data. For each longitudinal data series we initialized the nodes of the networks as shown above so that these initialized networks per AC, dose and data platform can be interpreted as the dynamically affected interactome. Network propagation then delivers a final ranking of all proteins  along with a subnetwork containing the major connected components of the drug response. Hotnet2 offers four different subnetworks depending on a threshold parameter δ, and in all cases we chose the subnetwork derived with the minimal threshold parameter.Numerical simulations: The mitochondrial model, represented schematically in Fig. 44. The reactions in the system were grouped into three subsystems, relating to the electron transport chain (ETC), ROS scavenging, and the tricarboxylic acid cycle (TCA). The model solutions seek to emulate the functional behavior of the mitochondrion subject to drug exposure by modifying the kinetic parameters of the baseline model22 as deduced from the in vitro experiments conducted on 3D cardiac microtissues. The solution of the differential equations then yield the predicted flux through each reaction in the mitochondrial theoretical model, and the procedure was repeated for each drug and dosage in order to map the evolution of the steady state over up to 14 days, as per the in vitro experiments. The effect of a given drug and dose level on individual reaction rates, at each time point, was estimated experimentally by applying the drug to the microtissues, following a PBPK-based protocol. The drug exposure was then implemented in the simulations by scaling the appropriate reaction rates based on the predictions derived from the experimental time-series data, fitted to a polynomial model.ode15s numerical solver of Matlab until a steady state was reached . Average fluxes through the ROS, ETC, and TCA subsystems are plotted in Supplementary Fig. For each such reaction rate configuration, the set of coupled differential ordinary differential equations was solved using the Functional measurements: The simulation results were compared with intracellular ATP concentrations measured in the microtissues using a CellTiter-Glo cell viability assay . The amount of ATP detected in the assay increases with the number of viable cells in the culture, as described in other studies86. The assay is a luminescence test involving a detergent component that dissociates the cells and disrupts the membrane. The released ATP then catalyzes the luciferase reaction, making the luminescence intensity proportional to the amount of ATP, as determined by comparison with a standard curve.Sensitivity analyses: sensitivity analyses were performed to assess the relative impact of the drug exposures to each reaction in the system (Supplementary Fig. Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Reporting Summary