A miniature pig model of ischemic mitral regurgitation (IMR) was developed by posterior mitral chordae tendinae rupture and implantation of an ameroid constrictor. A 2.5-mm ameroid constrictor was placed around the left circumflex coronary artery (LCX) of male Tibetan miniature pigs to induce ischemia, while the posterior mitral chordae tendinae was also ruptured. X-ray coronary angiography, ECG analysis, echocardiography, and magnetic resonance imaging (MRI) were used to evaluate heart structure and function in pigs at baseline and one, two, four and eight weeks after the operation. Blood velocity of the mitral regurgitation was found to be between medium and high levels. Angiographic analyses revealed that the LCX closure was 10–20% at one week, 30–40% at two weeks and 90–100% at four weeks subsequent ameroid constrictor implantation. ECG analysis highlighted an increase in the diameter of the left atria (LA) at two weeks post-operation as well as ischemic changes in the left ventricle (LV) and LA wall at four weeks post-operation. Echocardiography and MRI further detected a gradual increase in LA and LV volumes from two weeks post-operation. LV end diastolic and systolic volumes as well as LA end diastolic and systolic volume were also significantly higher in pig hearts post-operation when compared to baseline. Pathological changes were observed in the heart, which included scar tissue in the ischemic central area of the LV. Transmission electron microscopy highlighted the presence of contraction bands and edema surrounding the ischemia area, including inflammatory cell infiltration within the ischemic area. We have developed a pig model of IMR using the posterior mitral chordae tendineae rupture technique and implantation of an ameroid constrictor. The pathological features of this pig IMR model were found to mimic the natural history and progression of IMR in patients. Ischemic mitral regurgitation (IMR) is characterized by the backflow of blood from the left ventricle into the left atrium of the heart The dominate theories for mechanisms underlying IMR include: (i) imbalance of leaflet tethering and closing force, (ii) left ventricular remodeling, (iii) left ventricular dysfunction, (iv) dyssynchrony of the left ventricular electromechanical activity and (v) changes in spatial structure of the annulus IMR is associated with a worse prognosis after myocardial infarction and subsequent revascularization Since treatments for IMR remain controversial, the field has focused on developing animal models  to study IMR pathophysiology and test therapeutic approaches for IMR All animal procedures were in full compliance with the guidelines approved by the Animal Welfare and Ethics Committee in Fuwai Hospital, Chinese Academy of Medical Sciences, Beijing, China (permission number of 2012-1-40-BKJ).Male Tibetan miniature pigs that were 6 months of age (n = 13) and weighed 22–28 kg were obtained from the Beijing Paike biotechnology company. Pigs had free access to autoclaved water and were fed twice a day.2 at 100%. During this time, pigs were maintained with half doses of ketamine  and diazepam . Pigs were placed on their right lateral sides, using sterile techniques. Subsequently, the pericardium was opened at intercostal segments 2 and 3 by performing a small thoracotomy. A pericardial incision was performed and a 2.5-mm ameroid constrictor was placed around the left circumflex coronary artery (LCX), which resulted in myocardial ischemia in a region supplying blood to the LCX (including papillary muscle ischemia). At the same time, a moderate to severe mitral regurgitation was achieved by pulling off the mitral valve chordae tendinae through a mitral strain device. A pocket was sewn on the left atrial appendage and then the mitral strain device was put through a small incision on the left atrial appendage, which was deep within the left ventricle. Guided by epicardial ultrasound and using cutting barbs on the cutting rod ends, we selectively cut the mitral valve chordae behind P2 and P3 of mitral chordae. As a result, moderate to severe mitral regurgitation was induced in the pigs. In the sham group, pigs were treated the same as in the IMR group but did not undergo ameroid ring implantation and injury of the mitral valve. After the surgery, fentanyl  was administered as an analgesic. Penicillin , aspirin (100 mg per day for 1 week), hydrochlorothiazide (8 mg per day for 1 week) and potassium citrate (1.46 g twice per day) were also administered to all pigs.General anesthesia was intramuscularly administered in pigs with ketamine (35 mg/kg) and diazepam (1.5 mg/kg). A 14-French cuffed endotracheal tube was used for intubation in pigs and ventilation was administered at 15 ml/kg and FiOX-ray angiography was performed on pigs at baseline as well as one, two and four weeks after surgery using standard transfemoral Judkins techniques that were employed after right and left intracoronary administration of 10 ml iopromide . Cardiac coronary angiograms were obtained by dynamic scanning of the pig heart at a speed of 3 frames per second with an X-ray machine (Philips).Electrocardiograms were recorded as previously described Pigs were anesthetized as described above. Echocardiography was performed with a biplane scanner (Philips Sonos 7500) ultrasound system using a 2.5-MHz transducer . Ejection fraction (EF), left ventricular end diastolic volume (LVEDV), left ventricular end systolic volume (LVESV) and left ventricular mass were determined using the Cube formula Magnetic resonance imaging (MRI) was performed in all pigs at baseline as well as one and thirteen weeks post-surgery using a 1.5T whole body imager . Specific absorption rates (SAR) were limited to below 2.2 W/kg and the average SAR for the chest was 1.4 W/kg using fast gradient-echo sequences. MRI results were interpreted by a radiologist who was blinded to the animal groups.Cardiac troponin I  and B-natriuretic peptide  were measured by an enzyme immunoassay that uses pig specific enzyme-linked immunosorbent assay kits. The assay was performed according to the manufacturer's instructions.Hearts were harvested from pigs and fixed for 24-hours in formalin. Fixed hearts were embedded in paraffin. Serial coronal sections were obtained from the ventricle and evenly distributed into five slides. Cardiac sections were stained with nitro blue tetrazolium (N-BT) for 15 min at room temperature. A HPIAS-1000 medical imaging system  was used to measure the infarct area (no NBT staining) and non-infarcted area (NBT staining area). Myocardial infarct sizes and ratios were calculated by dividing myocardial infarct area to total ventricular area. Serial transverse sections were also obtained along the entire length of the embedded hearts. Cardiac sections were stained with hematoxylin and eosin  and Masson's trichrome stain. The histopathological severity of each heart was determined by scoring the number of inflammatory cells, area of infiltration, presence of interstitial edema, presence of neutrophil infiltration and the size of the necrotic area.For electron microscopy, hearts were fixed for 1 hour at 4°C in a cacodylate buffer containing 1% osmium tetroxide and subsequently treated with tannic acid as an en bloc stain to enhance the contrast of elastic laminae as described previously Data are presented as mean ± standard error of the mean. Statistical analyses were performed using the unpaired student t-test or analysis of variance followed by the Newman-Keuls test. A Chi-Square test was also performed to analyze differences between images. P-values found to be less than 0.05 were considered statistically significant. Statistical analyses were performed with the use of the SPSS 11.0 software .The ameroid constrictor was used to cause vessel closure, which resulted in 10–20% closure at one week, 30–40% closure at two weeks and 90–100% closure at four weeks post-implantation in pigs as well as using the angiographic guide wire as a scale (diameter: 0.21 cm) . ECG parThe cTnI concentration was measured in sera of pigs at baseline as well as 30 and 60 days post-surgery. There was a peak (106.8 ng/ml) in cTnI levels at 30 days after surgery, indicative of the severity of ischemic injury . HoweverColor Doppler flow imaging was used to detect eccentric “drop-like” jets of mitral valve regurgitation near the left atrial wall in all pigs. The abnormal movement of the mitral muscle could also be detected. We showed that the mitral ring gradually expanded in pig hearts with increasing left ventricular (systolic and diastolic) and atrial volumes . All of Although color Doppler analysis indicated structural changes to the heart, both the sV and EF were not significantly different at any of the observed time points . The E/ACardiac MRI was performed in pig hearts at baseline and eight weeks post-surgery . MRI resAnalyses of pig heart structure post-surgery revealed that the pig hearts displayed chordae tendinae rupture (Grade I or Grade II) of the mitral valve and “jet lesions”, which have been previously described in the left ventricle IMR is a unique valvular heart disease caused by MR resulting from rupture or extension of the papillary muscle after myocardial infarction. It is different from MR induced from systemic diseases that target the mitral valve such as rheumatic valvular disease and mitral valve prolapse. IMR is a multi-faceted and multi-stage disease that is characterized by local and global left ventricular remodeling Animal models and clinical studies have demonstrated the multifaceted and pathophysiological origins of IMR We have developed a novel pig IMR model using a posterior mitral chordae tendinae rupture technique and implantation of an ameroid constrictor. The pathological features of this pig IMR model mimicked the natural evolution of IMR in patients. We present an IMR model that should provide unprecedented opportunities to study pathogenesis and therapeutic interventions for IMR.Table S1Cardiac troponin I levels in plasma of operated pigs.(DOC)Click here for additional data file.Table S2B-natriuretic peptide levels in plasma of operated pigs.(DOC)Click here for additional data file.Table S3Cardiac dimensions, function and regurgitation parameters in control pig heart. Note: LVEDV: Left ventricular end diastolic volume, LVESV: Left ventricular end systolic volume, EF: Ejection fraction, LAEDV: Left atrial end diastolic volume, LAESV: Left atrial end systolic volume. The same with those in table D-I.(DOC)Click here for additional data file.Table S4ardiac dimensions, function and regurgitation parameters before surgery in operated pig heart.(DOC)Click here for additional data file.Table S5Cardiac dimensions, function and regurgitation parameters immediately after surgery in operated pig heart.(DOC)Click here for additional data file.Table S6Cardiac dimensions, function and regurgitation parameters one week after surgery in operated pig heart.(DOC)Click here for additional data file.Table S7Cardiac dimensions, function and regurgitation parameters two weeks after surgery in operated pig heart.(DOC)Click here for additional data file.Table S8Cardiac dimensions, function and regurgitation parameters four weeks after surgery in operated pig heart.(DOC)Click here for additional data file.Table S9Cardiac dimensions, function and regurgitation parameters eight weeks after surgery in operated pig heart.(DOC)Click here for additional data file.


Nutlin inhibits TP53-MDM2 interaction and is under investigation in soft-tissue sarcomas (STS) and other malignancies. Molecular mechanisms of secondary resistance to nutlin in STS are unknown. We performed whole-transcriptome sequencing (RNA-seq) on three pretreatment and secondary resistant STS cell lines selected based on their high primary sensitivity to nutlin. Our data identified a subset of cancer gene mutations and ploidy variations that were positively selected following treatment, including TP53 mutations in 2 out of 3 resistant cell lines. Further, secondary resistance to nutlin was associated with deregulation of apoptosis-related genes and marked productive autophagy, the inhibition of which resulted in significant restoration of nutlin-induced cell death. Collectively, our findings argue that secondary resistance to nutlin in STS involved heterogeneous mechanisms resulting from clonal evolution and several biological pathways. Alternative dosing regimens and combination with other targeted agents are needed to achieve successful development of nutlin in the clinical setting. MDM2 gene. In turn, MDM2 binds to TP53 and blocks its transactivation domain. MDM2 also serves as a TP53 ubiquitin ligase that targets TP53 for ubiquitin-dependent degradation in the proteasome   as an indicator of metabolically-active cells . Known nCell cycle distribution of sensitive and resistant cell lines was studied by DNA content by fluorescence-activated cells sorting and analyzed using the Cell Quest Pro software . After 48h of treatment with two different concentrations of RG7388, cells were centrifuged at 1500g for 5 min and washed twice with PBS. Cells were then fixed with 70% ethanol at 4°C overnight. Ethanol was removed and cells washed twice with PBS. 300μl of a propidium iodide, ribonuclease-containing solution were added to cells and then analyzed by FACS. Data were analyzed with FlowJo v.7.6.3. software and results expressed in terms of percentage of cells in a given phase of cycle.The mitochondrial membrane depolarization (ΔΨm) was measured by tetramethyl-rhodamine ethyl staining (TMRME). 5000 cells were seeded in 96-well plates, incubated 24h before adding an increasing concentration of RG7388 over 48h. Cells were loaded with TMRM (200nM) for 30 min at 37°C and then trypsinized and harvested in saline extracellular solution. Cells were then analyzed on a FAC scan flow cytometer .For apoptosis assessment, 1.105 cells were seeded in 6-wells plates and after 24h cells were treated by Nutlin for 72hr and exposed to FITC-Annexin and propidium iodide (PI) according to the manufacturer’s protocol . It allows distinguishing annexin V positive cells in early apoptosis, versus annexin V and PI positives cells in late apoptosis. Cells were analyzed in cytomerty using FL1 Annexin-V, whereas FL2 was used for Propidium iodide.Flow cytometry  data were analyzed with FlowJo v.7.6.3. software.Cells were trypsinized, washed in cold phosphate buffered saline (PBS) and lysed by 3 cycles of freezing in liquid azote and thawing in ice as described by Tansey . CellulaCells were seeded on coverslips and treated with RG-7388 for 72 hours. Slides were then washed twice with PBS, fixed in formaldehyde 4% and incubated with anti-LC3IIB monoclonal antibody  overnight, and then with a goat anti-rabbit Alexa fluor 488 antibody . Slides were then counterstained by 4,6-diamidino-2-phenylindole (Hoechst).RnaSeq was sequenced by the Centro nacional de análisis genómico  using standard Illumina  protocols. Briefly, the library from total RNA was prepared using the TruSeq™ Stranded mRNA Sample Preparation kit (Illumina Inc.) according to manufacturer’s protocol. 1μg of total RNA was used for poly-A based mRNA enrichment selection using oligo-dT magnetic beads followed by fragmentation by divalent cations at an elevated temperature resulting into fragments of 80-450nt, with the major peak at 160nt. First strand cDNA synthesis by random hexamers and reverse transcriptase was followed by the second strand cDNA synthesis, performed in the presence of dUTP instead of dTTP. Blunt-ended double stranded cDNA was 3´adenylated and the 3´-“T” nucleotide at the Illumina indexed adapters was used for the adapters’ ligation. The ligation product was amplified with 15 cycles of PCR.Each library was sequenced using TruSeq SBS Kit v3-HS, in paired end mode with the read length 2x101bp. We generated 135 million minimally-paired end reads for each sample run in one sequencing lane on HiSeq2000  following the manufacturer’s protocol. Images analysis, base calling and quality scoring of the run were processed using the manufacturer’s software Real Time Analysis (RTA 1.13.48) and followed by generation of FASTQ sequence files by CASAVA.https://github.com/jstjohn/SeqPrep) was used to remove sequencing adaptors from the raw reads. The application of these standard packages revealed a proportion of RNA fragments of smaller size, whose R1 and R2 paired end reads were overlapping. To keep exploiting these fragments without biasing the count of reads in reads overlapping regions, we developed a home-made script (awk language) that merged the overlapping R1 and R2 and split it into new non-overlapping read sequences.Raw RnaSeq sequences where quality controlled using a set of published tools in order to produce curated reads. Briefly, the package sickle  was usedhttp://broadinstitute.github.io/picard/).Curated read sequences were then aligned using TopHat2  on both The statistics of the number of raw, curated and aligned sequences and curated aligned sequences was that created with a home-made tool Positions in the transcriptome showing at least one alternative allele were detected via samtools mpileup  taking cPositions detected as variant in several samples (sensitive and resistance cell lines) were merged using a homemade script (bash awk).http://cancer.sanger.ac.uk/cancergenome/projects/cosmic). For variants in coding positions the impact of the aminoacid change in the protein domains is reported via the use of Sift [Variant positions were annotated using the Annovar tool found in sequences in at least one of the sensitive or resistant samples, of any of the three cell lines. The table resumes the variants that present at least 1 alternative allele counts in any sample, each base having sequencing quality (phred score) of at least 20, without additional filters. The table, containing 285,999 variants  allows checking the alternative and reference allele counts at a position, in any of the sequenced samples.http://cancer.sanger.ac.uk/cancergenome/projects/cosmic) and whose alternative allele frequency (AF) in the Caucasian population (CEU), as reported in 1000g, was lower than 5%. If a variant was neither reported in 1000g CEU nor dbSNP, it was included only if either SIFT or Polyphen2 HDIV functional interpretations were reported as “deleterious” for the protein. If a variant was not reported in 1000g CEU but reported in dbSNP, it was included only if both SIFT or Polyphen2 HDIV functional interpretations were reported as “deleterious” for the protein. Also, if a variation was located in a region of the genome that is annotated as Segmental Duplication [P< = 10–3, and difference between AAR < = 0.1). Variants whose differences in AAR were not significant and superior to 0.30 in both conditions were associated to the ancestral primary tumor clone, while the other variants identified the differences between the clones sensitive and secondary resistant to RG7388.We extracted from the Merged Variant table the variants detected in sensitive and resistant cells. We then included in the analysis only variants whose genotype was estimated as a non-homozygous reference in both samples, with more than 10 reads at the variant position, localized in coding regions and of non-synonymous type. We included all variants whose positions were reported in the COSMIC database  in order to predict the interactions between the genes and the potential effects on cellular pathways and networks.RnaSeq was used to estimate the ploidy of the tumor samples, where the term ploidy defines the total number of parental alleles in large genomic regions see . The RnaMDM2 gene copy numbers. In agreement with the mechanism of action of nutlins, treatment of wild-type TP53 (Sanger sequencing) STS cell lines with RG7388 showed an accumulation of the TP53 protein and its targets, P21 and MDM2, as revealed by Western blotting , but not of the 6 out of 11 cell lines with TP53 mutations . The mosblotting .One of the main cellular functions of activated TP53 is blocking cell cycle progression in the G1 and G2 phase. Treatment of exponentially proliferating STS cell lines with RG7388 for 48 hours led to a dose-dependent cell cycle block in G1 and G2/M phase and a depletion of the S phase compartment . One of Among the entire panel of STS cell lines tested, RG7388 displayed the highest cell growth inhibition effects in IB111, IB115 and IB128. In order to investigate mechanisms of secondary resistance to RG7388, IB111, IB115 and IB128 cells were treated with RG7388 for 3 days. The cells were then rinsed to remove RG7388, and the remaining cells were expanded in normal medium (minus RG7388). We repeated the process four times, and we obtained populations that survived 1–4 rounds of RG7388 treatment (P1 for one round—P4 for four rounds). We compared the extent to which IB111, IB115 and IB128 underwent apoptosis and cell cycle inhibition when treated for 3 days with RG7388. Results indicated that the selected populations became progressively more resistant to cell growth inhibition  to apoptIn order to assess specificity of secondary resistance to RG7388, we compare the sensitivity profiles of IB111, IB115, IB128 and their RG7388-resistant counterparts IB111P4, IB115P4 and IB128P4 to doxorubicin and gemcitabine, two cytotoxic drugs commonly used to treat STS patients with advanced disease. Doxorubicin and gemcitabine significantly inhibited the proliferation with the same extent in the parental cell lines and the RG7388-resistant sublines . This inTP53 gene.Several studies have shown correlations between gains and losses of chromosomes and cancer-specific drug resistance –29. We wTP53 gene was the only recurrent gene we identified as having mutations with significant changes in abundance between the secondary resistant and the parental cell lines. Indeed, we identified one TP53 mutation in the IB115 cell line (C275S) and three different TP53 mutations in the IB128 cell line . These mutations have all been previously reported in human tumor samples  and are located in the highly- conserved DNA binding domain of TP53. All these positions have been validated with ultra-deep DNA resequencing of the TP53 coding region (data not shown). No TP53 mutations were identified in the IB111 cell line.To identify changes in the mutation profiles of secondary resistant cells, we compared the abundance of somatic mutations found in the parental and secondary resistant cell lines. For each cell line, we examined a conservative list of mutations and used Fisher’s exact test to identify those specifically associated with secondary resistance see . In all ®,QIAGEN Redwood City, www.qiagen.com/ingenuity). The results showed that these genes were mainly enriched in proliferative, growth and movement networks  between the parental and secondary resistant cell lines in IB111, IB115 and IB128 respectively . We thennetworks . We focuand PUMA . Among td agents –39, we dThe new paradigm of targeted therapies has dramatically impacted oncology practice with the discovery and development of 'personalized' anti-cancer drug medicines that produce remarkable clinical responses in a subset of patients with advanced disease. While there is rapidly growing enthusiasm for this new paradigm, there is also increasing realization that such targeted therapies suffer from the same major limitation as traditional chemotherapy—clinical benefits are of limited duration due to secondary drug resistance.Establishing specific molecular mechanisms of resistance to classical cytotoxic drugs has been challenging due to the nonspecific nature of their antitumor mechanisms of action. On the other hand, identification of mechanisms of acquired resistance to targeted therapies is crucial since the discovery of such mechanisms can prompt the development of strategies specifically designed to overcome them. The design of new drugs to treat lung cancer patients who become resistant to EGFR-directed therapy due to the emergence of the T790M secondary mutations represents a perfect example of the importance of research efforts in the field of secondary resistance .In Phase I studies, RG7388 has been associated with disease control in a significant subset of sarcoma patients. However all of them had disease progression within a median of 6 months after treatment onset suggesting the occurrence of mechanisms of secondary resistance involved in tumor progression .We report here the first comprehensive analysis of genetic mechanisms involved in secondary resistance to nutlin using next-generation sequencing of isogenic pairs of nutlin-sensitive and nutlin-resistant STS cell lines. The aberrations involved in secondary resistance we described here can be considered specific to RG7388 given that no cross-resistance was noted with other drugs commonly used for the management of STS patients such as doxorubicin or gemcitabine.By performing deep sequencing of secondary resistant and parental sensitive cell lines, we showed here that secondary resistance to nutlin was associated with several mutations whose mutant allele fraction significantly changed between parental and secondary resistant cells. Among them, we identified TP53 mutations in the secondary resistant cells that were not present in the parental counterpart. Previous reports investigating mechanisms of secondary resistance to nutlin in neuroblastoma and osteosarcoma cell lines suggested that exposure to nutlin induces the emergence of TP53-mutated clones , 42. WheN-RAS in the IB128 resistant cells. Activation of the MAPK/ERK kinase (MEK)1/2 kinase pathway has been shown to impair TP53-dependent apoptosis in U20S cells treated with nutlin [ECT2 gene, the expression of which has been shown to be normally down-regulated by Nutlin in a TP53-dependent manner [ECT2 was shown to be sufficient to prevent cell death induced by ionizing radiation underlying its potential important role in resistance to cancer therapy resistance [Besides TP53 mutations, our data identified a subset of other gene mutations that were positively selected in secondary resistant cells, many of which have been previously involved in the control of cell growth or apoptosis. Some of them may represent ‘passenger’ mutations or false-positives, but some are likely to contribute to resistance to RG7388. We highlight two examples here. In IB128 resistant cells, we observed a significant increase in the abundance of an activating mutation in h nutlin . Recent h nutlin . The post manner . Inactivsistance .MDM2 (12q13-15) for IB111 resistant cells or TP53 (17p) for the IB128 resistant cells. This implies that some ancestral cells that carried this complement of ploidy aberrations emerged under RG7388 selection pressure. These additional ploidy aberrations demarcate the split between fully clonal versus subclonal ploidy variations.We have also found that secondary resistance to RG7388 was associated with few acquired ploidy variations. This observation fits with the results of a previous study showing that instable aneuploidy is associated with acquired drug resistance and that nutlin treatment of U2OS osteosarcoma cells and HCT116 colon cancer cells can result in the emergence of tetraploid cells . Here weIt was recently observed that tumor cells vary in terms of their apoptotic activity potential, and that alteration of the apoptotic machinery is a cause of chemoresistance and oncogenic transformation –49 By coIn conclusion, our results demonstrate that secondary resistance of STS cells to nutlin involves multiple and complex mechanisms and that the interplay between these mechanisms warrants extensive investigation. As such events do not appear to impact sensitivity to cytotoxic agents commonly given to STS patients, therapeutic strategies preventing their emergence should be designed. One of them could be administering nutlin for a restricted period, alternating with other drugs and then restarting, irrespective of disease progression status in the period between treatments.S1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 Fig(DOCX)Click here for additional data file.S1 Methods(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Sound waveforms convey information largely via amplitude modulations (AM). A large body of experimental evidence has provided support for a modulation (bandpass) filterbank. Details of this model have varied over time partly reflecting different experimental conditions and diverse datasets from distinct task strategies, contributing uncertainty to the bandwidth measurements and leaving important issues unresolved. We adopt here a solely data-driven measurement approach in which we first demonstrate how different models can be subsumed within a common ‘cascade’ framework, and then proceed to characterize the cascade via system identification analysis using a single stimulus/task specification and hence stable task rules largely unconstrained by any model or parameters. Observers were required to detect a brief change in level superimposed onto random level changes that served as AM noise; the relationship between trial-by-trial noisy fluctuations and corresponding human responses enables targeted identification of distinct cascade elements. The resulting measurements exhibit a dynamic complex picture in which human perception of auditory modulations appears adaptive in nature, evolving from an initial lowpass to bandpass modes  following repeated stimulus exposure. Amplitude modulations are considered the key carriers of intelligible information in auditory signals, and consequently it is of significant interest to discover how they are neurally analyzed and perceptually encoded. A dominant model has emerged from extensive experimental and theoretical studies of this phenomenon. This model posits that amplitude modulations are parsed into channels of different temporal rates via a bank of bandpass filters. Using exclusively data driven approaches with minimal assumptions about the structure of the model, the picture that emerges is of an adaptive process. Initially, human listeners in these tasks perceive modulations as if through a lowpass filter with very low cutoff frequency, which gradually evolves to become a broadly tuned bandpass process at higher modulation frequencies, reflecting the modulations of the target stimuli. This surprising dynamic characteristic emphasizes the plastic nature of modulation analysis in sensory perception. Natural sounds carry salient amplitude modulations (AM) essential for successful interpretation of auditory landscapes and robust source identification –5. The hendogenous adaptive strategies can influence the measurements of the modulation filter when no stimulus and/or task-related factors are concomitantly modified. Specifically, we sought to adopt a single stable measurement approach throughout (the reverse correlation method), reasoning that systematic changes in the subjects’ detection strategies would be unveiled as a dynamic evolution of the filter estimates during the experimental session  where w(0) is power at frequency 0 (DC) and w(f > 0) is all remaining power in the spectrum. Spectral slope ; for exposure-related analyses , they wen detailed above) and used it as proxy for the noise-induced DC perturbation of each stimulus. We then took the difference in noise DC content between the two stimuli presented on each trial, and computed the biserial correlation coefficient between this differential DC content and the binary response returned by listeners . Each simulation returned the difference between the AM content of the two traces over 1000 trials, and the average of 100 simulations is plotted in We also wished to verify that the slight amplitude deviations introduced by the randomly generated carrier  did not affect our calculations based on the notional AM perturbations specified before application of the carrier to generate the stimulus waveform. To this end, we applied the following energy-based recalibration to the stimulus samples in our dataset: each waveform as it was delivered to the listener was split into 9 segments, and RMS (root-mean-square) was computed from each segment to obtain a proxy AM vector equivalent to the 9-element vector specified by our amplitude modulation protocol. We then applied the same kernel estimation procedures used with the pre-specified noise samples to these RMS-corrected samples. The resulting kernel estimates are plotted in L1 applied via template-matching, followed by a threshold conversion to binary decision [L1NL2) cascade [L1 linear stage in the L1NL2 cascade involves convolution, not template matching; see Data from reverse correlation experiments are almost invariably interpreted with relation to a cascade that only incorporates one linear filter decision . However cascade  which, i cascade , 76 (notL1NL2 cascades as illustrated in L1 stage (blue in N) and a subsequent linear stage (L2). Established techniques in nonlinear system identification [L1 and L2 from first-order and second-order psychophysical kernels like those shown in Figs L1/L2 and psychophysical kernels).Both LED and MFB can be cast in the form of  blue in : low-pas blue in , primari blue in . Subsequfication , 64, comfication , can be L1 and L2 can be thought of as ‘front-end’ and ‘read-out’ filters. L1 is the component of primary interest for this study, as it supports the distinction between LED and MFB as being associated with low-pass versus band-pass characteristics respectively . This band-pass property is not an artefactual distortion induced by the target signal, because it is preserved when estimates are obtained from target-absent noise modulations alone . Estimainset to . As we dall cases, L1 estimate distributions (solid histograms in L2 estimate distributions (open histograms) fall within the low-pass range ; Q values (L1 (but not L2) are significantly larger  than expected for a uniform spectrum (indicated by dashed lines in −5) for L1 (indicative of band-pass/highpass characteristics) and smaller than 0 for L2 (p<0.05); the spectral slope (L1 (p<10−4) but negative for L2 (p<0.01).We quantify this band-pass finding using four different metrics . In all  studies ). More sQ values  for L1 : there was no statistically significant difference (at p>0.05) for any metric and for either L1 filter is band-pass; therefore, AM processing resembles the characteristics of the MFB more than the LED model. This conclusion is not the product of fitting either model to the data: it is based on non-parametric characterization of the front-end filter associated with a general framework cascade that encompasses both models  in the direction of greater band-pass for the late epoch. This shift in band-pass value was accompanied by a significant shift in filter centroid  of the noise samples presented on those same trials see . In the The filter bank proposed by recent versions of the MFB model , encompaTo gain better insight into the temporal evolution of the exposure-mediated effects, we obtained centroid estimates across 10 different epochs of data collection. Centroid estimates drifted exponentially towards higher values  matchingThe above-detailed modifications of filter structure were associated with only mild improvements of absolute efficiency in some listeners . This apFirst, learning effects on AM discrimination are small and difficult to expose . SecondPrompted by the above results, we re-analyzed data from a prior published study  to deterThis study represents the first targeted application of psychophysical reverse correlation to AM processing. Although related tools have been applied successfully in auditory neuroscience  and psyclowpass characteristics, with no evidence of bandpass AM filtering. Signatures of bandpass processing are exposed specifically by second-order kernels , allowing us to confidently treat our dataset as reflecting the properties of the same perceptual machinery throughout , 32 . This rasticity . For exaasticity  may needasticity ). This i−7) and late  epochs  Sensitivity is in the d′∼1 range (unity is indicated by dashed lines). (B) Bias is not statistically different from 0 (indicated by dashed lines). (C) Internal noise is within expected range  (grey sh(EPS)Click here for additional data file.S2 Fig Target-absent and target-present first-order kernels are plotted in A and B respectively (see (EPS)Click here for additional data file.S3 FigL1 (bigger symbols for estimates obtained from first row/column of second-order kernels (L2. Different symbol shapes refer to different listeners. Error bars plot ±1 SEM. As for performance metrics (Panels (A-D) display estimates from increment detection on x axis against those from decrement detection on y axis. Solid symbols refer to  kernels  and smal kernels ). Open s metrics , there w(EPS)Click here for additional data file.S4 Fig(A) Absolute efficiency  was simi(EPS)Click here for additional data file.S5 FigThe stimulus waveform (A) is mapped to its spectrotemporal representation (B) using standard cochlear parameters . It is t(EPS)Click here for additional data file.S6 FigL1 estimates from second-order kernels) before and after correcting for carrier-induced AM deviations (black/red versus blue/yellow respectively). More specifically, A-B plot the quantities estimated in As detailed in Materials and Methods, a new carrier was generated at the beginning of each blok of data collection. This additional source of randomness in the stimulus caused slight amplitude deviations from those specified by the AM noise deliberately introduced and controlled by the experimenter. To verify that these deviations had no impact on our results, we reconstructed the amplitude content of individual noise samples while taking into account the specific carrier waveform presented on those specific trials (see (EPS)Click here for additional data file.S7 FigWe re-analyzed data from a previously published study  in light(EPS)Click here for additional data file.


BRCA1 mutation carriers have an 85% risk of developing breast cancer but the risk of developing non-hereditary breast cancer is difficult to assess. Our objective is to test whether a DNA methylation (DNAme) signature derived from BRCA1 mutation carriers is able to predict non-hereditary breast cancer.BRCA1 mutation carriers and 72 BRCA1/2 wild type controls) blood cell DNA samples were profiled on the Illumina 27 k methylation array. Using the Elastic Net classification algorithm, a BRCA1-mutation DNAme signature was derived and tested in two cohorts: (1) The NSHD (19 breast cancers developed within 12 years after sample donation and 77 controls) and (2) the UKCTOCS trial (119 oestrogen receptor positive breast cancers developed within 5 years after sample donation and 122 controls).In a case/control setting (72 BRCA1-mutation DNAme signature applied to blood cell DNA from women in the NSHD resulted in a receiver operating characteristics (ROC) area under the curve (AUC) of 0.65  which did not validate in buccal cells from the same individuals. Applying the signature in blood DNA from UKCTOCS volunteers resulted in AUC of 0.57  and is independent of family history or any other known risk factors. Importantly the BRCA1-mutation DNAme signature was able to predict breast cancer mortality . We also found that the 1,074 CpGs which are hypermethylated in BRCA1 mutation carriers are significantly enriched for stem cell polycomb group target genes (P <10-20).We found that our blood-based BRCA1 carriers is able to predict breast cancer risk and death years in advance of diagnosis. Future studies may need to focus on DNAme profiles in epithelial cells in order to reach the AUC thresholds required of preventative measures or early detection strategies.A DNAme signature derived from  BRCA1/2 genes [Breast cancer is the most common cancer in women, affecting at least 1 in 10 women in the western world. The potential to predict breast cancer and offer preventive measures is an effective intervention in women with an inherited predisposition to breast cancer due to mutations in /2 genes , epidemi/2 genes .Predicting the likelihood of breast cancer development is therefore still challenging not only because the sensitivity of current strategies is low  but alsoBRCA1 is a key negative modulator of PRC2 and that loss of BRCA1 inhibits stem cell differentiation and enhances an aggressive breast cancer phenotype by affecting PRC2 function [It is known that epigenetic variation contributes to inter-individual variation in gene expression and thus may contribute to variation in cancer susceptibility -10. DNAmfunction . Severalfunction -32. It wfunction .BRCA1 mutation) carry a specific methylation signature in peripheral blood cells, which is also able to predict sporadic breast cancer incidence and death. We also tested whether this signature is tissue-specific.Here we tested the hypothesis that women with an extremely high breast cancer risk . The NSHD epigenetics study was approved by the Central Manchester Research Ethics Committee (REC reference: 07/H1008/168). UKCTOCS was approved by the UK North West Multicentre Research Ethics Committees (North West MREC 00/8/34). Ethical approval for this nested case control study was obtained from the Joint UCL/UCLH Committees on the Ethics of Human Research (REC reference: 06/Q0505/102).All studies were approved by the relevant research ethics committee or institutional review board. Informed consent was obtained by all volunteers and conforms with the Declaration of Helsinki. The The DNA from whole blood and tissues was extracted at UCL  and at GData from two of the studies in this manuscript have been deposited in the Gene Expression Omnibus repository under the accession numbers (GSE58119), (GSE57285), (GSE32396). The NSHD data are made available to researchers who submit data requests to mrclha.swiftinfo@ucl.ac.uk; see full policy documents at . ManagedBRCA1 study, differentially methylated CpGs, with false discovery rate (FDR) corrected P values, between BRCA1 mutant carriers and BRCA1 wild type samples were identified via a multivariate logistic regression that was adjusted for age, batch and the presence of cancer.From the The elastic net classification method was chosen for our study as it has been shown to be particularly effective when the number of predictors is far greater than the number of training points . The elaTo evaluate its predictive accuracy, the identified classifier was tested on two independent datasets: (1) NSHD, and (2) UKCTOCS. For each individual, risk scores, based on their methylation profiles, were estimated and correlated to their disease status. An AUC value was then obtained via Somers’ Dxy rank correlation  see Add.BRCA1 mutation and 72 women with no mutation in the BRCA1 or BRCA2 gene  were hypermethylated (hyperM) and 1,092 (43%) were hypomethylated (hypoM) in women who had a BRCA1 mutation . To arrive at a specific DNAme signature, which would allow classification of independent samples, we used the elastic net (ELNET) framework  [BRCA1-mutation signature demonstrated the association of BRCA1 mutation with promoter hypermethylation of PCGTs. Indeed, the top categories of genes, associated with the hypermethylated CpGs in BRCA1 mutation carriers, were significantly (P <10-10) enriched for stem cell PCGTs irrespective of the definition used   ,44 on thd Figure . In contBRCA1-mutation DNAme signature is able to identify women who will develop breast cancer we analysed one of the best available characterised longitudinal cohorts , yielded a breast cancer risk AUC = 0.65    Figure A. IntereBRCA1 status - was not able to predict invasive breast cancer  to be oestrogen receptor (ER) negative [BRCA1-mutation DNAme signature predicted the development of an ER positive breast cancer with an AUC = 0.57   Figure . ImportaBRCA1-mutation DNAme signature is able to predict not only incidence but also breast cancer mortality we performed ROC statistics in the UKCTOCS set comparing women who died from breast cancer (n = 10) during the follow-up period with women who did not develop breast cancer . In line with these findings women with a higher than average BRCA1-mutation DNAme signature score were 8.46 (95% CI 1.06 to 67.69) -fold more likely to die from breast cancer (P = 0.04) than those with lower than average scores  many years in advance of diagnosis. Our findings also show that genes encoding developmental transcription factors integral for stem cell differentiation and biology are hypermethylated in women predisposed to breast cancer.Here we have provided several novel lines of evidence indicating that DNAme profiles obtained in cells from women with a BRCA1-associated DNAme signature is a risk predicting signature rather than an early detection signature, because: (1) the DNAme signature was derived from WBCs in women with a known BRCA1 status and was adjusted for cancer status ; (2) the time from sample draw to diagnosis had no dramatic impact on the strength of association between DNAme and potential for breast cancer development; (3) the signature was validated in two independent cohorts; (4) we observed a very strong overlap of CpGs associated with BRCA1 mutation (BRCA1 study) and CpGs indicating future breast cancer risk (UKCTOCS); and finally (5) the signature was also associated with invasive non-breast cancers.Our data suggest that the BRCA1-mutation associated CpGs are highly enriched for PCGTs which we and others have previously shown to be an epigenetic hallmark of cancer tissue [BRCA1 defect leads to retargeting of the PRC2 and reduces cell differentiation.The observation that the top ranked hypermethylated r tissue -18 and wBRCA1-mutation DNAme pattern in the absence of a BRCA1 mutation? It is likely that a combination of risk factors or factors which we have not captured  contribute to epigenetic modifications which are in common to those associated with BRCA1 mutation. Second, is the BRCA1-mutation DNAme signature in WBCs functionally relevant or just simply an indicator of breast cancer risk? The fact that the signature is indicative of breast cancer mortality would support the view that subtle epigenetic mis-programming of immune cells may lead to general immune defects which in turn supports the development and proliferation of cancers. However, all these suggestions are highly speculative and need validation in further independent cohorts using well-defined subsets of blood cells or epithelial cells.Two key issues remain unclear. First, which factors lead to a BRCA1 mutation carriers, nor did we have access to independent prospective buccal cell data. Third, we used the 27 k array, instead of the 450 k array, to generate the BRCA1-mutation DNAme signature.There are limitations to this study. First, we analysed whole blood DNA or serum DNA representing whole blood DNA and not a specific subset of peripheral blood cells. Second, although we found some good preliminary evidence that DNAme profiles in buccal cells are better at predicting future breast cancer risk (data not shown), we did not analyse buccal cells from In summary, our data highlight DNAme analysis as a promising tool to predict future breast cancer development. Future epigenome-wide studies should focus on using epithelial cells like buccal - or epithelial cells from the uterine cervix which are hormone sensitive and more likely to capture an ‘epigenetic record’ of breast cancer risk factors. Such studies are more likely to provide the level of specificity and sensitivity which is required for a clinically useful risk prediction tool.BRCA1 carriers is able to predict breast cancer risk and death years in advance of diagnosis albeit with a modest AUC. Our data further support the notion that DNAme modification at stem-cell differentiation genes, even in unrelated tissues, is an early event associated with carcinogenesis.In summary, our DNAme signature derived from blood cells from AUC: Area under the curve; DNAme: DNA methylation; FDR: False discovery rate; GSEA: Gene Set Enrichment Analysis; hyperM: Hypermethylated; hypoM: Hypomethylated; ROC: Receiver operating characteristics.IJ had a consultancy arrangement with Becton Dickinson in the field of tumour markers and ovarian cancer. Both IJ and UM have a financial interest through UCL Business and Abcodia Ltd. in the third party exploitation of clinical trials biobanks which have been developed through the research at UCL. The remaining authors declare that they have no competing interests.MW and SA had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: MW. Acquisition of data: EOF, MZ, AW, AGM, RH, DC, DK, UM, IJ. Analysis and interpretation of data: SA, AJ, AET, MW. Drafting of the manuscript: MW. Critical revision of the manuscript for important intellectual content: AET, SA, EOF, MZ, AW, AGM, AJ, RH, DC, DK, UM, IJ. Statistical analysis: SA. Obtained funding: MW, IJ, UM, DK. Administrative, technical or material support: SA. Study supervision: MW, AET, DK, IJ, UM. All authors read and approved the final manuscript.Description of the BRCA1 Study (White Blood Cell DNA samples from the Charles University in Prague).Click here for fileCharacteristics of the sample from the MRC National Survey of Health and Development study (NSHD).Click here for fileCharacteristics of the samples used from the UK Collaborative Trial of Ovarian Cancer Screening (UKCTOCS). Statistical significance was tested using a two-sided, Fisher’s exact test. The missing values were not included in the analysis.Click here for fileSupplementary Information Document with further details of Materials and Methods.Click here for fileTop ranked 2514 CpGs in white blood cells associated with BRCA1 mutation. These differentially methylated CpGs were identified based on their β-value methylation profiles. This was done via a multivariate logistic regression that was adjusted for age, batch and the presence of cancer. To correct for multiple hypothesis testing, the false discovery rates (FDR) were estimated using the q-value analytical procedure available through the R qvalue Bioconductor package.Click here for fileBRCA1-mutation associated DNA methylation signature consisting 1829 CpGs. The signature was derived by combining, via stacked generalisation, 100 cross-validated classifiers using ElasticNet with an alpha = 0.1.Click here for fileGene Set Enrichment Analysis on the top 1074 hypermethylated CpGs of the BRCA1 DNA methylation signature.Click here for fileGene Set Enrichment Analysis on the top 755 hypomethylated CpGs of the BRCA1 DNA methylation signature.Click here for fileGene Set Enrichment Analysis on the 105 differentially methylated CpGs (FDR < =0.05) in white blood cells associated with BRCA1 mutation.Click here for fileROC curve for the identified signature in invasive non-breast cancer samples of the NSHD dataset.Click here for fileAverage number of differentially methylated CpGs between tissue types. UKOPS WB = whole blood from postmenopausal healthy women; T1D WB = whole blood from pre and postmenopausal women with type-1 diabetes.Click here for fileROC curve of the identified signature on the UKCTOCS dataset, separated around the diagnosis time of less than/greater than 2 years.Click here for fileTop ranked 5482 CpGs associated with future breast cancer risk from serum DNA in the UKCTOCS set.Click here for fileGene Set Enrichment Analysis on the top 3395 hypermethylated CpGs in the UKCTOCS cohort using a multivariate linear regression framework.Click here for fileGene Set Enrichment Analysis on the top 2087 hypomethylated CpGs in the UKCTOCS cohort using a multivariate linear regression framework.Click here for fileCharacteristics of the samples used from the UK Collaborative Trial of Ovarian Cancer Screening (UKCTOCS). The samples were categorised according to their individual risk scores. These risk scores are the product of the methylation profile with the regression coefficients of the signature. The statistical significance was assessed by a two-sided, Fisher’s exact test. The missing values were not included in the analysis.Click here for fileROC curve of the identified signature on the UKCTOCS dataset samples, without a family history of breast cancer, separated into breast cancer incidence and breast cancer mortality.Click here for fileROC curve of the identified signature on the UKCTOCS dataset samples, with a family history of breast cancer, separated into breast cancer incidence and breast cancer mortality.Click here for file


Gene therapy has strong therapeutic potential for the personalized treatment of type 1 diabetes. Local gene delivery methods for gene therapy are a promising strategy to increase the therapeutic genes concentration at the target location. Transient ultrasound can cause the local increase of cell membrane permeability thus promoting the penetration of transfection particles into the cell.of this work is to evaluate transfection efficiency and safety for intrahepatic polyplex-mediated gene delivery by sonoporation.Polyplexes of expression vector DNAs complexed to galactose-bearing polyethylenimine (L-PEI) have been used. Expression vectors are two plasmids containing expression cassette for full-size human preproinsulin gene controlled with CMV promoter and flanked by inverted terminal repeats of AAV (pTRhins) and the same cassette for marker gfp gene (pTRegfp). Animals were injected under US guidance with polyplexes in a dose of 40 µg pTRegfp /0.7 ml (rat) and of 15 µg pTRhins /0.15 ml (diabetic mouse) into the liver parenchyma of subdiaphragmal segments using 31 G needles. Afterwards injection locus in depth of 1 cm during 180 sec was insonated by 130 Db ultrasound using multifrequency 3-5 MHz probe.in vivo. Flow cytometry analysis of primary hepatocytes isolated from the liver of experimental rats showed that ultrasound -enhanced polyplex gene transfer was highly localized, and was superior to all controls. At least 42% of the liver cells in vivo can be transfected in this way with ultrasound exposure versus 1.2% without it. Hypoglycemic effect of the insulin gene delivery followed by 3 min US exposure was observed on the third day: glucose level of diabetic mice (hyperglycemic 6- week) decreased in average by 30%. Nevertheless sonoporation does not increase naked plasmid DNA (without L-PEI ) transfection efficiency. A week and a month after the procedure serial sections from rat liver injected with polyplexes containing 40 µg marker plasmid or saline solution alone exposed to US and without it was analyzed for the presence of inflammation. Pathomorphological and histological analysis of experimental livers revealed no inflammatory processes in tissues, and any detectable side effects of US-enhanced gene delivery were seen. Experimental mice and rat liver DNAs were positive in transgene PCR for 3 months after gene delivery.Sonoporation is able to increase polyplex gene transfer to liver cells Our results demonstrate that polyplex gene transfer by US exposure is effective, robust and feasible and illustrate the potential of ultrasound-triggered gene delivery technology for gene therapy.As a follow-up to this study, it is recommended to create project to study and implement diabetic gene therapy with drug delivery systems for personalized treatment of type 1 diabetes.


The ability of viruses to subvert host pathways is central in disease pathogenesis. Over the past decade, a critical role for the Ubiquitin Proteasome System (UPS) in counteracting host immune factors during viral infection has emerged. This counteraction is commonly achieved by the expression of viral proteins capable of sequestering host ubiquitin E3 ligases and their regulators. In particular, many viruses hijack members of the Cullin-RING E3 Ligase (CRL) family. Viruses interact in many ways with CRLs in order to impact their ligase activity; one key recurring interaction involves re-directing CRL complexes to degrade host targets that are otherwise not degraded within host cells. Removal of host immune factors by this mechanism creates a more amenable cellular environment for viral propagation. To date, a small number of target host factors have been identified, many of which are degraded via a CRL-proteasome pathway. Substantial effort within the field is ongoing to uncover the identities of further host proteins targeted in this fashion and the underlying mechanisms driving their turnover by the UPS. Elucidation of these targets and mechanisms will provide appealing anti-viral therapeutic opportunities. This review is focused on the many methods used by viruses to perturb host CRLs, focusing on substrate sequestration and viral regulation of E3 activity. Successful viral replication requires modifying and hijacking key cellular pathways within host cells. In particular, viruses must overcome host immune responses to successfully survive and propagate. To achieve this, viruses regularly usurp the host cells degradation machinery. The predominant degradation apparatus within host cells is the Ubiquitin-Proteasome System (UPS), which is responsible for targeted protein degradation. The UPS regulates proteins involved in most fundamental cellular processes including transcription, translation, DNA repair and the cell cycle. Critically, the UPS can rapidly respond to environmental changes such as viral infection . In thisComponents of the UPS are tightly regulated by exquisite mechanisms involving cycles of precisely timed E3 activation/inhibition and dynamic E3 protein complex remodeling. Substrate targeting and degradation is a highly selective process and commonly involves an upstream posttranslational pathway “tagging” a substrate marking it for degradation. The tagged substrate then interacts (in)directly with a specific E3, (one out of an expanding number of ~600 monomeric and multimeric enzymes), driving the ubiquitin transfer .Viruses have co-evolved to utilize the host UPS for their own requirements in many aspects of their life cycle, including driving virus egress from the host cell , increasA pivotal mechanism in hijacking the UPS is through host–viral interactions with ubiquitin E3 ligases (and their regulators)—as these enzyme complexes are directly responsible for substrate selection and turnover. There are a number of well-established examples, one of the best defined being the papillomavirus E6 protein. This viral protein binds cellular E3s redirecting them to degrade host proteins including p53, driving oncogenesis in infected cells .The largest families of E3 ligases—the multisubunit Cullin-RING E3 Ligases (CRLs)—are responsible for up to 20% of all ubiquitinated substrates within cells . In CRL Several key mechanisms specifically relating to viral exploitation of CRLs have been elegantly studied see . These sCRLs are modular complexes that constitute the largest class of E3 ligases  and are i.e., whether it is active/inactive) or if the identity of its substrate(s) are also known.Seven different cullin scaffold proteins exist in humans a–f, eachIt is possible to subdivide CRLs into clusters according to the interaction mode between the cullin scaffold subunits and their adaptors . CRL3s aThe regulation of cullins involves several alternate complex conformations. Activation occurs when the cullin subunit is covalently modified by NEDD8 , step 6 + [/JAMM ] motif, harbored in its catalytic subunit, CSN5  resulting in the dissociation of E2-ubiquitin complexes from the CRL  of possible substrates for each CRL and many more examples are likely to be uncovered. However, with our current understanding it is clear that viral hijacking of CRLs utilizes similar mechanisms to that used in host-CRL substrate targeting as well as uniquely evolved viral mechanisms to direct host factors for degradation as described below .As CRLs are responsible for the turnover of ~20% of ubiquitinated proteins , it is ni.e., DNA, RNA) and strandedness  ,2928,29]Preventing the apoptotic-inducing ability of p53 however has the unwanted effect of potentially causing extensive DNA damage within infected cells. Adenoviruses (like Ad5) circumvent this by additionally targeting the MRE11 subunit of the host DNA damage sensing MRN complex (consisting of MRE11A-RAD50-NBS1) for degradation using the same E4ORF6–EB155K–CRL5 . As mentThe E7 protein of the Papillomavirus (HPV), a virus that plays a causative role in numerous mucosal cancers, also alters CRL activity. The HPV E7 protein is responsible for host cellular oncogenic transformation and binds an active form of CRL2 via a direct interaction with Elongin C. This leads to the ubiquitination and degradation of the host pRB tumor suppressor. . DegradaKaposi’s sarcoma-associated herpesvirus (KSHV) is a Group-I rhadinovirus and the etiologic agent of several human malignancies that occur frequently in AIDS patients. KSHV proteins subvert many aspects of the host UPS (including CRLs) in several intricate cascades. Firstly, the KSHV latency-associated nuclear antigen (LANA) protein functions as a SOCS-box protein mimic within CRL5 complexes. The N-terminus of LANA contacts EloB/C while the C terminus interacts with Cul5 directly. This allows LANA to interact with host p53 and VHL (Von Hippel-Lindau Tumor Suppressor), driving their CRL5-dependent ubiquitination g 34]. K. K34]. KThe M-T5 protein of the Group I poxvirus Myxoma (a rabbit/hare virus) interacts with host CRL1 complexes. This interaction results in the ubiquitination and degradation of the cell cycle regulatory protein p27. Degradation of this CDK inhibitor overcomes the cell cycle arrest produced by infection and prevents infected cells from triggering their anti-viral responses, increasing infectivity .CRL1 is a target of the Group-I Baculovirus Autographa Californica Multicapsid Nucleo-polyhedrovirus (AcMNPV). AcMNPV encodes a viral FBP (Lef7) that alters the host DNA damage response—often a requirement for the replication of large DNA baculoviruses. Although the direct host ubiquitinated target for Lef7 has not yet been found, it is thought to be linked to histone phosphorylation .Murid herpesvirus-4 (MuHV-4), which causes mononucleosis in infected mice, expresses its ORF73 protein to target the NFκB family member RelA/p65 for proteasomal degradation. To do this ORF73, using a non-conventional SOCS-box motif, forms an active complex with CRL5. This inhibition of NFκB by RelA degradation is critical for the amplification of latent MuHV-4 and persistent infection within the host .Avian adenovirus CELO  expresses a GAM1 protein capable of recruiting both CRL2 and CRL5 complexes. The GAM1-CRL2/5 complexes subsequently target host SAE1 and SAE2  Activating Enzyme Subunit 1/2) for proteasomal degradation. SAE1/2 normally form the heterodimeric E1 for the posttranslational modification SUMO-1. Although the downstream functional impact of this degradation is as yet unknown, it is likely to severely down-regulate SUMO-1 modifications within infected cells .The polyomavirus SV40 (a simian/human virus) encodes the Large T antigen (LTAg)—a well-studied highly oncogenic viral protein. LTAg has recently been shown to bind to the carboxyl terminus of CRL7 h 41]. T. T41]. TIn addition to directly targeting CRL core complex components, Group-I viruses also target CRL regulators. A clear example of this is exhibited by Herpes Simplex Virus 1 . HSV1 expresses its ICPo protein, which is capable of transactivating many host cellular genes. Although ICPo does not directly bind to CRLs, it is dynamically associated with proteasomes and enhances the ability of Cdc34 (ubiquitin-E2 for CRLs) to bind its E3 presumably increasing CRL complex activity to drive infectivity .Perhaps, one of the most amazing examples of viral mimicry of host protein function is the recent discovery of how Epstein Barr virus  controls CRL activity. Gastadello and coworkers found the Epstein Barr BPLF1 protein was in fact a deneddylase with a similar enzymatic activity to the host CSN . In infeIn summary, Group-I viruses co-opt CRLs with varied outcomes including both inhibition and promotion of host factor degradation by direct CRL interaction, as well as specifically targeting CRL regulators, with indirect but robust effects on CRL substrate ubiquitination.Geminiviruses—members of Group-II, are plant viruses that cause extensive losses in crop production worldwide. Genomes of these viruses encode a C2 protein. It has been shown that in specific Geminiviruses ), C2 is capable of interacting with the catalytic subunit of the CSN complex (CSN5/Jab1). Expression of C2 in transgenic Arabidopsis plants decreases CSN activity and causes the accumulation of neddylated CRL1. As a result, pathways regulated by CRL1, including responses to hormones such as jasmonates, auxins, gibberellins, ethylene and abscisic acid, are altered in these plants . AnotherFinally, infection by parvovirus minute virus of mice (MVM) causes CRL4 (with the SR CDT2) to drive degradation of the host protein p21—a cell cycle regulator . SpecifiGroup-III viruses include Reoviruses and Rotaviruses, which cause gastrointestinal infections. Rotavirus nonstructural protein-1 (NSP1) causes the degradation of the β-TRCP, (Beta-Transducin Repeat Containing E3 Ubiquitin Protein Ligase) an FBP constituent of CRL1 complexes . OrdinarHepatitis E is an RNA virus that causes hepatoma. In a similar manner to the abovementioned Rotavirus, the HepE ORF2 protein directly associates with β-TRCP . This inin vivo.Another member of this family is the Beet Necrotic Yellow Vein Virus (BNYVV)—a plant virus that infects sugar beet. This Group-III virus encodes a P25 protein that binds a Kelch-type FBP, which is involved in controlling plant resistance to infection through an unknown mechanism ,52. InteAs outlined above, in order for viruses to replicate unhindered by the host immune system the interferon response has to be deregulated. A key step in the interferon response is the activation of the transcription factors STATs . The acquired immunity response activates just STAT1 whereas the innate immune response activates both STAT1 and STAT2. In cells stimulated with interferon STAT1 and STAT2 heterodimerize and interact with Interferon Regulatory Factor 9 (IRF9) to form a larger active transcription factor complex. This complex is capable of binding interferon stimulated response elements within the promoters of anti-viral genes driving their expression, an integral part of the host immune response. Group-V virus members have developed a mechanism co-opting CRLs to neutralize this host anti-viral response.The paramyxovirus virus family contains many human and animal pathogens, particularly well-known are the Nipah (NiV), Hendra virus and Simian Virus 5 (SV5). Paramyxovirus V proteins are capable of binding the DDB1 adaptor subunits of CRL4 complexes . Simian Viruses of this and other groups use several orthogonal approaches to down regulate the host anti-viral immune response. A clear illustration of this is within the livestock pathogen Rift Valley Fever virus, which expresses the non-structural, NSs protein. It has been recently shown that the direct binding of NSs to FBXO3 (an SR of CRL1) results in the degradation of a subunit of the TFIIH transcription factor ,59. ThisIn summary, Group-V viruses have evolved several mechanisms to inactivate specific arms of host immunity. Future targeting of this pathway will hopefully prove promising as an anti-viral target.(HIV) is a Group VI retrovirus and the causative agent of Acquired Immunodeficiency Syndrome (AIDS). HIV has a number of mechanisms by which it usurps CRL complexes to create a cellular environment conducive to viral replication: (1) HIV Viral infectivity factor (Vif)—binds primarily CRL5  via a direct interaction with the adaptor proteins Elongin B (EloB) and Elongin C (EloC) as well as contacting Cul5 , , 152], tAmong the CRLs targeted by viruses, two major themes are evident: (1) FBPs are targeted in many species by many viruses and (2) viruses themselves encode FBPs. The range of substrates targeted by these FBPs is not well catalogued. Identifying these substrates will be very important in looking for common viral strategies used to hijack the UPS and in identifying therapeutically targetable CRL–substrate pairs. Understanding why viral hijacking strategies converge on FBP proteins to alter host UPS pathways will be fascinating.Furthermore, as well as identifying potential host and viral ubiquitination targets, the type of ubiquitination linkage will be important in understanding the mechanistic outcomes. Most studies to date focus on the degradative ubiquitination but it is likely that many host–viral ubiquitination events are regulatory (and non-degradative) in nature—focusing on this subset will be very informative.Another level of understanding will also come with solving the structural mechanisms of virus–CRL interactions. Although recent structures have helped initiate these studies, very little is known about the stoichiometry, orientation and subunit contacts with viral proteins in CRL complexes with a few exceptions (such as the SV5-CRL4 interaction ).At a more functional level, how virus–CRL complexes integrate to modify the cellular environment is also not well deciphered. An example again is highlighted by the differential hijacking of adenovirus’ proteins of a number of CRL family members to target related host factors. This may be a general viral principle where perhaps there is not just one target for a host–viral CRL complex and moreover the same viral protein from different strains may have diverged targets. If so, then care must be taken in attributing a single substrate to an individual viral–CRL interaction or conformation.In conclusion, the field has demonstrated that viral proteins: (i) Serve as substrates themselves; (ii) redirect active host CRLs to degrade otherwise long-lived host proteins; (iii) are capable of inhibiting CRL ubiquitination activity; and (iv) indirectly affect CRLs by interacting with and modulating their regulators. These diverse and complex interactions all function to tip the balance of the host intracellular environment to drive viral pathogenesis. In many ways, uncovering these mechanisms has informed us as much about our own immune system as it has about infectious pathways. Our understanding of the intricacy of CRL biology will continue to expand as more such elegant mechanisms come to light.


These data provides valuable guidelines for generation of knock-in reporter human iPS cell lines for myogenic genes which can be used for disease modeling, drug screening, gene correction and future in vivo applications.Human iPS cells hold great promise for disease modeling and treatment of degenerative disorders including muscular dystrophies. Although a few research groups have used them for skeletal muscle differentiation, most were based on gene over-expression or long-term mesenchymal differentiation and retrospective identification of myogenic cells. Therefore, this study was aimed to generate a knock-in reporter human iPS cell line for MYF5, as an early myogenic specification gene, to allow prospective identification and purification of myogenic progenitors from human iPS cells. By using a CRISPR/Cas9 double nickase strategy, a 2A-GFP reporter was inserted before the stop codon of the MYF5 gene using homologous recombination. This approach allowed for highly efficient in-frame targeting of MYF5 in human iPS cells. Furthermore, in order to prove the reporter function, endogenous MYF5 expression was induced using a novel dead Cas9-VP160 transcriptional activator. Induced clones demonstrated appropriate MYF5-GFP co-expression. Finally, to confirm the differentiation potential, reporter human iPS clones were differentiated through embryoid body method and MYF5-GFP Skeletal muscle is the largest organ in the body with a tremendous regeneration potential. Indeed, its continuous growth and regeneration during life is exceptional, however, it is still prone to many pathologic conditions which might occur at different ages1235in vitro disease modeling, drug screening, gene correction and finally as a cell based therapy for muscle disorders78910in vitro differentiation of iPS cells.Fortunately, with the recent advancements of generating induced Pluripotent Stem Cells (iPS cells) from somatic cells, different lineage progenitors can be generated from patient samples which can be used for For this reason, a few research groups including us have started working on human iPS cells to develop strategies for differentiation toward skeletal muscle. A majority of these efforts were based either on transient myogenic genes over-expression  or differentiation toward mesodermal/mesenchymal lineage12131415161718171819Therefore, in the current study, we planned to generate a knock-in reporter human iPS cell line for an early myogenic gene (such as MYF5). This will allow us and other scientists to use this approach for directed differentiation of human iPS cells toward myogenic progenitors and to study temporal emergence of myogenic progenitors during differentiation using a prospective strategy.We chose MYF5 as it is one of the earliest myogenic determination genes in the somite and its unique transcriptional isoform, makes it suitable for our targeting strategy2122+ myogenic cells26in vitro myogenic differentiation of human iPS cells.Furthermore, since homologous recombination (HR) targeting efficiency in human iPS cells is low, we used a Cas9 double nickase (Cas9n) method to introduce a double-strand break (DSB) in DNA to facilitate HR and thus improve the targeting efficiency24In order to design the sgRNAs for Cas9n pairs, the human MYF5 gene was analyzed to identify suitable target sites. 2123To generate the Cas9 mediated knock-in reporter lines for MYF5 in human iPS cells, first we analyzed the 100 bp regions flanking the stop codon in the MYF5 exon 3. As shown in a) and a 2A-GFP reporter along with the selection cassette was inserted using a blunt-end ligation (b). Later on, the backbone vector was switched into a gateway vector which contained the negative selection (tk) along with unique linearization sites (c). The final vector was validated by sequencing to ensure in-frame positioning of the reporter/selection cassette.Next we made the targeting vector for MYF5. As described in After confirmation of the Cas9n pairs and targeting vector, human iPS cells were used to generate reporter lines for the MYF5. For this reason, a well characterized integration-free human iPS cell line (DF19-9 human iPS cell line generated by Dr. James Thompson’s lab obtained from WiCell) was chosen for generation of the reporter clonesHuman iPS cells were expanded in feeder-free Matrigel coated plates in conditioned medium and after harvest, electroporated with targeting vector cocktails containing Cas9n pairs and the HR targeting vector for the MYF5. The cells were plated after electroporation and positive and negative drug selection was started three days later for the duration of two weeks. During this time, individual resistant targeted iPS clones started to grow and expressed RFP as demonstrated in In order to test in-frame positioning of the reporter cassette, we have used a PCR screening method for the transgene. As shown in Positive clones then were evaluated for homozygous cloning of the reporter cassette using a PCR screen. Interestingly, as shown in In order to confirm in-frame positioning of the reporter cassette and exclude any possible indels at the recombination sites, the 1.6 kb PCR product from the outside 5′ homology arm up to the 2A-GFP region  was tested for the reporter activity. For proof of principle, the cells were tested using a deadCas9 (dCas9-VP160) activator approach to activate the endogenous MYF5 geneIn order to do this, after identification of the open chromatin region for the MYF5 gene using DNase I hypersensitivity data , target Based on RT-PCR results, the cells were transfected again for MYF5-GFP activation in reporter iPS clones using confirmed activators . As demonstrated in Moreover, the sorted GFP positive and negative cells (from MB Mix activation) were fixed and stained for MYF5 and evaluated for co-expression of GFP and MYF5. As data on in vitro differentiation, the human iPS MYF5-GFP reporter clones were differentiated using an EB differentiation method. In order to activate the myogenic differentiation, the EBs were treated with a Wnt agonist  for four days and analyzed for GFP activity as well as the MYF5 expression during an EB differentiation time-course.Finally, in order to prove the MYF5 reporter activity during As gene expression data in in vitro differentiation of human iPS reporter cells.Furthermore, to confirm the myogenic activity of the cells, positive and negative cells from EB day 12 were sorted and expanded in EB medium on Matrigel-coated plates for a few days and were tested for myotube differentiation by switching medium to low-nutrient myotube induction medium (2% horse serum in DMEM). These data is demonstrated at in vitro disease modeling, drug screening, gene correction studies and eventually regenerative purposes7101213141516171819Directed differentiation of the pluripotent stem cells toward desired tissue progenitors is one of the important goals in the stem cell biology. Especially with the availability of iPS cells, derivation of the tissue specific progenitors provides an excellent opportunity for By using a CRISPR/Cas9 double nickase mediated homologous recombination (HR), we have demonstrated a highly efficient approach for generation of the knock-in reporters in human iPS cell lines. Although other site-specific endonuclease systems such as ZFNs or TALENs are also available, Cas9-nickase system has a clear advantage due to its easy design and construction and its reduced risk of off-target side-effects242532333435Another aspect of this study is the successful application of Cas9-mediated gene activation for MYF5 induction in the reporter clones26in vitro differentiation setting, we have demonstrated a time-course EB differentiation approach for the evaluation of myogenic induction using gene expression and FACS analysis/sort. As indicated in the results, by using a Wnt agonist (CHIR99021) to induce myogenesis in EBs, the MYF5 gene expression as well as the GFP reporter activity can be easily identified using a gene expression assay and flow cytometry. Furthermore, sorting for MYF5-GFP positive fraction confirmed their myogenic potential as demonstrated by their in vitro differentiation into multinucleated myotubes.Finally in order to prove proper reporter activity in an in vitro differentiation.Taken together, this study demonstrates successful generation of a knock-in MYF5-GFP reporter in human iPS cell line using a Cas9n mediated HR targeting with high efficiency. The generated reporter lines can be used for evaluation of directed differentiation of human iPS cells toward myogenic lineage using chemical screening as well as to study temporal emergence of myogenic progenitors during Cas9 target sites were identified using the online CRISPR design tool (crispr.mit.edu)Assembled Cas9n pairs were tested on 293T cells using transfection. Briefly, 0.5 μg of each plasmid was used for transfection of 293T cells. Three days after the transfection, DNA from the targeted and non-targeted cells was harvested and PCR-amplified for the targeted region. Then, the PCR products were hybridized using a thermocycler to allow heteroduplex formation from the targeted and non-targeted cells DNA. Later on, 400 ng of hybridized DNA was treated with the SURVEYOR nuclease and DNA fragments were analyzed using agarose gel electrophoresis to identify the presence of the expected bands.tk) as well as unique linearization sites. The final vector was linearized before its use for targeting.BAC clone for the human MYF5 was obtained from CHORI (BAC RP11-626E8). For construction of the targeting vector, we used a highly efficient recombination based cloning method in bacteria which was developed by Dr.Capecchi’s group7 cells were electroporated  using 10 μg of Cas9 vectors plus 50 μg of the linearized targeting vector for MYF5. After the electroporation, cells were expanded for a few days and selection was done by adding positive and negative selection (0.75 μg/ml Puromycin and 200 nM FIAU) for 2 weeks. After the selection, RFP positive clones were picked up for expansion and PCR screening.After the validation of Cas9n pairs using the SURVEYOR assay, a fully characterized integration-free human iPS cell line PCR primers were designed to span the outside 5′ homology arm up to 2A-GFP region to ensure proper site-specific targeting. This region (1.6 kb amplicon) was PCR-amplified and sequenced to ensure in-frame positioning of the reporter/selection fragment and to rule out the presence of indels at recombination site. 3′ end was also amplified using PCR (0.8 kb amplicon) and sequenced for analysis. Furthermore, to identify the clones with both allele (homozygous) reporters, PCR primers were designed to identify intact MYF5 as well as the targeted allele.+ membrane as described in details beforeIn order to prove single integration of the transgene, a southern blot strategy was designed using an internal probe within the GFP region. The genomic DNA from the Myf5 reporter hiPS cells was extracted and verified by PCR. 5 μg of genomic DNA from each cell line was digested with EcoRV, separated in a 0.9% agarose gel and transferred to a Hybond NAfter characterization of homozygous clones, the selection cassette was removed by transient expression of a Cre plasmid (pCAG-Cre-IRES2-GFP Addgene # 26646) and was validated using PCR screening and direct fluorescent imaging.In order to evaluate reporter activity in targeted iPS clones, we have used a nuclease-dead Cas9 activator system 5 cells/well of 24 well plate with 70–90% confluency on the day of transfection) were transfected into human iPS cells using Lipofectamine LTX transfection reagent . Three days after the transfection, cells were harvested for RNA extraction. cDNA was made using reverse-transcription and RT-PCR was performed using Taqman assays for human MYF5 to identify the best activators. After choosing the best activators, MYF5 -GFP knock-in human iPS cells were transfected again and were harvested for GFP analysis using a FACS Aria system and were gated against non-induced cells. For immunostaining, sorted cells were sorted and fixed using 4% PFA/PBS and after blocking, were stained for MYF5 , GFP  and DAPI. For secondary antibodies, we have used the appropriate Alexa Fluor antibodies . For myotube staining, fixed cells were stained for myosin heavy chain-MHC  overnight in 4 degree and later counterstained with appropriate secondary antibody.dCas9-VP160 activator constructs were tested on human iPS cells to validate their efficiency for gene activation. Individual or pooled constructs  and expanded according to its protocol. For EB generation, cells were harvested using EDTA and EBs were formed on a shaker as described beforeHow to cite this article: Wu, J. et al. Generation and Characterization of a MYF5 Reporter Human iPS Cell Line Using CRISPR/Cas9 Mediated Homologous Recombination. Sci. Rep.6, 18759; doi: 10.1038/srep18759 (2016).


MTOR and its upstream regulator RHEB (Ras homolog enriched in brain) in patients with clear cell renal cell carcinoma (ccRCC), the most common histology of kidney cancer. Interestingly, we found a prominent cluster of hyperactivating mutations in the FAT (FRAP-ATM-TTRAP) domain of mTOR in renal cell carcinoma that led to an increase in both mTORC1 and mTORC2 activities and led to an increased proliferation of cells. Several of the FAT domain mutants demonstrated a decreased binding of DEPTOR (DEP domain containing mTOR-interacting protein), while a subset of these mutations showed altered binding of the negative regulator PRAS40 (proline rich AKT substrate 40). We also identified a recurrent mutation in RHEB in ccRCC patients that leads to an increase in mTORC1 activity. In vitro characterization of this RHEB mutation revealed that this mutant showed considerable resistance to TSC2 (Tuberous Sclerosis 2) GAP (GTPase activating protein) activity, though its interaction with TSC2 remained unaltered. Mutations in the FAT domain of MTOR and in RHEB remained sensitive to rapamycin, though several of these mutations demonstrated residual mTOR kinase activity after treatment with rapamycin at clinically relevant doses. Overall, our data suggests that point mutations in the mTOR pathway may lead to downstream mTOR hyperactivation through multiple different mechanisms to confer a proliferative advantage to a tumor cell.Aberrations in the mTOR (mechanistic target of rapamycin) axis are frequently reported in cancer. Using publicly available tumor genome sequencing data, we identified several point mutations in  MTOR in various cancers, though it remains to be assessed if these are driver mutations causally implicated in oncogenesis . By. By24]. in vitro kinase assay in that mutants with higher in vitro kinase activity  have high RICTOR levels in immunproprecipitates. Notably, the R2505P mutant did not demonstrate an increase in RICTOR binding. These data indicate that mutations in the FAT domain of mTOR may lead to an overall increase in mTORC2 kinase activity due to better recruitment of mTORC2 substrates via enhanced RICTOR association. As previously reported, we observed a loss of DEPTOR binding in several of the FAT domain mutants relative to wild-type mTOR  activates and phosphorylates AKT, that in turn leads to the downstream phosphorylation of TSC2 and inhibits its GAP activity towards RHEB, allowing RHEB to activate mTOR , 39. AmiTo assess the role of a point mutation in RHEB at the conserved Tyr35 site, we generated a point mutation (Y35N) and transiently over expressed wild-type and mutant RHEB in HEK293T cells. As RHEB is a key mediator of the amino acid sensing mechanism of the cell, we examined the effect of this mutant in the presence and absence of amino acids. Mutant RHEB caused promoted mTORC1 activation under nutrient containing conditions, an effect that remained under nutrient deprived conditions as demonstrated by persistent phosphorylation of S6K and 4E-BP1 Figure . RHEB stThe TSC1/TSC2 complex interacts with RHEB and inactivates it through the activity of a GAP domain in TSC2. Thus we performed immunoprecipitation experiments to analyze the interactions of wild-type and mutant FLAG-tagged RHEB with TSC2. The RHEB mutation did not affect interaction with TSC2 Figure . RHEB haRHEB activation of S6K and 4E-BP1 is known to be rapamycin-sensitive . To detemTOR deregulation is observed in multiple sporadic cancer types; however, it also plays a causative role in various familial tumor syndromes such as Cowden syndrome, Peutz-Jeghers syndrome and Tuberous Sclerosis as recently summarized . In sporRecent cancer genome sequencing efforts have led to the identification of point mutations in mTOR. In this study, we chose to characterize a group of mutations in the TRD1 and TRD2 regions of the FAT domain that cluster distal from the kinase domain. While mutations in these regions could directly disrupt the binding sites for mTOR complex proteins that interact with the FAT domain, these mutation sites are not surface exposed. The FAT domain is comprised of helices arranged in α-α-helical repeats that form a C-shaped solenoid that surrounds the kinase domain. These mutation sites are found at the interfaces between adjacent helices, thus we propose these RCC-associated mutations are likely to destabilize the structure of the FAT domain. This may directly deregulate mTOR kinase activity as well as affect the binding of mTOR complex proteins. The binding of DEPTOR, which interacts with the FAT domain, was impaired by four of the six mutations characterized in this study. The side chain of L1433 in α3 is involved in hydrophobic interactions with the alkyl region of K1452 in α4, which appears to stabilize the orientation of helices 3 and 4, and would be disrupted by the L1433S or K1452N mutations. Likewise, the side chains of Y1463 in α5 and C1483 in α6 are intimately involved in the α5-α6 interhelical packing, which could be disrupted by loss of an aromatic ring (Y1463S) or steric clashes (C1483F). L1460 is in the middle of α5, thus its mutation to the helix-breaker residue proline would be expected to destabilize these helical bundles, as would the adjacent mutation A1459P. A1519, located in α8, packs into a small cavity formed by α7 that would not accommodate a larger sidechain. Thus the α7-α8 this bundle would likely be disrupted by the A1519T mutation. Mechanistically, we find that in addition to altering mTORC1 and mTORC2 complex assembly, FAT domain mutations also lead to a decrease in PRAS40 and DEPTOR binding, suggesting that multiple mechanisms may act in concert to activate downstream mTOR targets.RHEB that caused mTORC1 hyperactivation by conferring resistance to the GAP activity of TSC2. Mutations in RHEB are uncommon in many cancers, despite its central role in regulating downstream mTOR activation. In RCC, a recurrent mutation in RHEB at the Tyr35 residue has been reported, though it is unclear if this mutation is mutually exclusive to mutations in TSC1 or TSC2 as the mutation frequencies amongst these genes is low. The recurrent mutation of RHEB Y35 in cancers underscores the importance of this residue, and illustrates that the unique biochemical properties of RHEB lead to a different mutational hotspot relative to RAS, in which the majority of mutations affect G12, G13 and Q61. Indeed, the equivalent positions in RHEB are either already substituted in the wild-type sequence (R15 and S16), or lack a catalytic function (Q64). It is notable that while oncogenic RAS mutations synergistically impair both intrinsic hydrolysis and GAP sensitivity, the RHEB Y35N mutation enhances intrinsic hydrolysis but abolishes sensitivity to the GAP. Overall, our results suggest that mutations in the TSC-RHEB-mTOR signaling axis may lead to a loss of inhibitory inputs thus conferring a survival advantage to a dividing tumor cell.Additionally, we examined a recurrent point mutation in MTOR mutations are associated with worsened outcomes has prognostic relevance and suggests that this event may be a significant contributor to tumor progression. Consistent with this possibility is our data demonstrating the first evidence that cancer-associated MTOR mutations promote a proliferative advantage over wild type MTOR. The TCGA data set are from primary RCC tumor samples. In the setting of RCC, the morbidity and mortality is primarily associated with disease outside the kidney. Hence, the incidence of MTOR mutations may be much higher in metastatic tumor sites which would further support a role for this genetic alteration in tumor progression. Additionally, our data may have therapeutic implications. Approved mTOR inhibitors temsirolimus and everolimus serve as important therapeutic options within the current RCC treatment paradigm, though they primarily target the mTORC1 pathway. Biomarkers for rapamycin sensitivity/resistance are lacking. Our results suggest that FAT domain mutations may potentially lead to resistance to rapalog therapy in patients with ccRCC. Notably, a recent case report in anaplastic thyroid cancer suggests that MTOR mutation is a mechanism of acquired resistance to rapalog therapy [TSC2 mutation. After significant response to everolimus, the patient developed resistant tumor that had a MTOR kinase domain mutation (F2108L). Such resistance may occur in the setting of FAT domain mutations and could occur via several mechanisms as supported by our data. First, higher doses of rapamycin may be required to ablate rapamycin sensitive outputs of mTORC1. Prior in vitro studies have examined the rapamycin sensitivity of mTORC1 activation mediated by mTOR point mutants. However, in many cases, the doses used are much higher than serum rapalog levels in patients undergoing treatment in the setting of advanced RCC. The recent case report in thyroid cancer demonstrated persistent mTORC1 activity of the F2108L mutant at physiologically relevant rapamycin concentrations is consistent with our studies of FAT domain mutants. Additionally, it is now well established that there are mTORC1 activities that are not inhibitable by rapamycin. Our data demonstrate enhanced phosphorylation of such (e.g. 4E-BP1 Thr 37/46). Finally, mTOR FAT domain mutants can promote mTORC2 kinase activity-a critical finding given that rapalogs generally do not ablate mTORC2 activity [MTOR mutation. Dosing adjustments could be considered. Alternatively, strategies that target rapamycin resistant activities of mTOR mediated by mTORC1 or mTORC2 (e.g ATP competitive inhibitors) could be considered.Our findings have several implications for patients with RCC. The finding that  therapy . Sequencactivity . Hence, In summary, our findings shed new insight into the biology of mTOR as it pertains to kidney cancer. While mTOR is one of the largest genes in the human genome, the mutations in mTOR are clustered within finite regulatory domains suggesting that these mutations lead to a gain of function which is currently an area of active investigation. Characterizing these mutations and their sensitivity to targeted agents could thus be clinically relevant, especially with the increasing use of whole genome sequencing to direct therapy in patients with advanced cancer.HEK293T cells were obtained from American Type Culture Collection (ATCC) and maintained in DMEM supplemented with 10% heat-inactivated fetal bovine serum at 37°C in a humidified 5% CO2 atmosphere. HEK293T cells were transfected with myc-epitope tagged wild-type mTOR or mutant mTOR cDNA in their expression vectors, followed by whole cell lysis 48 hrs after transfection. For mutant RHEB experiments, HEK293T cells were transfected with FLAG-epitope tagged wild-type RHEB or mutant RHEB cDNA in their expression vectors, followed by whole cell lysis 48 hrs after transfection. Cells were starved of nutrients by gently rinsing the cells in serum free DMEM once and incubating them in serum free DMEM overnight. For amino acid starvation, cells were incubated in amino acid-free RPMI for 50 minutes as previously described .Rat wild-type myc mTOR cDNA was obtained from Dr. David Sabatini (Whitehead Institute - MIT) via Addgene(#1861). Human wild-type Flag RHEB cDNA was obtained from Dr. Fuyuhiko Tamanoi (UCLA) via Addgene (#19996). Protein G sepharose beads were obtained from GE Healthcare Lifesciences. Rapamycin was purchased from Selleckchem. Inactive, N-terminal His-tagged recombinant full-length human Akt1 for use in kinase assays was obtained from EMD Millipore (#14-279).All mTOR and RHEB point mutations were generated in the parental vectors using site-directed mutagenesis with the QuikChange II XL kit according to the manufacturer's protocol. Point mutations were verified using Sanger sequencing.Six replicates each of 3000 cells were seeded into 96-well plates and assayed using Cell Titer Glo according to the manufacturer's protocol.Cells were rinsed once with ice-cold PBS and lysed in ice-cold lysis buffer . * represents a 15N RHEB was expressed in in E. coli BL21 DE3 Codon+ grown in M9 minimal media supplemented with 15N ammonium chloride, and purified on glutathione-Sepharose, released from the resin by thrombin cleavage followed by gel filtration chromatography on Sephadex-75 resin . To assay GTP hydrolysis, 15N-Rheb was loaded with GTP in the presence of EDTA, and excess nucleotide and EDTA were removed by passing the sample through a desalting column  (as previously described [15N RHEB-GTP (residues 1-169) in NMR buffer  were snap frozen and stored at −80°C. The assay was initiated by thawing a 35 μl sample, and successive 15N 1H HSQC spectra  were collected at 15°C on a 600 MHz Bruker Avance III NMR spectrometer equipped with a 1.7 mm microcryoprobe. Pairs of GTP/GDP-specific peaks from several residues were used to evaluate the fraction of GTP-bound RHEB remaining at each time point to obtain the half-life and exchange rate as described previously [15N RHEB-GTP sample prior to collection of 15N 1H HSQC spectra.Constructs encoding wild-type and Y35N human RHEB (residues 1-169) were subcloned into PGEX2T to generate thrombin-cleavable GST fusion proteins. As described previously , 15N RHEescribed , and alieviously . To assa