Retinitis pigmentosa (RP) represents a genetically heterogeneous group of retinal dystrophies affecting mainly the rod photoreceptors and in some instances also the retinal pigment epithelium (RPE) cells of the retina. Clinical symptoms and disease progression leading to moderate to severe loss of vision are well established and despite significant progress in the identification of causative genes, the disease pathology remains unclear. Lack of this understanding has so far hindered development of effective therapies. Here we report successful generation of human induced pluripotent stem cells (iPSC) from skin fibroblasts of a patient harboring a novel Ser331Cysfs*5 mutation in the MERTK gene. The patient was diagnosed with an early onset and severe form of autosomal recessive RP (arRP). Upon differentiation of these iPSC towards RPE, patient-specific RPE cells exhibited defective phagocytosis, a characteristic phenotype of MERTK deficiency observed in human patients and animal models. Thus we have created a faithful cellular model of arRP incorporating the human genetic background which will allow us to investigate in detail the disease mechanism, explore screening of a variety of therapeutic compounds/reagents and design either combined cell and gene- based therapies or independent approaches. Retinitis pigmentosa  with a prevalence of 1 in 3,500 individuals is the most common form of hereditary retinal disorder affecting the working age group. RP is characterized by progressive dysfunction and death of mainly the rod photoreceptor cells (PR) of the retina however in some cases retinal pigment epithelium (RPE) cells are also involved, often resulting in permanent blindness. So far 54 genes have been implicated in this disease coding for proteins involved in a myriad of functions such as phototransduction signaling cascade, retinoid cycle, cell-cell adhesion or the cytoskeletonin vitro and differentiated into any type of mature cell in the human body, without the ethical and immunogenicity issues associated with ESC346in vitro such as phagocytosis, secretion of vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) and epithelial barrier formation. Cellular models of hereditary retinal dystrophies have been successfully created in vitro in Best disease, and RP where patients’ fibroblasts were reprogrammed to iPSC and then converted to RPE8Patient-derived induced pluripotent stem cells (iPSCs) provide an unprecedented opportunity to recapitulate disease pathogenicity without the need for genetic manipulation and creation of gene targeted animal models. Human iPSCs, similar to embryonic stem cells (ESC), can be expanded indefinitely MERTK was found to be disrupted in RCS rats16merkd miceMERTK mutations. Indeed, the reduced retinal thickness and debris detected in the sub-retinal space in patients harboring the MERTK –splice-site -mutation resembles the observed phenotype in the RCS ratMERTK mutations without any systemic disease or defects of phagocytosis by macrophages, indicating a specialized function of this protein in the RPE cells. In contrast to the detailed clinical understanding of the disease, the mechanism by which MERTK acts during the phagocytosis remains partially unveiled. Outer segments are known to bind to the integrin receptor αvβ523Mer tyrosine kinase receptor (MERTK) belongs to the Tyro3/Axl/Mer (TAM) receptor tyrosine kinase family of proteins distinguished by a conserved intracellular kinase domain and extracellular adhesion molecule-like domain. TAM receptors regulate a variety of processes such as cell proliferation/survival, adhesion, migration, inflammatory response, in a cell- microenvironment- and ligand- specific mannerin vitro model of RP caused by the mutation of MERTK gene using human iPSC technology. We report the generation of a cellular model of MERTK-associated RP, which recapitulates the disease phenotype described in animals and in patients and provides a tool to advance our understanding of the signaling pathways activated by MERTK. Importantly this can be studied in detail in the context of the human genetic background, a crucial feature of the cellular model. Equally important is that it provides us with a unique opportunity for the testing of novel therapeutic agents in the presence of the human background, which has the potential to significantly reduce the cost of preclinical studies in animal models. The derivation of iPSCs from a RP patient’s fibroblasts and the generation of a RPE cell line expressing the molecular defect makes an important contribution in our ability to undertake translational research.In this study we describe the generation of an MERTK. Sequence analysis allowed identification of a novel homozygous frameshift mutation c.992_993delCA (p.Ser331Cysfs*5) in MERTK, segregating with the disease in the family  at the age of 7. Ophthalmological examination at 32 years of age showed severe constriction of visual fields in both eyes and loss of VA to 20/70 in right eye and finger counting in the left eye. The fundus showed typical changes of RP with pale disc, narrowed retinal vessels and bone-spicule pigmentations in the peripheral retina. For this study, a healthy individual unrelated to this family and no family history of blindness was taken as a healthy control. The healthy individual had a VA of 20/20 at the time of skin biopsy.MERTK gene is composed of 19 coding exons with two immunoglobulin-like C2-type (Ig-like) domains, two fibronectin type III (FN3)-like domains, a transmembrane (TM) domain and a tyrosine kinase (TK) domain (UniProt Q12866). The deletion of CA in exon 7 (NM_006343.2), at position 992_993 causes the frameshift resulting in a premature stop codon at position 335 . Three iPSC lines per individual were sub-cultured and analyzed at cellular and genetic level to confirm successful reprogramming. Pluripotency was assessed by immunocytochemistry for pluripotency markers such as octamer-binding transcription factor 4 (OCT4), NANOG, SRY (sex determining region Y)-box 2 (SOX2), and in situ staining to TRA-1-81. The alkaline phosphatase is known to be more active in iPSCs and the colorimetric assay depicting its activity confirmed that the selected iPSC colonies are indeed pluripotent  mice. Eight weeks after injection, tumor was formed and extracted. Histological sections showed that the tumor contained derivatives of all three germ layers including gut-like tissues (endoderm), neural cells and retina (ectoderm) and striated muscle, bone, cartilage and adipose tissue (mesoderm)  . The heaesoderm) .in vivo. Intercellular junctional complexes are visible in appropriately aligned sections of iPSC-RPE of both individuals. The integrity and function of the RPE monolayer depend on its basolateral cellular junctions, which include the tight junctions, adherens junctions and membrane interdigitations  are raised over a 1000 fold in iPSC-RPE cells from both individuals with respect to the fibroblasts, while CRALBP and RPE65 are just detectable by qRT-PCR in iPSCs.To validate the identity of generated RPE cells, RT-PCR of selected RPE specific genes was performed to compare the expression levels throughout the differentiation process ). This iMERTK by RT-PCR using primers spanning exons 14–17 and also by qRT-PCR assay using primers spanning exon junction 18–19 , respectively, is a hallmark of RPE cells and was found in both healthy and MERTK p.Ser331Cysfs*5 iPSC-RPE cells , a tight-junction marker, indistinguishable in both individuals . Apico-lPE cells .Immunocytochemical detection of MERTK in iPSC-RPE monolayer from healthy individual showed its expression apically as previously describedExpression of MERTK and mature RPE specific markers was confirmed by western blot throughout the differentiation process of two iPSC lines per genotype . The exp15MERTK deficient phenotype described in animal models is defective phagocytosis, a daily process of OS uptake, which renews the light–damaged membranes of PR. The process of OS phagocytosis can be mimicked in cultured RPE cells by feeding the isolated OS derived from bovine or pig retina6We have thus generated patient-specific RPE cells that lack the expression of MERTK and consequently recapitulate the MERTK deficient phenotype i.e. absence of phagocytosis. These data show that patient-specific, mature and functional RPE can be generated from control and RP patients via iPSCs. The monolayers derived from the two individuals as reported here are morphologically identical and indistinguishable by RPE-specific marker expression while preserving patient’s identity as observed by distinctive MERTK expression and its associated function.CHM gene deficient mice (model for choroideremia) which are lethalRetinal dystrophies comprise a group of eye diseases caused by mutations in a spectrum of genes with a specific function in photoreceptors or RPE cells or in many cases mutations in ubiquitously expressed genes that exclusively give rise to a retinal phenotype. While retinal tissue is inaccessible for the studies of molecular mechanism, unrelated tissues such as blood is not truly representative of molecular events in the retina. Animal models remain an invaluable tool to understand disease mechanism, however in some cases they are not fully representative of the disease pathophysiology, owing to differences in neuronal development, physiology and the genetic background. In some cases, such as the Usher-1 mouse model, the animal lacks the disease phenotypeMERTK gene causing RP, the majority being missense/nonsense mutations (11), splicing-site mutations (3), small deletions (3), insertions (1) and gross deletions (2) as reported in the HGMD public database . We show here the successful generation of iPSC from a RP patient bearing a novel p.Ser331Cysfs*5 mutation in MERTK which has not been described before. Adult dermal fibroblasts were reprogrammed with non-integrative virus delivering pluripotency genes and the generated iPSCs maintained genomic integrity throughout the culture period of over 30 passages. It should be noted that genomic alterations including numerical and structural alterations are not uncommon in iPSC cultures. In our experience the use of Sendai virus as a vehicle for gene delivery resulted in the absence of genomic alterations in contrast to integrative viral strategies. As demonstrated here, iPSCs established from the control and the affected patient were identical in relation to reprogramming efficiency and exhibited similar kinetics and efficiency of differentiation. Both iPSC-RPE were morphologically indistinguishable, the polygonal morphology and pigmentation was displayed by control and patient RPE. The RPE specific marker expression such as CRALBP, RPE65, BEST1 and ZO-1 was similar in both iPSC-RPE monolayers. A distinctive functional feature of the native RPE is the cellular polarity based on the polarized localization of RPE markers. We show polarized distribution of ZO-1, a tight junction marker, and Na+/K+ ATPase pump protein expressed on the apical membrane. This is a distinctive RPE feature since Na+/K+ ATPase is reported to be expressed basolaterally in other transporting epitheliaMERTK mutations account for approximately 1% of arRP and were reported in several families with retinal dystrophy2031323334MERTK expression raises the possibility that the patient’s mRNA partially escapes nonsense–mediated decay and perhaps the transcript is translated into an abnormal 334 amino acid protein lacking the kinase domain. However, we do not detect the truncated protein in patient’s iPSC-RPE extracts by western blot nor by immunocytochemistry in the iPSC-RPE monolayer, suggesting that it may be degraded after being translated.In order to confirm that the difference between healthy and diseased cells is due to disease mutation and not due to inter-lineage variability described for iPSCs37MERTK mutations.Our results prove that patient-specific iPSC reproduce the disease phenotype reliably and this should allow faithful modeling of degenerative disease of the RPE in the future with more subtle phenotypes and completely unknown disease mechanisms. In addition, the generated iPSC-RPE represents clinically reliable setting to test therapies in order to reverse or delay the disease onset in patients harboring A consanguineous Spanish family (RP-0503) consisting of three affected siblings  was recrWhole-genome single nucleotide polymorphism (SNP) microarray analysis was performed using Illumina HumanCyto-12 SNP array, containing 298,199 SNPs markers . Arrays were processed according to the manufacturer’s protocols. Linkage analysis was performed, using the Gene Hunter program (version 2.1r5) in the easyLINKAGE plus software package (version 5.08), to identify regions that may contain the causative mutation. As the affected siblings were born of a consanguineous marriage, only the homozygous regions were taken into account.MERTK gene for mutations. Exons and exon–intron boundaries of MERTK were analyzed using oligonucleotide primer pairs designed using Primer3 software (http://frodo.wi.mit.edu/). Sequences and annealing temperatures are available from the authors on request. The PCR conditions and primer sequences are available on request. The PCR products were enzymatically purified with ExoSAP-it  and both strands sequenced using Big Dye Terminator Cycle Sequencing Kit version 3.1 . The PCR products were purified in a 96-well multiscreen filter plate  and resolved on an automated sequencer .Bidirectional automatic sequencing was performed to screen the MERTK upstream and downstream of c.992_993 were as follows: 5′CGAAGAGGTTCTAAGAGAGG3′ and 5′CCATTTTCATCAGTCGCCTC3′ .Direct sequencing in fibroblasts and iPSCs: Genomic DNA from fibroblasts and iPSCs was isolated using the QIAamp DNA Blood mini kit (Quiagen). Primers used for amplification and directed sequencing of 2. After 3–4 weeks, fibroblasts outgrowing from the biopsy pieces cover most of the dish and cells were passaged and plated in a T-75 flask. After reaching 80% confluency fibroblasts were further expanded and cryostored in liquid N2.Skin biopsy of a healthy individual and an affected patient (with a confirmed molecular diagnosis of early-onset RP) was taken under sterile conditions following informed consent. The skin biopsy sample was placed in ~25 ml of sterile PBS at room temperature (20–25 °C) for shipment. Inside a tissue culture hood, the PBS was replaced with PBS containing 10× penicillin/streptomycin (500 U/ml penicillin and 500 μg/ml streptomycin) and 10× Fungizone (25 μg/ml amphotericin B) and left at room temperature for 15 min, with occasional mixing by inverting the tube. This step was repeated twice and finally the biopsy sample was washed with PBS for 15 min. The sample was cut into small pieces and cultured in 60-mm plastic dishes (8–10 pieces per dish) in DMEM  at 37 °C under 5% COThe iPSC were derived from patient’s fibroblasts using Sendai virus  according to manufacturer’s instructions. Single colony subcloning was performed for 5 passages and the presence of Sendai virus transgenes was detected by RT-PCR  with pri2 on Matrigel coated transwell inserts . The RPE cells were cultured for 30–60 days until they reached high pigmentation. Two iPS cell lines per each genotype were induced to differentiate toward RPE and no differences were observed with respect to kinetics of differentiation, degree of pigmentation or characteristic RPE morphology between the iPS cell lines from one individual nor between healthy and affected genotype.To obtain RPE cells the iPSCs colonies were mechanically removed and cultured in low attachment plates as floating aggregates in the iPSC medium with 15% KSR and deprived of bFGF. When dark patches formed on the aggregates they were mechanically excised, treated with trypsin and plated on Matrigel  coated plastic culture dishes. Depending on the confluency of plating, 3–5 weeks later, monolayers of cells with RPE characteristics  appeared. After excising the aggregated portions, the RPE-like monolayer was dissociated with trypsin–EDTA (0.05%), passed through a 70 μm strainer and plated at 200,000 cells/cmFor teratoma assay the colonies from a fully confluent 6-well plate were cut mechanically and resuspended in 200 μL of ihPSCs media and matrigel. The suspension was immediately injected into 5 week-old SCID nude mice subcutaneously on the dorsal part. After about 8 weeks, teratomas of 1 cm diameter were formed and these were excised. Teratomas were fixed in 4% PFA for 3 days, washed 3 times with PBS, sectioned in half and observed macroscopically for the presence of cysts. Then the samples were dehydrated using 70–80–95% ethanol and xylol and embedded in paraffin. Continuous 6 μm sections and hematoxilin/eosin staining were performed at the Histology Unit, CABIMER. Pictures were taken with Leica DM6000B, connected with Leica DFC 350 FX camera.et al., 1987OS labeling: Two mg/ml stock solution of FITC isomer 1  in 0,1M sodium bicarbonate at pH 9.0–9.5, was prepared, filter-sterilized, and stored in aliquots at −20 °C. For labeling with FITC, OS were suspended in solution containing 10% sucrose, 20 mM sodium phosphate, and 5 mM taurine, and incubated with FITC for 1,5 h at room temperature, rotating in the dark. FITC-labeled OS were washed 5 times in buffer containing 10% sucrose, 20 mM sodium phosphate and 5 mM taurine, suspended in DMEM, aliquoted and stored at −80 °C until use.Photoreceptor OS were obtained from InVision BioResources  or prepared in-house according to Molday iPSC-RPE cells were incubated with OS for 2 h/37 °C, after which the OS were removed by triple washing with PBS. Cells were fixed by 4% PFA for 15 min/RT, washed and permeabilized by 0.1% Triton-X. Phallodin staining was performed for 30 min, the samples were then mounted with vectashield mounting media with DAPI. The samples were visualized by Leica confocal microscope TCS SP5 using HCX PL APO lambda blue 63X/ 1.4 OIL objective.RPE cultured cells were fixed in 4% paraformaldehyde, 2% glutaraldehyde in 0.1 M sodium phosphate buffer  for 2 h, washed with the same buffer, and then postfixed in 1% OsO4 in PB. After gradual dehydration in ethanol series, the pieces were embedded in EPON 812. Semithin sections stained with 1% toluidine blue in 3% sodium tetra-borate were analyzed with a Leica DMR light microscope (Leica Microsystems). Semithin and ultrathin sections were obtained in an ultramicrotome . After staining with lead citrate and uranil acetate, ultrathin sections were examined in a JEM-1400 Plus electron microscope .How to cite this article: Lukovic, D. et al. Human iPSC derived disease model of MERTK-associated retinitis pigmentosa. Sci. Rep.5, 12910; doi: 10.1038/srep12910 (2015).


Current cell based treatment for articular cartilage and osteochondral defects are hampered by issues such as cellular dedifferentiation and hypertrophy of the resident or transplanted cells. The reduced expression of chondrogenic signalling molecules and transcription factors is a major contributing factor to changes in cell phenotype. Gene modification of chondrocytes may be one approach to redirect cells to their primary phenotype and recent advances in nonviral and viral gene delivery technologies have enabled the expression of these lost factors at high efficiency and specificity to regain chondrocyte function. This review focuses on the various candidate genes that encode signalling molecules and transcription factors that are specific for the enhancement of the chondrogenic phenotype and also how epigenetic regulators of chondrogenesis in the form of microRNA may also play an important role. ES cells have been shown to differentiate into chondrocytes in a two-step process, where initially the stem cells change their phenotype to chondrogenic progenitors, followed by differentiation of these progenitor cells into chondrocytes. In vitro differentiation of these stem cells is highly efficient when combined with a three-dimensional microenvironment, with the addition of growth factors to enhance differentiation [The self-healing capacity of articular cartilage is minimal when damaged, due to the avascular nature of the articular joint surface. This reduces the potential for wound healing progenitor cells to access the site of injury for regeneration of the articular tissue . The curntiation .Both MSCs and chondrocytes are permissive to gene transfer and as such are excellent candidates for gene modification to enhance their chondrogenic phenotype and promote proliferation, avoiding detrimental cellular dedifferentiation, and senescence . Gene deThe introduction of foreign DNA encoding a gene of interest directly into a living cell results in the degradation of the naked DNA and therefore requires an efficient carrier for its delivery to the cell nucleus for gene transcription and subsequent protein expression . PhysicaA major advantage in using nonviral gene delivery techniques is the lack of transgene integration in the host genome. However, the delivery methods themselves are challenging due to the high level of cytotoxicity seen during plasmid DNA ransfections with liposome based transfection systems . Increas in vitro models of chondrogenesis with high doses of viral vector [Efficient transgene delivery into MSCs and articular chondrocytes has been achieved using a variety of viral based vectors including adenovirus, recombinant adeno associated virus (rAAV), retrovirus, and lentiviral vectors. Each of these viral vectors has inherent advantages and disadvantages associated with their function . The mosl vector , 20. On l vector .Retroviral vectors predominantly based on the murine leukaemia virus (MLV) transduce dividing cells and also integrate transgenes into the host genome which allow continued transgene expression but can potentially lead to insertional mutagenesis as experienced in an early human trial . SimilarPseudotyping lentiviral vectors with a heterologous envelope such as the commonly used glycoprotein derived from vesicular stomatitis virus (VSV-G) can provide structural integrity to the virus particle and also enhance tropism for a wide variety of cell types . NumerouA distinct advantage of using a reporter gene primarily in the form of a fluorescent protein is the relatively easy quantification of transduction efficiency and visualisation of transgene localisation. Delivery of the commonly used green fluorescence protein (GFP) reporter gene has been extensively used to document and track the efficiency of gene delivery by both viral and nonviral systems to MSCs and chondrocytes.Viral based gene delivery to adipose derived multipotent stem cells has been efficiently performed using adenoviral, retroviral, and lentiviral vectors expressing enhanced green fluorescent protein (eGFP) . All thrThe efficiency of gene delivery is also relative to the cell type used. For example it has been shown that viral gene delivery to primary human chondrocytes could be carried out successfully with the use of an AAV vector to deliver GFP which was expressed in these cells at a high efficiency of 93.7% at 7 days after transduction and gene expression was maintained for up to 28 days after transduction . ImportaIn contrast, GFP delivered via AAV vectors to MSCs have shown a maximum transduction efficiency of 65% . Importa col2a1 gene in both differentiating MSCs and chondrocytes [Enhancement of the chondrogenic phenotype can be achieved by ectopic expression of transcription factors to regulate subsequent expression of their target genes which are often lost due to cellular dedifferentiation or in pathological conditions such as osteoarthritis. Representative studies related to the use of these factors are listed in drocytes .β receptor mediated chondrogenic induction pathway and important in the activation of Sox9 transcription. Adenoviral transduction of human MSCs with Smad3 cDNA resulted in strong upregulation of extracellular matrix protein secretion [ col2a1 gene encoding collagen II directly influencing its expression [Smad3 is an important downstream transcription factor in the TGFecretion . The asspression . in vitro by lipofection [Efficient ectopic expression of Sox9 has been demonstrated by nonviral transfection of Sox9 cDNA into mouse MSCsofection . Subsequ Sox9 has also been achieved with adenovirus, rAAV [ Sox9 gene efficiently for both in vitro and in vivo applications. A comparative study into the use of these viruses to deliver Sox9 to human osteoarthritic chondrocytes in vitro showed reduced dedifferentiated phenotype and the formation of hyaline like cartilage. At a transduction efficiency of 85%, lentiviral transduction was identified as an appropriate vector system for Sox9 delivery for translational applications such as autologous chondrocyte transplantation [The overexpression ofus, rAAV –37, retrus, rAAV , and lenus, rAAV . In geneantation . In vitro AAV mediated Sox9 overexpression in human bone marrow derived mesenchymal stem cells resulted in a significant reduction in hypertrophy markers. Nonviral delivery of Sox9 plasmid DNA has been shown to enhance chondrogenesis of human MSCs when codelivered with siRNA to the osteogenic transcription factor Cbfa-1 [ Sox9 delivery by adenoviral vector into rabbit MSCs has shown that the ectopic expression was maintained when transduced cells were implanted into osteochondral defects in rabbits resulting in successful integration of the graft tissue [ Sox9 transfection system using a biodegradable scaffold for chondrogenic stimulation has also been used to enhance the phenotype of rat chondrocytes. This combined approach may bring Sox9 mediated gene therapy closer to clinical application [The delivery of sox9 by AAV vectors has also been shown to be efficient in explant cultures of human cartilage, with enhanced expression of extracellular matrix components . In vitrr Cbfa-1 . Sox9 det tissue . A tetralication . In vitro retroviral transduction of mouse embryonic MSCs with the transcription factor Barx2 showed an increase in cell aggregation prior to chondrogenic differentiation indicating its capacity to enhance cell-cell interaction [ col2a1 through Barx2 interaction with Sox9. This study may indicate the potential for the cotransduction of this novel transcription factor with Sox9 for enhancing the expression of collagen II in adult stem cells or dedifferentiated chondrocytes.The homeobox transcription factor Barx2 has also been recognised as a regulator of chondrogenesis during embryonic development.eraction . Retroviβ superfamily of growth factors are therefore potential candidates for gene therapy applications.Growth and differentiation factors are important in chondrogenic differentiation of MSCs and in the maintenance of the chondrogenic phenotype. Particular members of the TGF BMP-2 resulted in higher chondrogenic potential, evidenced by both increased levels of cell proliferation and collagen II matrix protein secretion. The degree of chondrogenic differentiation was observed to be highly influenced by the cell type. Perichondrium derived cells demonstrated the highest chondrogenic capacity followed by bone marrow derived stem cells and fat derived stem cells. Importantly the formation of fibro cartilage was observed moderately in bone marrow derived stem cells and significantly higher in fat derived stem cells highlighting the importance in the choice of cell type for genetic modification in translational clinical applications. In agreement with this study Pan et al. [ BMP-2 enhanced chondrogenesis through plasmid DNA delivery of BMP-2 cDNA which resulted in an increase in Sox9 expression, the major transcription factor in the regulation of chondrogenesis. A recent study has also demonstrated the same effect and has shown that BMP-2 expression is lowered in Sox9 enhanced chondrogenesis [Bone morphogenetic protein-2 (BMP-2) has mainly been used to enhance osteogenesis and facilitate repair of critical size defects in bone; however, some studies have also shown increased chondrogenesis upon ectopic expression of this factor . Adenovin et al.  showed togenesis . This fiBMP-7) overexpression by adenoviral transduction in equine chondrocytes has shown that articular cartilage matrix generation occurs earlier than untransduced control cells when transplanted in vivo [ BMP-7 transduced cells when compared to controls. However, at 8 months the difference in matrix formation between the BMP-7 treated and control cultures was not significant, indicating that BMP-7 might initially accelerate wound healing process. Furthermore, the compressibility of the defects treated with BMP-7 transduced chondrocytes showed a 2- to 10-fold reduction in weight bearing capacity, suggesting the need to activate alternative chondrogenic pathways to enhance mechanical properties. Transgene silencing associated with adenoviral transduction was not observed and immunological response to the virus was significantly low in articular joints, supporting the use of adenovirus for efficient gene transfer and expression. Gavenis et al. [ BMP-7, enabling long-term culture expansion, while retaining the hyaline cartilage phenotype of the cells and high resistance to dedifferentiation.Interestingly, bone morphogenetic protein-7  (PCL) woven scaffolds to transduce human bone marrow derived MSCs [β-3 protein.β-3 TGF has also been delivered to adipose derived stem cells (ADSCs) using a baculoviral vector [β-3 expressing cells displayed enhanced collagen II and matrix protein expression improved biomechanical properties.TGF culture , 50. Howved MSCs . Transdul vector , in whic IGF-1 into rabbit articular chondrocytes has shown enhanced glycosaminoglycan production increasing over time [ IGF-1 in human MSCs did not enhance chondrogenesis and inhibited collagen II expression [ IGF-1 function in different cell types. IGF-1 plasmid DNA immobilised onto collagen II-glycosaminoglycan scaffolds has been delivered to canine articular chondrocytes to facilitate slow release of the plasmid DNA at sustained therapeutic levels [ IGF-1 plasmid DNA transfected rabbit articular chondrocytes have been used to treat in vivo cartilage defects in combination with fibroblast growth factor-2 (FGF-2) resulting in articular cartilage regeneration with low collagen I expression [Insulin-like growth factor-1 (IGF-1) enhances cell proliferation and differentiation in articular chondrocytes during growth plate development . Nonviraver time . In contpression . This obc levels . The resc levels . IGF-1 ppression .FGF-2) has been delivered to rabbit chondrocytes by transfection of plasmid DNA and the transplantation of the transfected cells demonstrated enhanced collagen II expression. Importantly the expression of fibrocartilage marker collagen I was detected; however, the level of expression was lower than that of the control [ FGF-2 has also been studied in human MSCs demonstrating strong chondrogenic differentiation potential with enhanced cartilage matrix production and exhibiting low fibrocartilage and osteogenic markers [Fibroblast growth factor-2  targeting the col1 gene. This resulted in enhanced expression of the type II collagen and a significant reduction in collagen I expression after culturing transduced cells encapsulated in an alginate hydrogel for subsequent in vivo transplantation. This ex vivo strategy was used to eliminate the carryover of immunogenic viral components by subsequent passaging to allow their dilution in culture.To enhance chondrogenic differentiation in synovium derived MSCs by growth factor and short hairpin RNA expression, Zhang et al.  used bic BMP-2 [β-1 TGF, and IGF-1 [ in vitro with insulin-like growth factor (IGF-1) andβ-1 TGF or BMP-2 has been shown to have synergistic effects in terms of chondrogenic enhancement with increased collagen II expression [Individual treatments of BMP-2 , TGFβ-1,nd IGF-1  have beepression , 20. Imp IGF-1 and FGF-2 in chondrogenic differentiation have also been shown in ovine adipose derived stem cells with adenovirus transduced cells demonstrating very low expression of fibrocartilage marker collagen I or hypertrophic marker collagen X [The synergistic effects ofllagen X .Sox9 and BMP-2 have also been delivered in a bicistronic nonviral plasmid vector into dedifferentiated human chondrocytes expressing a weak chondrogenic phenotype [henotype . OverexpOct4, Klf4, C-Myc, and Sox2) were capable of reprogramming mouse dermal fibroblast to obtain a pluripotent stem cell phenotype.As a successful expression system, retroviral transduction has been used to deliver transcription factors important in the reprogramming of somatic cells to obtain stem cell like phenotypes. Previously Takahashi and Yamanaka  demonstr Sox9, Outani et al. [ Klf4 and c-Myc with Sox9 yielded the highest efficiency in chondrogenic differentiation resulting in extracellular matrix secretion, with physical and histological properties as those of articular cartilage. This result has also been shown in the direct differentiation of human dermal fibroblasts to chondrocytes with the same factors [Combining these transcription factors along with chondrogenic transcription factori et al.  were abl factors . The codhTeRT) gene has been performed [ hTeRT has been coexpressed with Bmi-1 oncogene with a lentiviral vector to prolong the lifespan of the immortalized cells [ hTeRT expression in human MSCs, a Tet-On inducible system has been used previously which showed that the transduced cells ceased proliferation when doxycycline was removed [ hTerRT has also shown that the chondrogenic differentiation capacity of MSCs is retained with its forced expression [A reduction in the rate of proliferation in chondrocytes, as with most somatic cell types, is greatly affected by telomere shortening as these cells divide, affecting the total number of healthy cells available for chondrocyte transplantation. As a comparatively novel approach for obtaining sufficient human chondrocyte numbers by culture expansion, retroviral vector mediated forced expression of the human telomerase reverse transcriptase . This mature microRNA is formed by the cleavage of the parental microRNA transcript H19, and its overexpression has been shown to result in Sox9 enhanced col2a1 expression in vitro but not in Sox9 induced expression of Col9a1 [ col2a1. The target could potentially be histone deacetylase I or II, of which inhibition resulted in the elevated expression of collagen II in arthritic chondrocytes [ col9a1 may suggest a link in their regulatory mechanism.MicroRNAs (MiRNAs) are a group of noncoding short RNA molecules that bind to the 3′ untranslated region of target mRNA, resulting in the repression of gene translation and can be delivered to cells by means of plasmid DNA encoding the parental form of the microRNA which after enzyme cleavage yields the mature and functional microRNA . A list f Col9a1 . The tardrocytes . The ina in vitro. miR-145 targets Sox9 protein directly resulting in a decrease in cartilage matrix proteins [The repression of Sox9 has been demonstrated with the forced expression of microRNA-145 (miR-145) in the C3H10T1/2 cell lineproteins . Inhibitproteins . However in vitro [MicroRNAs have also been associated with the prevention of senescence and apoptosis in chondrocytes. MicroRNA-21 (miR-21) has been shown to enhance cell proliferation in rat articular chondrocytes when deliveredin vitro . The maiHighly specific to cartilage tissue, microRNA-140 (miR-140) has been studied extensively for its ability to inhibit the expression of chemokine (C-X-C Motif) ligand 12 (CXCL12) and A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS-5) that lead to articular cartilage degradation in osteoarthritis . MicroRNADAMTS-5 is a cleavage enzyme that degrades the major cartilage protein aggrecan and is highly expressed in osteoarthritic cartilage [ ADAMTS-5. In vivo expression of miR-140 in transgenic mice with the transcript driven by the col2a1 promoter has shown specific articular cartilage matrix expression and resistance to arthritis [artilage . It has rthritis .The potential for microRNAs to control the chondrogenic process may have future translational application; in particular the overexpression of candidate miRNAs such as miR-140, miR-21, and miR-675 may enhance the chondrogenic potential of both MSCs and culture expanded chondrocytes. IL-1Ra against inflammation caused during rheumatoid arthritis [ IL-1Ra. Intra-articular injections of these cells resulted in significantly lower inflammation relative to the control group and importantly no adverse effects were observed in patients undergoing treatment. At the time of writing only one clinical trial involving gene therapy to treat arthritis had been listed, with allogeneic human chondrocytes expressingβ-1 TGF to be used for the treatment of cartilage defects . Future advancement in gene therapy techniques leading to clinical trials may require highly efficient gene transfer systems, such as self-complimentary AAV vectors (scAAV), providing more efficient gene transduction compared to traditional single stranded vectors with high levels of safety. Recent work demonstrates their efficiency in vivo [The first and only gene therapy human trial for treating an osteochondral defect has been the use of the interleukin-1 receptor antagonistrthritis , where s in vivo .A wide variety of transgenes and gene delivery vectors have been used to enhance chondrogenic differentiation in adult stem cells and to reduce dedifferentiation and hypertrophy in articular chondrocytes. The current challenge for the clinical applications of gene modified cells is enhancing the safety of gene delivery vectors while maintaining therapeutic levels of transgene expression. Advances in higher efficiency, scaffold employing nonviral gene delivery systems, and enhanced safety features in viral based gene delivery systems have shown great potential for clinical application of such technologies.


Measurement-based care is an evidence-based practice for depression that efficiently identifies treatment non-responders and those who might otherwise deteriorate . HoweverThis study is a dynamic cluster randomized trial of standardized versus tailored measurement-based care implementation in Centerstone, the largest provider of community-based mental health services in the USA. This prospective, mixed methods implementation-effectiveness hybrid design allows for evaluation of the two conditions on both clinician-level  and client-level (depression symptom change) outcomes. Central to this investigation is the focus on identifying contextual factors  that mediate MBC fidelity and optimize client outcomes.This study will contribute generalizable and practical strategies for implementing systematic symptom monitoring to inform and enhance behavioral healthcare.NCT02266134.Clinicaltrials.gov The online version of this article (doi:10.1186/s13012-015-0313-2) contains supplementary material, which is available to authorized users. Depression is one of the world’s leading causes of illness burden , costingand transdiagnostic  relevance [Conversely, measurement-based care (MBC) is a relatively simple evidence-based intervention framework . MBC, byelevance . We concelevance ) as it melevance .In terms of utility, MBC aligns with the Patient Protection and Affordable Care Act (P.L. 111–148) by focusing on monitoring outcomes and satisfying meaningful use requirements . MBC preDespite MBC’s demonstrated effectiveness, reach, and utility, recent estimates suggest that fewer than 20 % of PhD level clinicians, master level clinicians, and psychiatrists routinely measure mental health symptoms prior to each session . MBC hasThe long-term goal of this research project is to provide generalizable and practical recommendations about strategies that promote MBC implementation, fidelity, and sustainment in CMHCs. To realize this goal, this study will test a standardized versus a tailored approach to implementing MBC at Centerstone, the largest provider of community-based mental health services in the USA. In this study, MBC will center on administration of the Patient Health Questionnaire-9 Item Version . The PHQAlthough touted as superior, tailored implementations have rarely been compared to standardized approaches , 31. MorFramework for Dissemination [The mination  is the sThe first three domains affect the willingness and ability of stakeholders to implement and sustain new interventions . Norms aFramework for Dissemination organizes the evaluation of key contextual factors that may promote or hinder the implementation and sustainment of EBPs in CMHCs [Taken together, the context of diffusion within the in CMHCs . As suchDynamic Sustainability Framework . The DSs et al.  contend s et al. ), whereaAim 1: To compare the effect of standardized versus tailored MBC implementation on clinician-level (1a) and client-level (1b) outcomes. We hypothesize that tailored implementation will outperform standardized in terms of (H1a) MBC fidelity and (H1b) reducing client depression severity.Aim 2: To identify contextual mediators of MBC fidelity. We hypothesize that contextual mediators  will be leveraged in the tailored condition, but serve as barriers in the standardized condition.Aim 3: To explore the impact of MBC fidelity on client outcomes as a preliminary test of the Dynamic Sustainability Framework [ramework . We hypoCenterstone is the largest provider of community-based mental health services in the USA – delivering psychotherapy, social work services, psychiatric services, and health promotion/disease prevention interventions to over 140,000 children, families, and adults each year. Centerstone has a physical presence in five-states  and hosts the Knowledge Network . Centerstone also maintains the largest repository of community mental health data in the USA—containing data from over 350,000 patients, 20 million service records, and 2 million prescriptions. This “data warehouse” exists to facilitate mental health services research and populates an innovative analytics platform  to guide clinical decision-making and agency management. The clinical research, program evaluation, informatics, and grant writing arm of Centerstone—Centerstone Research Institute (CRI)—works with the clinical enterprise to prioritize the standardization of care through EBP delivery. Centerstone initiated a partnership with the principal investigator (CCL) in 2011 to develop clinical pathways for depression. A workgroup, comprised of three regional directors from clinics across Indiana and Tennessee and the CRI Director of Clinical Research, convened for monthly meetings over the course of 2 years. MBC was identified as the EBP of focus given its relevance and potential utility for improving client outcomes across diagnoses—a necessary feature given the highly complex and comorbid presentations of Centerstone clientele.Framework for Dissemination [Both conditions (standardized and tailored) will employ the same blended protocol of implementation strategies  to remomination ), the bemination , 36–41),mination .Table 1Sresponsive application of implementation strategies and content matched to determinants of practice  identified via a needs assessment and formative evaluations. The same strategies outlined in the standardized condition will be employed in the tailored condition  are randomized to either early or later stage implementation in four cohorts (2–4 sites per cohort spaced 5 months apart), with half the sites randomized to the standardized and half to the tailored condition  and rural versus urban status. Twelve sites were matched using three characteristics: number of clinicians, number of adult depression diagnoses, and urban versus rural status. A dynamic cluster randomization trial was selected to enhance feasibility of capacity management ; the desRandomization was implemented following Chamberlain et al.  and BrowN = 150). The number of sites and clinicians was determined by a simulation-style power analysis to detect a small effect size. Within each enrolled site, clinicians will have the option to participate if they (1) are at least 40 % full-time equivalent, (2) provide individual psychotherapy, (3) treat adults with depression, and (4) conduct sessions in English.A total of 187 clinician participants will be recruited across 12 sites (target Table N = 500) will be recruited for enrollment in this study; approximately three clients will be recruited per participating clinician. As this is a pragmatic trial [A total of 625 depressed adult clients . Client PHQ-9 responses will be entered into the electronic health record by clinicians to enable review of scores over time  and to facilitate discussion of symptom trajectories in session. Given that this is an implementation trial, there is potential for variance in the completion/administration of the PHQ-9 instrument. To enhance data quality, enrolled clients in both conditions will complete the PHQ-9 at baseline and at week 12 of treatment in a phone call with study staff. The 12-week window reflects a commonly used time period in randomized clinical trials during which depressive symptoms are expected to remit. We will also supplement active research data collection with regularly collected administrative data  captured in intake and progress review reports in the electronic health record. Finally, to substantiate the clinician self-report of PHQ-9 integration into the session, the client will be asked to respond to five-item text message surveys immediately following each session inquiring about clinician MBC fidelity.H1a) MBC fidelity and (H1b) reducing client depression severity.We hypothesize that tailored implementation will outperform standardized in terms of (Data pertaining to MBC fidelity will be collected via three mechanisms: (1) clinician report in the electronic health record; (2) client report via text messaging; and (3) objective coding of a subsample of clinician-client sessions. The trichotomous fidelity outcome variable will be computed using the following data from the electronic health record as follows: (a) clinician recorded client PHQ-9 scores ; (b) a report to indicate whether the clinician looked at the scores ; and (c) clinician self-report of whether they discussed the PHQ-9 scores with the client . Overall MBC fidelity scores can range from 0 to 3 reflective of “0 = none”, “1 = mild”, “2 = moderate”, and “3 = excellent” fidelity. This fidelity outcome variable will be triangulated with client post-session text message surveys and objective session coding as the three reports  may not align , 48.In addition to the PHQ-9 serving as the central focus of the MBC (symptom monitoring), client scores on the PHQ-9  at baselMultilevel generalized linear models will be used to assess hypotheses for standardized versus tailored MBC implementation for both models of clinician and client outcomes. The multilevel generalized linear model framework includes continuous outcomes and extends the linear model to accommodate nonlinear outcomes by including a distributional assumption and link function . Specifically, we will examine the effect of the implementation condition on MBC fidelity  measured across time. Multilevel models account for the non-independence of repeated measurements within participants and non-independence due to sites . Models d for growth models [d = 0.46 for the clinician models (N = 150) and effect sizes as small as d = 0.30 for the client models (N = 500).A power analysis for the Aim 1 multilevel models was conducted using Monte Carlo studies in which power is the proportion of significant effects (2-tailed α = 0.05) for a parameter of interest across all simulated data sets fit with the same model , 52 usinh models . Using tWe hypothesize that contextual mediators  will be leveraged in the tailored condition but serve as barriers in the standardized condition.Framework of Diffusion as potential mediators, assessed at baseline (AT1), 5 months into the implementation process (AT2), and 15 months into the implementation process (AT3). See Table The dependent variable for Aim 2 is the implementation outcome of MBC fidelity as described in Aim 1. The independent variables summarized in Table rocess AT. See TabFramework for Dissemination domains highlighted above  after the active implementation phase AT2) and at 10 months post active implementation phase (AT3). Focus group participants will be selected in collaboration with the clinic director using purposive sampling for extreme variation . Focus g and at 1a), (2) the mediator predicts growth change in MBC use (path b), (3) condition predicts change in the outcome (path c), and (4) whether the implementation condition’s effect on MBC use becomes significantly weaker when controlling for the mediator (path c’) [a and b) will be tested using biased-corrected bootstrapped confidence intervals [We will examine mediation models in which contextual factors  , 57. We path c’)  to demonntervals .Framework of Dissemination [a priori and through emergent themes analysis.Rapid ethnography  will be mination . InductiFramework for Dissemination [Mixed methods will be used to integrate findings from Aim 2 using a QUAN + QUAL structure (wherein both types of data are collected simultaneously) to achieve the function of data expansion for the purposes of evaluation and elaboration . Using tmination , we willκ2 effect size for mediation  was computed [κ2 = 0.16 for the clinician models and effect sizes as small as κ2 = 0.10 for the client models.For Aim 2, we conducted power analyses for the hypothesized mediation effects with the same assumptions described above but with the use of effective samples size estimates (an ICC corrected sample size) based on a design effect adjustment  in ordercomputed . We are H3) that adapted MBC protocols (tailored condition) will outperform weekly administration of PHQ-9s (standardized condition) with respect to clinically significant change in depression severity from intake to week 12.We hypothesize (We will assess the impact of MBC fidelity (standardized versus tailored) on clinically significant change observed in each client between intake and session 12 using generalized linear mixed models. Models will represent the three-level structure  with a binary outcome, representing whether a client exhibited clinically significant change using the reliable change criterion , modeledr = 0.27 for clinicians and as small as r = 0.21 for clients. The planned sample sizes are consistently sufficient for detecting medium-effect sizes for clinician outcomes and small effect sizes for client outcomes, with the power to treat site as a random effect based on six sites per condition [For Aim 3 analyses, we are sufficiently powered to detect effect sizes as small as ondition .The Indiana University Institutional Review Board has approved all study procedures. The Pre-Implementation phase (Phase 0) of the study was completed in May 2015, and Phase 1 subject recruitment and data collection began in June 2015 with the first cohort of clinic sites.This study is innovative in at least three ways. First, our focus on testing strategies to implement MBC in community mental health is innovative because this simple MBC framework may be the minimal intervention needed for significantly reducing the burden of depression on society . For theSecond, we will modify the electronic health record system to provide clinically relevant data to providers who use MBC, such as symptom trajectory graphs, alerts when suicidality is endorsed, etc. With electronic health record prevalence increasing, this approach presents a generalizable and cost-effective method for engaging in systematic outcome monitoring that maximizes therapeutic benefit and aligns with meaningful use requirements .a priori selected implementation strategies that generally neglect contextual tailoring of the interventions. A qualitative analysis of 22 implementation studies revealed that few focused on matching strategies to determinants of practice [Third, the majority of existing implementation research has focused either on descriptive studies that explore barriers and facilitators (determinants of practice) or on comparisons of practice . A critipractice  by experpractice , 70, butIn addition to these and other strengths, this approach to implementation of MBC has limitations. First, this implementation effort focuses on outcomes for standardized and tailored MBC as implemented within multiple sites of a single behavioral health organization. While it is anticipated that many of the strategies necessary to facilitate MBC implementation will align with previous research efforts, the degree of generalizability of the standardized and tailored approaches to other sites and settings is unknown. Second, clinician and client turnover across participating clinic sites may limit the sample size obtained, despite researcher efforts to exceed recruitment targets. Fortunately, literature suggests that participatory approaches to organizational change in practice patterns result in lower clinician attrition . Third, Measurement-based care aligns with priorities delineated in the Patient Protection and Affordable Care Act (P.L. 111–148), notably systematic evaluation of services  and mean


Saccharomyces cerevisiae, origins are defined at discrete sites. Regular spacing and diverse firing characteristics of origins are thought to be required for efficient completion of replication, especially in the presence of replication stress. However, a S. cerevisiae chromosome III harboring multiple origin deletions has been reported to replicate relatively normally, and yet how an origin-deficient chromosome could accomplish successful replication remains unknown. To address this issue, we deleted seven well-characterized origins from chromosome VI, and found that these deletions do not cause gross growth defects even in the presence of replication inhibitors. We demonstrated that the origin deletions do cause a strong decrease in the binding of the origin recognition complex. Unexpectedly, replication profiling of this chromosome showed that DNA replication initiates from non-canonical loci around deleted origins in yeast. These results suggest that replication initiation can be unexpectedly flexible in this organism.Eukaryotic DNA replication initiates from multiple sites on each chromosome called replication origins (origins). In the budding yeast  Saccharomyces cerevisiae, origins contain defined discrete DNA sequences termed autonomously replicating sequences (ARSs), so-named because of their ability to maintain plasmid propagation S. cerevisiae have enabled extensive origin mapping and characterization studies by many researchers, which have led to a detailed understanding of the replication landscape in budding yeast DNA replication in eukaryotic cells initiates from multiple sites on each chromosome called replication origins (hereafter origins). In the budding yeast S. cerevisiae chromosome III with a greatly reduced number of canonical origins can be replicated relatively normally. However, how replication of the origin-deficient chromosome is accomplished is not understood.Although pre-RCs are formed at all origins that have the ability to fire in the following S phase, each origin exhibits different characteristics in timing (when in S phase the origin fires) and efficiency (how likely in a given S phase the origin fires). It is generally believed that these properties, along with proper origin spacing, are essential for chromosome stability, as dormant origins  can act as backups to early-firing, efficient origins if replication forks collapse in early S-phase S. cerevisiae chromosome VI. We found that the strain exhibits no detectable growth defects, even in the presence of DNA damaging agents and replication inhibitors. Unexpectedly, we detected initiation of DNA replication from loci around deleted origins, despite the fact that origin deletions cause sharp decrease in ORC binding at deleted origins. Our results suggest that initiation of DNA replication can be unexpectedly flexible in S. cerevisiae, an organism that normally initiates DNA replication from discrete sites.In this study, we aimed to establish an independent system to investigate how an origin-deficient chromosome is replicated by systematically deleting seven well-characterized origins on the left arm of rad5 mutation in the original W303-1a was corrected 7oriΔ strain was constructed by deleting confirmed ARS sequences identified by www.yeastgenome.org). The exact regions of deletion are listed in ARS600::LEU2, ARS606::ADE2, ARS601, 602::loxP, ARS605::loxP, ARS603::loxP, ARS604::loxP, ARS603.5::KanMX. The loxP alleles were created by looping out the KanMX cassette using the Cre-lox system as described 7oriΔ strains exhibit the same growth phenotypes. In addition, through a large number of genetic crosses, we confirmed that the deleted origins co-segregate at an expected rate.Strains used in this study were created using standard yeast manipulations and are listed in Orc-bound DNA was isolated from Orc2-2Xlinker-3XFlag epitope-tagged cells arrested with nocodazole (15 µg/ml) for 2 hours at 30°C. Samples were cross-linked with 1% formaldehyde for 20 min at room temperature, and cross-lining was stopped with 0.125M glycine. Chromatin was sonicated using a Bioruptor sonicator bath (Diagenode), to an average fragment size of 250 bp. Soluble chromatin was immunoprecipitated as previously described DNA was harvested from cells harboring a single integrated BrdU incorporation vector Enriched and input DNA was UDG fragmented and terminally labeled with Cy5 and Cy3, respectively, and competitively hybridized to custom tiling arrays (Roche NimbleGen) as previously described 7oriΔ strain, a yeast mutant in which seven annotated and confirmed origins on the left arm of chromosome VI were deleted .To establish a system to investigate how an origin-deficient chromosome can be replicated, we created the  deleted ; Fig. 1aher rich  or synthnditions . UnexpecRAD97oriΔ strain for interaction with genes known to contribute to these and other pathways. Interestingly, we found no genetic interaction with DNA damage pathway genes including RAD9, MEC1, and RAD53; the S-phase checkpoint genes MRC1, TOF1, and RRM3; and other cell cycle genes including CLB5, MAD3, and SIC1, even in the presence of DNA damaging agents (data not shown). This is likely partly due to the fact that the chromosome III loss assay in the presence of an extra copy of the chromosome is more sensitive than growth assays. We also cannot exclude the possibility that the effects of origin depletion are somewhat different between chromosomes III and VI, and/or that the presence of an extra copy of chromosome III sensitizes the system.An extra copy of origin-depleted chromosome III has been reported to exhibit increased chromosome loss rates when combined with mutations in replication checkpoint and DNA damage pathway genes, including 7oriΔ chromosome is replicated, we first measured how origin deletion affected the binding of the ORC across the left arm of chromosome VI, as the deleted sites have been confirmed to be essential for ORC binding and origin function 7oriΔ mutation on ORC binding, we subtracted Orc2 signals in the 7oriΔ strain from those in wild-type cells. This resulted in clear Orc2 peaks at every deleted origin on chromosome VI  ARS602.5 and ARS603.1, which have been recently identified as functional origins 7oriΔ strains. The distinct peaks of replication at ARS602.5 and ARS603.1 in wild-type cells in the presence of HU are consistent with a report by Knott et al, who also used the BrdU-IP method 7oriΔ strain, and to our knowledge, has never been reported before.To determine how replication of the 7oriΔ chromosome VI initiates at multiple loci near deleted origins, where ORC is bound at low levels. Importantly, we did not observe any detectable increase in Orc2 ChIP signals around deleted origins in the 7oriΔ strain, suggesting that the background levels of ORC binding seen in wild-type cells is sufficient for origin firing when canonical origins fail. The robust growth of the 7oriΔ mutant, even in the presence of replication inhibitors, demonstrates that initiation of replication from these sites is sufficient for efficient S phase progression.To more closely examine the correlations between the ORC binding pattern and origin activity, we aligned the Orc2 ChIP signals and BrdU replication profiles around deleted origins . This reARS606 was dependent on the ADE2 marker, as no origin firing was detected when ADE2 was replaced with the KANMX marker (data not shown). This is consistent with a recent report that a point mutation in ARS606 largely diminishes replication initiation from this origin ADE2 gene on the pRS402 plasmid, which was used as a template for generating a PCR fragment to replace ARS606, contains a partial fragment of ARS1516, although the predicted ACS sequence was not included ARS603, 604 and 605, which have a 34 base pair loxP sequence as a replacement. Because loxP sequence does not have noticeable sequence homology with the ACS sequence, it is unlikely that a loxP site alone can act as an ARS. These results collectively suggest that DNA sequences surrounding the endogenous origins ARS603, 604, 605 and 1516 have an innate ability to support origin firing. It was previously shown that deletion of multiple origins from disomic S. cerevisiae chromosome III allowed proper replication and segregation of this chromosome 97% of the time de novo origin firing similar to what we observed contributed to these results as well. Our results suggest that, although replication normally initiates from discrete sites in S. cerevisiae, initiation site selection of DNA replication can be unexpectedly flexible in budding yeast, and that yeast chromosomes are capable of initiating replication from non-canonical sites when multiple origins fail.Interestingly, the origin activity around the deleted


Drosophila) as a critical target and effector of cAMP in injured axons. DLK/Wnd is thought to function as an injury ‘sensor’, as it becomes activated after axonal damage. Our findings in both Drosophila and mammalian neurons indicate that the cAMP effector kinase PKA is a conserved and direct upstream activator of Wnd/DLK. PKA is required for the induction of Wnd signaling in injured axons, and DLK is essential for the regenerative effects of cAMP in mammalian DRG neurons. These findings link two important mediators of responses to axonal injury, DLK/Wnd and cAMP/PKA, into a unified and evolutionarily conserved molecular pathway for stimulating the regenerative potential of injured axons.A broadly known method to stimulate the growth potential of axons is to elevate intracellular levels of cAMP, however the cellular pathway(s) that mediate this are not known. Here we identify the Dual Leucine-zipper Kinase (DLK, Wnd in DOI:http://dx.doi.org/10.7554/eLife.14048.001 Adult mammals typically cannot repair damage to the nerve fibers in their brain or spinal cord. This is because these nerve cells cannot generally grow new nerve fibers. However this inability to regenerate nerve fibers is not set in stone. Instead, it can be unlocked by a second injury in nerves elsewhere in the body, the so-called “peripheral nervous system”. This process relies on an enzyme called DLK, which becomes activated in damaged nerve fibers.But how does DLK ‘sense’ damage to nerve fibers? Injuring the peripheral nervous system causes the levels of a molecule called cAMP to increase in the damaged nerve cells, and the elevated cAMP levels stimulate the nerve fibers to regenerate. However, it was not known if cAMP activates DLK, or if the two act independently of each other.By looking at the regeneration of damaged nerve fibers in fruit fly larvae, Hao et al. now show that the cAMP and DLK signaling pathways are clearly linked. Further experiments with nerve cells from mice and human cells revealed more detail about this link. Together the results showed that another enzyme called PKA activates DLK directly when cAMP levels are high. These findings reveal a unified pathway that is the key to unlocking the regenerative potential of injured nerve fibers, which has been conserved for hundreds of millions of years of evolution. Further work could now ask if the DLK enzyme is involved in the other known roles of cAMP signaling in nerve cells; or if cAMP and PKA activate DLK in other forms of nerve damage, including injuries where nerve fibers normally fail to regenerate.DOI:http://dx.doi.org/10.7554/eLife.14048.002 Repair of lost axonal connections generally fails to occur after neuronal injury in the adult mammalian central nervous system (CNS). This failure is not only a reflection of the growth inhibitory nature of CNS tissue , but alsin vivo , mediates protective effects of a conditioning lesion in Drosophila motoneurons  or activation of PKA by overexpression of the catalytic subunit (PKACA) (Drosophila motoneuron axons after nerve crush injury (3 to 200 µm3 15 hr after injury. PKA activation led to a 1.5 fold increase in this volume compared to control (WT) axons   led to ah injury . The newh injury . In contT) axons . The enhT) axons . These oignaling . In addiol axons . cAMP anDrosophila for monitoring the activation of Wnd and downstream nuclear signaling. Wnd signaling induces expression of the c-Jun N-terminal Kinase (JNK) phosphatase puckered (puc), which can be measured as lacZ expression using fly lines that contain the puc-lacZ enhancer trap reporter  version (GFP-WndKD) that contains a point mutation in the kinase domain , we have generated phospho-specific antibodies against a phosphorylated peptide corresponding to activation loop of mouse DLK (KELSDKpSTKMpSFAGTV). The phospho-DLK antibodies detected a strong band in HEK293 cells that overexpress WT mouse DLK, but show no reactivity for mutant DLKS302A . RemarkaS302 DLK , even whS302 DLK . SimilarS302 DLK .in vitro  conditional knockout (KO) mice and littermate controls. The effects of forskolin on neurite outgrowth were abolished in DLK conditional knockout DRG neurons . These fThe Wnd/DLK kinase is likely to function as a sensor of axonal damage. Depending upon the context, its activation can lead to either axonal regeneration or cell death and degeneration . The facC. elegans, PKA gain-of-function mutations can induce axonal outgrowth even in dlk mutants  and UAS-moody-RNAi (VDRC 100674) were from the Vienna RNAi center  and UAS-dnc-RNAi (27250) were acquired from Bloomington stock center.The following fly strains were used in this study: Canton-S (WT), m12-Gal4 , BG380-GUAS-Wnd, , UAS-GFPWnd,  was diluted 1:100. Anti-dsRed polyclonal antibody  was diluted 1:1000. For secondary antibodies, A488- or Cy3-conjucated goat anti-mouse or rabbit  were used at 1:1000.Confocal images were collected on an Improvision spinning disk confocal system, consisting of a Yokagawa Nipkow CSU10 scanner, and a Hamamatsu C1900-50 EMCCD camera, mounted on a Zeiss Axio Observer with 40X (1.3NA) oil objectives. Similar settings were used for imaging of all compared genotypes and conditions. Volocity software (Perkin Elmer) was used for intensity measurements and quantification of all confocal data.A quantification of the sprouting response in injured motoneuron axons was measurement of the change of total volume of regenerating axonal membrane, labeled by mCD8-GFP, within a 100 µm distance from the injured tip. Pixels within the most distal 100 µm of the injured proximal stump were selected based on mCD8-GFP intensity criteria of >3 standard deviation above the mean, and then summed to measure total membrane volume. Puc-lacZ expression was quantified by measurement of mean intensity for lacZ staining in the nuclei of motoneurons located along the dorsal midline (segments A3-A7) of the nerve cord of third instar larva. The mean intensity of GFP-WndKD within segmental nerves was quantified by measuring GFP intensity within 100 µm distance of each nerve at the site of exit from the ventral nerve cord.S302A (K185A (directly generated from Flag-DLK) and PKACA (HEK293 cells were cultured in DMEM/F12 (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin (Gibco). For transient transfection, 1 µg of given vectors were transfected into 3.5 cm dish using Lipofectmine 2000 (Invitrogen). 20 hr after transfection, cells were processed for Western blotting. Plasmids used for transfection were Flag-DLK , Flag-DLS302A , Flag-DLnd PKACA .Cortical neurons were dissected from E18 rat embryos. The cortex was digested by incubating with 0.5% trypsin-EDTA (Gibco) and DNAse I (Roche) at 37°C for 10 min. Following digestion, neurons were washed twice in DMEM medium (Gibco) containing 10% FBS before resuspension in neuronal growth media which containing neurobasal (Gibco), Glucose (Sigma), Glutamax (Gibco), penicillin-streptomycin and B27 supplement (Gibco). All the plates were coated with 100 µg/mL ploy-D-lysine  for 2 hr. Neurons were then triturated and plated at a final concentration 400,000 cells/mL.in vitro (DIV)1, half of the media was removed and fresh media containing AraC  was added. Drug treatment and replating were performed as described previously (2). Trypsin was removed and fresh culture media was added to the cells which were then gently pipetted and transferred to culture slides. 18 hr after replating, neurons were fixed (4% PFA) and stained for βIII-tubulin . At least 100 neurons were imaged per group using either Leica DFC310 FX or DFC7000T color fluorescence cameras and longest neurite was traced using NeuronJ plugin for ImageJ. For replating experiments with PKA inhibitor , vehicle or inhibitor were added at the same time as DMSO and forskolin.For adult DRG experiments, DRG neurons were collected from either CD1(Charles River), or Map3k12 F/F; Advillin-Cre-/- (WT), or Map3k12 F/F; Advillin-Cre+/- (DLK KO) mice. WT and DLK KO mice were age-sex matched. Neurons were prepared as previously described . Brieflyeviously . On DIV4For detection of Wnd protein levels in larva nerve cords, the whole brains were carefully dissected from third instar larva. The two brain lobes were removed before they were frozen in liquid nitrogen and processed for western blotting (25 nerve cords per lane).For western blots using HEK293 cells or cortical neurons, cells are lysed by incubating on ice for 10 min with RIPA buffer  supplemented with Complete protease inhibitor cocktail (Roche) and PhosSTOP phosphatase inhibitor (Roche). Protein concentrations were measured by BCA assay kit (Thermo Scientific).Equal amount of protein samples were loaded on each lane of NuPAGE 4–12% Bis-Tris gels (Invitrogen) and subject to electrophoresis separation in MOPS buffer (Invitrogen). Blots were visualized with SuperSignal chemiluminescent substrate (Thermo Scientific) and exposure to either film or ChemiDoc (Bio-Rad). Bands intensities were determined using software ImageJ (NIH) using the gel analysis plug-in.The following antibodies were used for Western blotting: anti-Wnd A3-1,2 at 1:700 ; anti-DLS302 antibodies were raised by immunization of rabbits with the peptide KELSDKpSTKMpSFAGTV and affinity purified before use at dilution 1:100. While the antibodies were raised against a dually phosphorylated peptide , no difference in reactivity was noticed for DLK S298A mutants (data not shown).Anti-phospho-DLKForskolin  was applied to either HEK293 cells or cortical neurons at the final concentration of 30 µM for 6 hr. H-89  was used at the final concentration of 5 µM for cortical neurons for 6 hr. JNK inhibitor VIII  was used at the final concentration of 10 µM on HEK293 cells for 6 hr. Torin1  or Rapamycin (LC laboratories) was applied to the cells at 1 µM  for 2 hr before harvest.2 (NEB) and either 1000 U lambda protein phosphatase (NEB) or equal amount of glycerol (control) in RIPA buffer for 30 min at 30°C. Beads were then removed from the reaction buffer and Flag-DLK was eluted by boiling in SDS sample buffer for 10 min. Equal amount of samples were analyzed by western blot.24 hr after transfection of the given constructs, HEK293 cells in a 6 cm dish were washed by ice-cold PBS and harvested in ice-cold RIPA buffer supplemented with EDTA-free protease inhibitor (Roche). The cell lysates were incubated on ice for 30 min and centrifuged at 14,000 rpm at 4°C for 10 min. The soluble lysate was incubated with 3 µg anti-Flag antibody pre-bound with 15 µl Dynabeads Protein G (Novex) for 1 hr at 4°C. After removal of supernatants the beads were washed 3 times with RIPA buffer, and then incubated with 1X PMP buffer (NEB), 1X MnCl2, 0.01% Triton X-100, 2 mM DTT, and 30 µM ATP). 10,000 U recombinant human full length PKA catalytic subunit alpha (NED Millipore) was added to one of the tubes, while the control tube were added with glycerol. Both tubes were incubated at 30°C for 90 min. Flag-DLK was eluted from beads by boiling in the SDS sample buffer. Equal amounts of samples were analyzed by western blotting with anti-DLK and anti-pDLKS302 antibodies.HEK293 cells transfected with Flag-DLK were washed by ice-cold PBS and harvested using ice-cold cell lysis buffer  and 30 µM MG132 (Sigma)). Flag-tagged DLK was immunoprecipitated from HEK293 cell lysates using anti-Flag M2 antibody (Sigma) and Protein G Dynabeads (Novex). The DLK-bound beads were then washed with 3 times with lysis buffer and incubated with 2000 U lambda protein phosphatase (NEB) for 30 min at 30°C to remove all the phosphate groups. After incubation, the beads were washed with wash buffer containing cocktail phosphatase inhibitor (Roche) and split equally into two tubes containing kinase reaction buffer  has previously been implicated in invertebrate and mammalian systems to be essential for neuronal axon regeneration. DLK is enriched in axons after axonal injury, and DLK is required for injury-induced axonal regeneration in neurons of mammals, flies and worms. However, the specific mechanism by which DLK is activated is currently unknown, but multiple signaling pathways are likely to activate axonal regeneration. The authors focus on the cAMP-PKA signaling pathway, which stimulates axonal elaboration in multiple models systems of axon growth/regeneration. Based on an evolutionarily conserved PKA phosphorylation site residing in the kinase domain of DLK, the authors hypothesized that DLK phosphorylation lies downstream of the cAMP-PKA pathway to modulate DLK-dependent axonal regeneration. Consistent with this hypothesis, the authors demonstrate that DLK is subject to direct PKA phosphorylation at the proposed PKA phosphorylation site (S302). Additionally, the authors demonstrate in HEK293 mammalian cultured cells that DLK phosphorylation is enhanced (phospho-specific DLK antibody) when the PKA signaling pathway is activated either pharmacologically or by expression of constitutively activated PKA. The authors correlate DLK phosphorylation with enhanced DLK abundance in HEK293 cells and implicate a previously described JNK feedback pathway that affects DLK ubiquitination and degradation. These observations are consistent with their well-executed in vivo experiments demonstrating #1) a genetic interaction in Drosophila between PKA and Wnd in mediating axonal regeneration after injury, #2) enhanced levels of Wnd protein and activity after axonal injury or genetically activating the PKA pathway, and #3) failure to rescue axonal regeneration by expressing the phosphorylation incapable Wnd S302A in the wnd mutant compared to WT controls.Hao et al. describe an evolutionarily conserved mechanism mediating cAMP axonal regeneration via the phosphorylation of dual leucine zipper-bearing kinase (DLK). DLK  levels are reduced.I recommend that the authors perform a few experiments to assess whether elevating cAMP levels in neurons mimics the expression of PKACA is attenuated in the hiw mutants (by Westerns OR by imaging Wnd-GFP in axons). The results of this experiment will be interesting and relevant to this study regardless of the outcome! At the very least, the authors should discuss the stabilization of Wnd by PKA in the context of their previous findings regarding Hiw.Given that PKA activation leads to a strong upregulation of Wnd levels, I wonder whether Hiw and PKA function in the same pathway. I recommend that authors examine whether the increase in Wnd levels by PKADrosophila after expression of PKACA and/or axonal injury using westerns and/or immunostaining? Not sure if the antibody works across species, so this might not be an option. If not, has the antibody been tried in forskolin treated cultured cortical or DRG neurons? Would be a very nice positive addition to support the model, though not required given how difficult phospho-antibodies can be.Is endogenous DLK/Wnd phosphorylated by PKA? The authors generated a beautiful DLK S302 phospho-specific antibody and successfully used it in transfected HEK293 cells expressing DLK. Can this antibody be used to demonstrate enhanced S302 phosphorylation of endogenous DLK/Wnd in S302A mutant exhibited decreased overall DLK abundance by enhancing DLK degradation. Consistent with these previous findings, the current authors observed PKA-induced enhancement of DLK levels are blocked by JNK inhibitors without affecting S302 phosphorylation , why do they behave differently with JNK inhibitors? The authors' statement that "The induction of DLK activation loop phosphorylation independently of its own signaling abilities places PKA firmly upstream of DLK, as an upstream regulator/activator” (Results) seems true, but what does this data mean for the proposed model . That is, DLK downstream activation of the JNK pathway was proposed to result in a feedback loop in which JNK→DLK phosphorylation results in increased DLK abundance by reducing DLK degradation. There, the DLKrylation . Intereshibitors , suggestir model  and preved model ? Finallyeriments , as theyDrosophila rescue experiments in the supplemental figures, but I'm not sure why these are not in the main text. As mentioned in #2, failure of the DLKS302A rescues to enrich DLK in axons after PKACA expression or axon injury would strengthen the authors model that PKA-dependent phosphorylation of DLK modulates its local stability to promote regeneration programs. Finally, are the DRG experiments amenable to rescue experiments with WT and DLKS302A rescue experiments?The authors placed the WT and WndS302A wnd levels in axons than cell bodies. Does this mean PKA is activated by injury in axons but not cell bodies? This should be possible to test with a PKA activity reporter, and possibly also the phospho-specific wnd antibody.Axons vs. cell body. PKA activity has much more effect on  Essential revisions:CA with regard to Wnd-dependent axonal sprouting and puckered induction. For instance, the authors could utilize loss-of-function alleles of the gene encoding the cAMP phosphodiesterase, Dunce. It will also be worthwhile to evaluate whether axon sprouting and puckered induction in dunce mutants are sensitive to Wnd knockdown. Conversely, the authors could examine if injury dependent axon sprouting and puckered induction are diminished if adenylyl cyclase (for e.g. Rutabaga or AC78C) levels are reduced.I recommend that the authors perform a few experiments to assess whether elevating cAMP levels in neurons mimics the expression of PKADunce in Drosophila motoneurons, which leads to enhanced axonal sprouting response . Note that this previous study already showed that the enhanced regeneration in phosphodiesterase mutants required dlk-1 function.In our revision we have included new data with knockdown of phosphodiesterase response . These rDrosophila motoneurons cause increased puckered expression in uninjured animals . The results of this experiment will be interesting and relevant to this study regardless of the outcome! At the very least, the authors should discuss the stabilization of Wnd by PKA in the context of their previous findings regarding Hiw.Given that PKA activation leads to a strong upregulation of Wnd levels, I wonder whether Hiw and PKA function in the same pathway. I recommend that authors examine whether the increase in Wnd levels by PKAWe agree that the relationship between Hiw and PKA is very interesting to understand, particularly since some biochemical data in other species suggest that Hiw/Pam may regulate cAMP levels by inhibiting adenylate cyclase activity  . Our data thus far suggest that the relationship is complex, so including data/experiments on this issue would be both distracting to the current story, and would require many more experiments to address the relationship properly. So instead we are following the reviewer’s suggestion to discuss the regulation of Wnd by PKA in the context of what is previously known for Hiw, (in the Discussion section).Is endogenous DLK/Wnd phosphorylated by PKA? The authors generated a beautiful DLK S302 phosphospecific antibody and successfully used it in transfected HEK 293 cells expressing DLK. Can this antibody be used to demonstrate enhanced S302 phosphorylation of endogenous DLK/Wnd in Drosophila after expression of PKACA and/or axonal injury using westerns and/or immunostaining? Not sure if the antibody works across species, so this might not be an option. If not, has the antibody been tried in forskolin treated cultured cortical or DRG neurons? Would be a very nice positive addition to support the model, though not required given how difficult phospho-antibodies can be.We have tested the pS302-DLK antibody in flies and unfortunately it does not work across species. Over the past two months, we have tried hard to get a decent signal for endogenous DLK in cultured neurons, including scaled up cultures and immunoprecipitations to enrich for the protein. This has been very challenging technically – we think that the levels of endogenous DLK are very low, and that the antibodies may also not be very sensitive. We prefer to leave this experiment out of the paper. As the phosphorylation of S302 is important for DLK to function as a kinase, our functional data combined with our biochemical data in HEK293 cells strongly suggests that S302 is indeed phosphorylated on endogenous DLK.S302A mutant exhibited decreased overall DLK abundance by enhancing DLK degradation. Consistent with these previous findings, the current authors observed PKA-induced enhancement of DLK levels are blocked by JNK inhibitors without affecting S302 phosphorylation (K185A kinase dead version of DLK remains enhanced AND phosphorylated at S302 with PKA activation even in the presence of JNK inhibitors (K185A and DLKS302A mutants lack kinase activity and fail to activate downstream effector pathways (e.g. JNK), why do they behave differently with JNK inhibitors? The authors' statement that "The induction of DLK activation loop phosphorylation independently of its own signaling abilities places PKA firmly upstream of DLK, as an upstream regulator/activator” (Results) seems true, but what does this data mean for the proposed model . That is, DLK downstream activation of the JNK pathway was proposed to result in a feedback loop in which JNK→DLK phosphorylation results in increased DLK abundance by reducing DLK degradation. There, the DLKrylation . Intereshibitors , suggesthibitors  and preved model ?We thank the reviewers for these points. We have carried out and included some additional experiments  and still have a great deal of troubleshooting to carry out in order to implement the reporter in a new preparation and verify that it is working properly. The PKA reporter would require even further validation and controls. We think this is an interesting future project, but is not practical for this current story. An important additional caution is that even if any of these suggested assays work, a negative result in our injury assays could still have the caveat that the method of detection is not sensitive enough to detect functional changes that indeed occur.We agree that it is very interesting to know whether PKA regulates DLK locally in axons, however this is currently a very technically challenging question. The phospho-DLK antibodies do not work in flies, and our recent efforts lead us to conclude that they are also intractable for IHC in mammalian neurons (due to a high degree of non-specific staining). While a FRET reporter has been used for PKA in flies , it has never been used before in our larval motoneuron preparation. We have begun to work with a cAMP FRET reporter that is more widely used in


Axitinib is a potent inhibitor of the vascular endothelial growth factor (VEGF) receptor family with clinical activity in patients with metastatic renal cell carcinoma (mRCC). Given this biochemical potency, the clinical activity of subsequent treatment with targeted therapies in patients progressing on axitinib is of interest.Patients with advanced renal cell carcinoma of any pathologic subtype treated with at least one cycle (four weeks) of axitinib followed by at least one subsequent targeted therapy were investigated in a retrospective analysis. Patient characteristics, duration of treatment and clinical outcomes were analyzed for axitinib and each subsequent line of therapy by Response Evaluation Criteria in Solid Tumors (RECIST).Twenty-five mRCC patients who received at least one approved targeted agent following axitinib were identified. Eight percent of patients achieved a partial response (one patient each to sunitinib and pazopanib) and 42 % had a best response of stable disease to the first therapy after axitinib. The estimated median duration of therapy was 4.4 months . Twelve patients received a second post-axitinib targeted therapy. Six out of 11 evaluable patients (55 %) had a best response of SD. The estimated median duration of treatment was 4.8 months .Objective responses and stable disease is observed to post-axitinib targeted therapies and prospective studies are needed for validating role of predictive biomarkers. VHL) gene mutations cause overexpression of vascular endothelial growth factor (VEGF) and increased tumor angiogenesis has led to development of multiple agents targeting this protein and its receptor.Renal cell carcinoma (RCC) is a biologically heterogeneous disease with distinct genetic and metabolic defects . Over thCurrently approved therapies for treatment of patients with mRCC include bevacizumab , a humanized monoclonal antibody that inhibits the VEGF ligand, and the multi-targeted receptor tyrosine kinase inhibitors, sunitinib, sorafenib, pazopanib and axitinib (VEGFr- TKIs) –7. Each P < 0.0001). Secondary endpoints included overall response rate (ORR), overall survival (OS), and safety and tolerability. ORR was 19.4 % (95 % Cl 15.4–23.9 %) versus 9.4 % (95 % CI 6.6–12.9 %) for axitinib and sorafenib, respectively. In the sub-group of sunitinib-refractory patients, median PFS was 4.8 months for patients treated with second-line axitinib and 3.4 months for patients treated with second-line sorafenib (P = 0.0107). In the subgroup of cytokine-refractory patients, median PFS was 12.1 months for patients treated with second-line axitinib and 6.5months for patients treated with second-line sorafenib (P < 0.0001) [The most recently Food and Drug Administration (FDA) approved agent for mRCC is axitinib, a second-generation, indazole derived molecule that binds selectively to the adenosine triphosphate (ATP)-binding intracellular domain of VEGFR-1, 2, and 3 at sub-nanomolar concentrations. The AXIS trial that led to the approval of axitinib was a phase 3, randomized controlled study comparing two VEGFr TKIs, axitinib and sorafenib, in patients whose disease progressed on initial systemic therapy . Patient 0.0001) . The lon 0.0001) . This suKnowing that axitinib is the most biochemically potent of the approved VEGFr inhibitors, and that there is possibility of cross-resistance with sequential VEGF-targeting therapy, the response to therapy after progressing on axitinib is of clinical interest.A retrospective review of patients from the Cleveland Clinic Taussig Cancer Center (CCF) and MD Anderson Cancer Center (MDACC) was thus undertaken to characterize and evaluate the response to subsequent systemic therapy in patients who had progressed on axitinib.n = 3), ongoing axitinib therapy (n = 16), lack of subsequent systemic therapy (n = 13), lost to follow up (n = 11), receiving non targeted or investigational agents post-axitinib (n = 11) or were not evaluable (n = 2). All patients were enrolled in axitinib clinical trials given that axitinib therapy pre-dated FDA approval.Patients from CCF or MDACC were identified through prospectively maintained databases and included in the study, which was approved by Cleveland Clinic and UT MD Anderson institutional review board. The initial criteria for case identification included a diagnosis of metastatic RCC of any pathologic subtype and treatment with at least one cycle (four weeks) of axitinib. Eighty-one patients treated with axitinib between November 2003 and August 2010 were initially identified; 25 patients (17 (68 %) from CCF and 8 (32 %) from MDACC) received at least one approved targeted agent following axitinib and were included in this analysis. The remaining patients were excluded due to ≤ 4 weeks on axitinib Twenty-five patients (17 (68 %) from CCF and 8 (32 %) from MDACC) received at least one approved targeted agent following axitinib and were included in this analysis. Patient characteristics at the start of axitinib were typical of an advanced RCC population and included 72 % male, median age 59 ; 96 % clear cell; 92 % prior nephrectomy; 72 % previously-treated. Patients had favorable (30 %) or intermediate (65 %) risk disease based on Heng criteria . The oven = 18) or mTOR inhibitors (n = 7). Overall, 8 % of patients achieved a partial response (one patient each to sunitinib and pazopanib) and 42 % had a best response of stable disease (nine patients to VEGF receptor inhibitors and four patients to mTOR inhibitors). The estimated median duration of subsequent therapy was 4.4 months   and in patients who had received no therapy prior to axitinib . Ten patients (55 %) had a best response of stable disease in the group of patients who had received axitinib as second-line or later therapy as compared to three (42 %) in the latter group. One patient achieved partial response in both groups Table .Table 3Pn = 3) or mTOR inhibitors (n = 8). No partial responses were observed. However, six of 11 evaluable patients (55 %) had a best response of SD to mTOR inhibitors. The estimated median duration of treatment was 4.8 months .Eleven of twelve patients who received a second post-axitinib targeted therapy were treated with VEGF receptor inhibitors  achieved a PR on their next systemic therapy, 7 (54 %) had a best response of stable disease, and 4 (31 %) progressed.p = 0.04). All of these seven patients had clear cell RCC without sarcomatoid features and six of these seven patients received sunitinib or pazopanib as first subsequent therapy and only one received temsirolimus.Further, Fig. The standard of care in metastatic RCC is the empiric and sequential use of systemic therapies, most of which target VEGF. Previous retrospective and prospective evidence points to lack of complete cross-resistance to sequential VEGF-targeting therapies , 15, altIt has been hypothesized that residual VEGF signaling after VEGF-targeted therapy could account for the activity of VEGF-targeted agents in this setting. Given the increased biochemical potency of axitinib, it was hypothesized that therapy  after axitinib treatment may not result in clinical benefit. Our retrospective data suggests that clinical effect is seen in patients with metastatic RCC who receive systemic therapy after axitinib, not only as second but also as third line. Two thirds of our patients had received systemic therapy prior to axitinib and we still observed objective responses on subsequent therapies, median duration of therapy was over four months and seven patients remained on first subsequent therapy longer than axitinib. This may be explained by the therapeutic effect of different target engagement by the various VEGF multikinase inhibitors , 17. OurThere are limitations to this analysis. This is a retrospective review of a small number of patients at two specialized institutions with inability to overcome selection bias. Individual treating physicians at each institute did the tumor assessment, thus limiting the reliability and consistency of the percent changes in tumor burden and the assessment of objective response.Further prospective trials are evaluating second-line treatment with approved or investigational agents in patients with mRCC who were refractory to previous treatment with a targeted agent. Other trials are evaluating the optimal sequence of targeted agents in treatment-naive patients with mRCC. Results of these trials may give us some insight into efficacy of sequenced VEGFR – TKIs and cross – resistance in metastatic renal cell carcinoma.Objective responses and stable disease is observed with post-axitinib targeted therapy, although efficacy is generally less than that seen with axitinib. Prospective studies will help us understand if prior response or resistance to axitinib predicts for clinical benefit to subsequent therapy. Variable patient response to drugs in the same class warrants prospective studies to validate role of predictive biomarkers in guiding individualized therapies and improving outcomes.Waived.Non-applicable.The dataset supporting the conclusions of this article is available at request from the corresponding author.