DLK1 and/or RTL1. The up-regulation of PARK7 was identified in hypertrophied muscles by microarray analysis and further validated by quantitative PCR. The expression of PARK7 in hypertrophied muscle of callipyge lambs was confirmed to be up-regulated at the protein level. PARK7 was previously identified to positively regulate PI3K/AKT pathway by suppressing the phosphatase activity of PTEN in mouse fibroblasts. The purpose of this study was to investigate the effects of PARK7 in muscle growth and protein accretion in response to IGF1. Primary myoblasts isolated from Park7 (+/+) and Park7 (−/−) mice were used to examine the effect of differential expression of Park7. The Park7 (+/+) myotubes had significantly larger diameters and more total sarcomeric myosin expression than Park7 (−/−) myotubes. IGF1 treatment increased the mRNA abundance of Myh4, Myh7 and Myh8 between 20-40% in Park7 (+/+) myotubes relative to Park7 (−/−). The level of AKT phosphorylation was increased in Park7 (+/+) myotubes at all levels of IGF1 supplementation. After removal of IGF1, the Park7 (+/+) myotubes maintained higher AKT phosphorylation through 3 hours. PARK7 positively regulates the PI3K/AKT pathway by inhibition of PTEN phosphatase activity in skeletal muscle. The increased PARK7 expression can increase protein synthesis and result in myotube hypertrophy. These results support the hypothesis that elevated expression of PARK7 in callipyge muscle would increase levels of AKT activity to cause hypertrophy in response to the normal IGF1 signaling in rapidly growing lambs. Increasing expression of PARK7 could be a novel mechanism to increase protein accretion and muscle growth in livestock or help improve muscle mass with disease or aging.Callipyge sheep exhibit postnatal muscle hypertrophy due to the up-regulation of  DLK1-DIO3 imprinted gene cluster Delta-like 1 (DLK1) and Retrotransposon-like 1 (RTL1) in hypertrophied muscles Dlk1 exhibited increased muscle mass and myofiber diameter Dlk1 in the mouse resulted in reduced body weight and skeletal muscle mass due to reductions in myofiber numbers Dlk1 in culture was shown to inhibit myoblast proliferation and enhance myotube differentiation Callipyge sheep exhibit postnatal muscle hypertrophy, with higher rates of protein accretion and lower rates of fat deposition compared to normal sheep semimembranosus muscle of callipyge and normal lambs Parkinson Protein 7  expression was up-regulated in hypertrophied muscles. PARK7 encodes a ubiquitously expressed, highly conserved protein that has been shown to be involved in diverse biological processes including oxidative stress response, transcriptional regulation and cell survival modulation. A mutation causing a loss of function of PARK7 was found to be responsible for a recessive early-onset form of Parkinson's disease Park7 was originally identified as an oncogene that transforms NIH3T3 cells in cooperation with the activated Ras gene Microarray analysis of gene expression identified 199 genes that were differentially expressed in Igf1 caused muscle hypertrophy in mice Igf1 receptor impaired muscle growth due to reduced muscle fiber number and size Akt in muscle fibers results in significantly larger fiber size Akt in muscle exhibit rapid skeletal muscle hypertrophy Akt in mice leads to a smaller body size and shorter life span The PI3K/AKT pathway is known to positively regulate muscle growth PARK7 in hyper-muscular animals suggests a hypothesis that increasing PARK7 gene expression can increase muscle growth and protein accretion. Differential expression of PARK7 was examined in this study using Park7 (+/+) and Park7 (−/−) mouse models to investigate the effects of Park7 gene expression in muscle growth and protein accretion in response to IGF1.Though extensively studied, a role for PARK7 in muscle growth had not been reported until recently. In addition to callipyge lambs Semimembranosus and supraspinatus muscle samples were collected from 3 callipyge and 3 normal lambs at 30–35 days of age when muscle hypertrophy is detectable by muscle mass. The semimembranosus from the pelvic limb undergoes the largest magnitude of hypertrophy and supraspinatus from the thoracic limb does not become hypertrophied semimembranosus of callipyge (+/C) relative to normal (+/+) lambs. There were no differences between genotypes in the supraspinatus. Elevated levels of DLK1 protein were also confirmed in the callipyge semimembranosus. No detectable DLK1 expression was found in the muscles of normal animals and non-hypertrophied muscles, along with a reduced PARK7 expression in these same animals and tissues.Park7 (−/−) mouse was obtained from Jackson Laboratory and back crossed with C57BL6 mice to generate Park7 (+/+), (+/−) and (−/−) littermate mice. Live animal body weights were monitored and collected from 3 – 6 weeks of age. Mice were euthanized at 6 weeks and carcass (muscle and skeleton) weight and internal organs weights were collected. The phenotypic data from male and female mice was analyzed by linear regression separately to account for gender differences. No significant differences were found between the three genotypes for analysis of live weight by age, carcass weight by live weight, or organ weight by live weight analysis in males or females  mice (Park7 (+/+) and Park7 (+/−) animals, but no PARK7 protein was detectable in Park7 (−/−) muscles.The expression of PARK7 protein in the /−) mice . The expPark7 (+/+) and Park7 (−/−) animals and fused into myotubes in order to model the effect of Park7 levels on growth and myosin expression. The relative sizes of myotubes were determined after 72 hours of differentiation in fusion media (Park7 (−/−) and (+/+) cultures (Park7 (+/+) myotubes were significantly larger  than Park7 (−/−) myotubes  myotubes having about 10% higher myosin expression than Park7 (−/−) myotubes, regardless of IGF1 treatment (Park7 (+/+) myotubes (25 ng/mL) relative to Park7 (−/−) myotubes (100 ng/mL).Primary myoblasts were isolated from on media . The fuscultures . Park7  in Park7 (+/+) myotubes, relative to Park7 (−/−) myotubes  and 30% (p = 0.0001) respectively in Park7 (+/+) myotubes. IGF1 also stimulated Myh7 and Myh8 mRNA expression, but only at the lower concentration (10 ng/mL) with 23% increase in Myh7 and 10% increase in Myh8 respectively, relative to the treatment without IGF1. At the high concentration of IGF1 (50 ng/mL), Myh7 expression dropped 12% and Myh8 expression dropped 16% compared to the treatment without IGF1. Myh3 was the only isoform measured that showed no genotype effect or IGF1 effect. There was no significant genotype by IGF1 interactions for the four myosin isoforms.Quantitative PCR was used to detect the effect of myotubes . The mRNmyotubes . The addPark7 regulation on myotube size and myosin gene expression, a series of experiments were conducted to assess the activation of AKT by phosphorylation at S473 in presence or absence of Park7. It has been demonstrated that PARK7 can suppress PTEN phosphatase activity, which would have the effect of increasing receptor tyrosine kinase signaling through AKT Park7 genotype on the phosphorylation of AKT. The level of phosphorylation of AKT was elevated in Park7 (+/+) myotubes relative to Park7 (−/−) myotubes in 5% horse serum differentiation media without added IGF1 (Park7 (+/+) myotubes at all levels of added IGF1. To investigate if the activity of PARK7 could be mimicked by inhibition of PTEN function, VO-OHpic trihydrate, a known effective PTEN inhibitor Park7 (−/−) myotubes and eliminated the differences between the two Park7 genotypes (Park7 (+/+) and Park7 (−/−) myotubes.In order to explore the mechanism of ded IGF1 . The phoenotypes . There wenotypes  or RNA ePark7 (+/+) myotubes, the timing and duration of the elevated phospho-AKT was tested. Myotubes were treated with IGF1 for 15 minutes and protein was collected at several time points up to 12 hours. Western blots showed increased phosphorylation of AKT in Park7 (+/+) myotubes (Park7 (+/+) myotubes relative to Park7 (−/−) myotubes with no added IGF1 (Park7 (+/+) myotubes reached its maximum at 45 minutes at 3-fold higher signal intensity than Park7 (−/−) myotubes after removal of IGF1. Park7 (−/−) myotubes had maximum phosphorylation at 90 minutes and the decreases in phosphorylation over time were similar with Park7 (+/+) myotubes, generally maintaining approximately 20% higher signal intensity through 12 hours.Since increased phosphorylation of AKT was found in myotubes . Quantitded IGF1 . The levOverall, the present results indicate that differential expression of the PARK7 protein can result in increased myosin gene expression, protein accretion and myotube growth through an altered response to IGF1/AKT signaling. This study focused on Park7′s effect on AKT phosphorylation due to its prominent role in regulating muscle growth, other reported biological activities in oxidative stress, anti-apoptosis and several regulatory pathways Park7 (−/−) mice are viable and lack obvious developmental abnormalities. No differences were found between Park7 (+/+) and (−/−) animals in vivo when comparing body weight, carcass weight and internal organ weight which indicate that Park7 is not a dominant regulator of live animal growth. These results were consistent with other findings, which indicated no significant differences in body weights between the two genotypes from birth to 12 months The PARK7 expression in callipyge sheep at the transcriptional level in vitro cell culture model was used in this study to investigate the effects of differential expression of Park7 on myogenesis. The in vitro comparison of myotube size showed Park7 (+/+) myotubes had a bigger size and higher myosin expression than Park7 (−/−) myotubes, but no significant change in the fusion index, which indicates Park7 expression affects myotube hypertrophy (increased myofiber diameter), not hyperplasia (increased myofiber number). These results support the proposed models that elevated PARK7 expression is part of the physiological mechanism for increased muscle growth in callipyge lambs and myostatin null mice and cattle A previous study showed elevated Park7 (+/+) myotubes had increased phosphorylation of AKT compared to Park7 (−/−) after IGF1 stimulation, which induced significantly more total sarcomeric protein accretion. Notably, this increase of protein accretion reached saturation at lower IGF1 concentrations in Park7 (+/+) relative to Park7 (−/−) myotubes. The expression of Myh4 showed the largest increases although Myh7 and Myh8 mRNA expression were also elevated in Park7 (+/+) myotubes indicating that both myosin transcription and translation were affected by Park7 expression. Muscle contractile characteristics partially depend on the expression of myosin heavy chain isoforms. The hypertrophied muscles in callipyge sheep have an increased size of fast twitch glycolytic myofibers along with increased MYH4 expression and a decreased frequency of oxidative fibers Akt had been reported to promote myofiber growth with no effect on fiber specification in regenerating skeletal muscle Akt transgene in mice resulted in the hypertrophy of glycolytic myofibers, but not oxidative myofibers Myh4 and the enhanced activation of AKT in the presence of Park7. Interestingly, double-muscled cattle, in which the expression of Park7 gene is also elevated, have an increased proportion of fast-twitch glycolytic fibers as well The PI3K/AKT pathway is one of the primary targets of IGF1 signaling and has been well characterized for its positive regulation on muscle growth Park7 (+/+) and Park7 (−/−) myotubes was eliminated when treated with higher concentrations of PTEN inhibitor, but the total amount of PTEN had not been changed, suggesting PARK7 acts through inhibition of PTEN phosphatase activity, not total PTEN expression. This result was consistent with the findings of Kim et al. Park7 transfected NIH3T3 cells, but no change was detected; indicating the inhibition of PTEN phosphatase activity by PARK7 is not affected by phosphorylation of PTEN. The mechanism by which PARK7 inhibits PTEN phosphatase activity remains unclear. Some studies have proposed that PARK7 can directly bind to PTEN to confer an inactive conformation by forming a disulfide bond between C71and C124 of PTEN upon oxidative stress The differences in phosphorylation of AKT in Park7 in the regulation of skeletal muscle growth. Park7 expression can modulate IGF1/AKT signaling, increase myosin gene expression and protein synthesis and increase myotube size. These results support the hypothesis that elevated expression of PARK7 is part of the physiological mechanism for muscle hypertrophy in callipyge lambs and increased muscle mass in double muscled cattle. Elevated expression of PARK7 in the muscles of callipyge lambs could lead to increased muscle growth in response to the normal IGF1 signaling present in young growing lambs. The prominent increase in Myh4 gene expression in myotubes was also consistent with changes in myosin fiber types in callipyge muscle. DLK1 is one of the causative genes for the callipyge phenotype due to the inheritance of the mutation PARK7 expression could be considered a downstream response to DLK1 signaling. Identifying how PARK7 gene expression is regulated in callipyge induced muscle hypertrophy could provide new mechanisms to increase livestock muscle growth and production efficiency or prevent the loss of muscle mass due to disease or aging.The present study provides a novel insight into a role for Sheep were maintained at the Purdue Animal Sciences Research and Education Center under standard agricultural housing and management. Lambs were euthanized for tissue sampling using three lethal procedures of captive bolt stunning, exsanguination and pneumothorax. This procedure was approved by Purdue Animal Care and Use Committee (protocol #1112000493). All mice were fed with standard chow food and water, and housed at 22–23°C with nesting material with 12 hour light/dark cycle. Cage bedding was changed weekly by professional personnel. Mice were euthanized by cervical dislocation and pneumothorax for dissection of muscle tissue for the growth trial and a source of primary satellite cells. Mice housing and euthanasia was performed according to protocols approved by the Purdue Animal Care and Use Committee (protocol #1112000440).Park7 locus inactivated by gene targeting [Park (−/−)] are available from JAX mice® . The initial C57BL6 Park7 (−/−) mouse was crossed with C57BL6 Park7 wild type (+/+) to produce heterozygous (+/−) Park7 mice for breeders. Matings of heterozygous Park7 mice were conducted to generate litters of mice with all three possible Park7 genotypes (+/+) (+/−) and (−/−). Twenty females with 4 Park7 (+/+), 12 (+/−) and 4 (−/−) animals and twenty-five males with 4 Park7 (+/+), 11 (+/−) and 10 (−/−) from seven litters were used to analyze live animal growth. Live animal weights were measured at 6 weeks of age to the nearest 0.01 g. The carcass and internal organ weights were measured on a total of 18 males (4 Park7 (+/+), 6 (+/−) and 8 (−/−) animals) and 16 females (4 Park7 (+/+), 8 (+/−) and 4 (−/−) animals) to the nearest 0.0001 g. Carcass weights were measured by removal of head, tail, skin and fur, internal organs and visceral fat of the mouse. The general linear model and genotype specific models for regression analysis are given in Supplementary Mice with the Rplp38) was used as the housekeeping gene control for TΔC calculation [TΔC  =  (TC of the target gene) – (average TC of housekeeping genes)]. Primer sequences were listed in −ΔΔCT methodsT  =  (TΔC of the treatment sample) – (TΔC of control treatment samples) with no added IGF1 as control treatment and normalized to 1.RNA from cultured cells was extracted and purified using Nucleo Spin RNA II columns . First strand cDNA was synthesized from RNA using random hexamer and oligo dT priming and moloney murine leukemia virus reverse transcriptase  to produce random primed cDNA. Quantitative PCR measurements were performed using the SA Bioscience SYBR Green Supermix  reagents on an iCycler Real-Time PCR Detection System . Each reaction was carried out in 15 μl reaction volumes of SA Bioscience SYBR Green Supermix with 5 pM of each primer and diluted first-strand cDNA. All cDNA samples were measured in duplicate. Ribosomal protein large protein 38 , minced and digested in type I collagenase and dispase B mixture . The digested muscle pulp was then filtered through a 100 μm filter  to remove large muscle fiber debris and then plated on culture dishes. In 3 days, the cells together with the small debris were collected and digested with 0.025% trypsin for 10 minutes with agitation. Cells were seeded in growth media  on rat-tail collagen (Roche Applied Science)-coated dishes with preplating on non-coated plates for 45 minutes to deplete fibroblasts as previous described Semimembranosus and supraspinatus muscles were extracted from callipyge and normal lambs at 30–35 days. Samples were frozen immediately after dissection and homogenized in RIPA buffer . Samples were centrifuged at 10,000 × g for 10 minutes at 4°C. The supernatant was transferred into ultracentrifuge tubes and centrifuged at 50,000 × g for 15 minutes at 4°C.Proteins were quantified by BCA assay  per manufacturer's recommendation. A total of 60 μg of protein from each muscle sample was diluted in SDS loading buffer, denatured by heating for 5 minutes in boiling water and loaded in the 6%–18% gradient SDS-PAGE gel. The gel was transferred to PVDF membrane by semi-dry transfer using 20% methanol, 40 mM glycine, 0.15% ethanolamine, 0.2% SDS at 180 mA for 1 h 45 min. PVDF membrane was blocked overnight with agitation in 5% sodium caseinate in PBS with 0.1% Tween 20. Antibodies used for blotting were goat polyclonal anti-Park7 , anti-bovine DLK1 , mouse monoclonal anti-tubulin  and secondary antibodies were rabbit anti-goat antibody conjugated HRP, goat anti-rabbit conjugated HRP, or goat anti-mouse conjugated HRP respectively . The blots were incubated in primary antibody 2 hours at room temperature with a dilution of 1∶1000. After washing 3 times with DPBS, the blots were incubated with secondary antibody with dilution of 1∶20000. Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific Pierce) was used to detect HRP on immunoblots. The blots were visualized using Gel Logic 2200 PRO (Carestream Molecular Imaging System) with exposure times of 2–6 minutes. Carestream Molecular Imaging Software was used to quantify the chemiluminescent signal intensity in arbitrary units. The signal intensities for PARK7 were compared using a t-test without normalization.Primary myotubes were fixed in 4% formaldehyde and permeabilized in 0.5% Triton X-100. Cells were blocked in 1% bovine serum albumin (BSA) in PBS for 1 hour at room temperature and stained with 1∶1000 anti-myosin heavy chain antibody  for 2 hours. Cells were washed in PBS, and then incubated in 1∶1000 Alexa Fluor 594-conjugated donkey anti-mouse IgG secondary antibodies (Life Technologies) for 1 hour. Finally, cells were mounted with Prolong Gold antifade reagent with DAPI (Life Technologies). The IP Lab software  and Leica DM6000 microscope were used to acquire pictures.The fusion index was defined as the ratio of nuclei within the myotubes (>2 nuclei) to the total number of nuclei. The average number of nuclei per myotube was determined by counting over 500 nuclei from randomly chosen MHC-positive cells. Myotube diameters were measured at 5 points along the entire tube. A total of 150 myotubes were examined for each genotype in the experiment.Primary myoblasts were put into Matrigel coated-96-well plate at a high density with four replicate wells for each genotype by IGF1 treatment combination. The myoblasts were fused into myotubes for 3 days in fusion media with different concentration  of IGF1. The mature myotubes were fixed in 4% formaldehyde in PBS for 5 minutes at room temperature, washed 3 times with PBS and permeabilized with 0.5% TritonX100 for 5 minutes. After washing 3 times with PBS, the cells were blocked for 1 hour in 1% BSA in PBS. Cells were incubated with anti-myosin heavy chain antibody  diluted in 0.1% BSA overnight at 4°C, and washed 5 times in PBS. Secondary antibody  were diluted in 0.1% BSA and filtered through 0.2 μM filters. Cells were incubated in secondary antibody for 1 hour and washed 5 times with PBS. The fluorescence signals were read on Tecan Genios Pro  plate reader.Cultured primary myoblasts or myotubes were used to examine AKT phosphorylation. Different concentrations of IGF1 were added into culture for 15 minutes to induce phosphorylation of AKT. For the PTEN inhibitor experiment, different concentrations  of VO-OHpic trihydrate (Sigma-Aldrich) were added into culture and incubated with myotubes for 15 minutes after removal of IGF1. For testing sustained activity of phosphorylation of AKT, IGF1 was removed after 15 minutes incubation with myotubes. The cells were incubated in normal differentiation conditions and cell lysis was collected at the indicated time points . Cells attached to plates were rinsed with ice-cold DPBS, and scraped on ice in cell lysis buffer  and 1 X complete protease inhibitor cocktail. Cells were collected in 1.5 mL microcentrifuge tubes, rotated for 45 minutes, and centrifuged at 12,000 × g for 10 minutes at 4°C to remove insoluble debris.A total of 45 μg of protein from each cell sample was diluted in SDS loading buffer, and loaded in the 6%–18% gradient SDS-PAGE gel. The gel electrophoresis and transfer to PVDF membranes were the same as described above. The PVDF membranes were blocked in 5% non-fat milk in Tris-Buffered Saline (TBS) with 0.1% Tween 20 for 3 hours. Antibodies used for blotting were rabbit polyclonal anti-AKT , anti-phospho-AKT (Ser473) , anti-PTEN . The primary antibody was incubated at 4°C overnight at a dilution of 1∶1000. The goat anti-rabbit conjugated HRP was used as secondary antibody and was incubated for 1 hour at 4°C at a dilution of 1∶20000. The visualization of blots was the same as described above.Park7 genotypes used in the mouse growth trials are provided in Supplementary All of the statistical analysis presented in this work was conducted using SAS 9.2 software  with assistance from the Statistical Consulting Service at Purdue University. The linear regression models for pairwise comparisons of the three Figure S1Analysis on live body weight by age in Park7 (+/+), (+/-) and (-/-) mice. Twenty females with 4 Park7 (+/+), 12 (+/-) and 4 (-/-) animals and twenty-five males with 4 Park7 (+/+), 11 (+/-) and 10 (-/-) were examined to analyze the live animal growth. Animals live body weights were collected from 3 weeks to 6 weeks old. Live body weights were regressed on age. Equations were given for each genotype. There were no genotype effect on the slopes and intercepts of any regression lines in both male and female.(DOCX)Click here for additional data file.Table S1Regression analysis of growth in Park7 (+/+), (+/-) and (-/-) mice.(DOCX)Click here for additional data file.Table S2Quantitative PCR primers.(DOCX)Click here for additional data file.Checklist S1(DOC)Click here for additional data file.


Virus isolates from all patients had the H275Y mutation in the neuraminidase gene. One isolate had the I117M mutation. Of the 11 patients, 6 were  The Korea Centers for Disease Control and Prevention asked clinicians to report all patients with suspected cases of drug-resistant pandemic (H1N1) 2009 when these patients showed treatment failure for oseltamivir or had unusually prolonged viral shedding (defined as >5 days after the onset of symptoms)  2009 virus infection. A total of 225 patients (0.03%) died of disease related to pandemic (H1N1) 2009. During this period, physicians in local clinics and tertiary hospitals sent specimens from 67 patients who were suspected of having drug-resistant pandemic (H1N1) 2009 to the Korea Centers for Disease Control; 11 patients (16%) had drug-resistant virus . After cTo investigate whether virus contained genetic markers associated with resistance to antiviral drugs, a conventional genotyping assay (sequencing) was performed, and neuraminidase (NA) and matrix 2 genes were sequenced. All 11 patients had virus with a histidine-to-tyrosine mutation at residue 275 of the NA protein (H275Y); 1 patient also had virus with an I117M mutation . Detaile<59 months of age and 5 had underlying immunosuppressive conditions; only 1 patient was immunocompetent and >59 months of age  2009 virus before the 11 patients were infected. Influenza-like illnesses developed in the 11 patients a median of 2 days (range 1–7 days) after the 8 contact persons were confirmed as having pandemic (H1N1) 2009. Five of the 8 contact patients were children; none had an immunosuppressive condition or were given oseltamivir chemoprophylaxis before illness; and 7 of 8 were All 11 patients were initially given the usual dose of oseltamivir (75 mg 2×/day in adults). After detection of oseltamivir resistance, treatment regimens were as follows: 3 patients were given high-dose oseltamivir (150 mg 2×/day in adults), 3 patients were given combination therapy ; 3 patients were given zanamivir nasally; and 2 patients continued to receive oseltamivir.Seven of the 11 patients had complications during treatment: 6 had viral or secondary bacterial pneumonia and 1 had acute respiratory distress syndrome . Three p<59 months of age) or immunocompromised. All isolates had the H275Y mutation in the NA protein, and 1 isolate also had the I117M mutation in the same protein.Our nationwide surveillance of drug-resistant pandemic (H1N1) 2009 in South Korea indicated that most patients were children  2009, particularly if there is treatment failure with oseltamivir or prolonged viral shedding is evident.Our finding that oseltamivir resistance developed in immunocompromised patients is consistent with those of recent case reports that described development of oseltamivir resistance in immunosuppressed patients receiving this drug  2009. Clinical trials have reported oseltamivir resistance in <5.5% of children with seasonal influenza  2009 in South Korea. However, our study was based on the nationwide surveillance system for drug resistance among patients with suspected oseltamivir treatment failures, which is different from other surveillance systems, in which all isolated viruses are tested. Thus, we cannot report the prevalence of drug-resistant pandemic (H1N1) 2009 and risk factors for drug-resistant virus infection in South Korea. Further studies are needed to monitor oseltamivir resistance, especially in immunosuppressed or pediatric patients when treatment failure with oseltamivir or prolonged viral shedding occur. Additional studies are also needed to determine proper oseltamivir dosing for children.


The key steps involved a Wittig reaction between aldehyde 5 and the ylide derived from phosphonium salt 6 to give enyne 17 and condensation of the same ylide with aldehyde 7 to afford enyne 11. Desilylation of 11 followed by hydrozirconation and iodination gave the vinyl iodide 4 and Sonogashira coupling between this compound and enyne 3 provided alkyne 18. Acetonide deprotection, partial reduction and ester hydrolysis then gave resolvin D2 (1).The total synthesis of the endogenous inflammation resolving eicosanoid resolvin D2 ( In 2002, a new family of endogenously generated lipid mediators involved in the resolution of inflammation named the resolvins (resolution phase interaction products) were identified by Serhan and co-workers in the inflammatory exudates of aspirin treated mice –3. The rd D1 (2)  derived 1) prevents the adherence of polymorphonuclear leukocytes (PMN) to the blood vessel wall by promoting the shedding of L-selectin from PMNs thus preventing binding to E selectin on the endothelial cell lining of the blood vessel [1) promotes the influx and phagocytic activity of macrophages, facilitating clearance of dead cells and microbial pathogens, allowing resolution of inflammation and infection [2) [RvD2 (d vessel . Furthernfection . This sunfection . Resolvition [2)  has beention [2) . Whilst S,16R,17S)-RvD2 (1) was communicated by Spur in 2004 [1 for a biological study [1) for biological evaluation but without detailed synthetic sequence to follow and given the very high cost [1 we elected to develop an alternative route to provide this important compound and analogues for further biological evaluation. Herein we describe a synthesis of RvD2 (1) which includes full experimental details so that other researchers can produce useful amounts of this important compound as well as novel isomers.The first total synthesis of  is shown in 1 could be secured via a Sonogashira coupling to form the C11–C12 bond followed by partial reduction. This is similar to the endgame of the reported syntheses of 1 [1 could arise from enyne 3 and vinyl iodide 4 which could both be obtained by Wittig extension using the common linchpin phosphorus ylide derived from phosphonium salt 6 [5 and 7 [A retrosynthetic analysis of RvD2 . Removal of the TIPS group with TBAF gave terminal alkyne 12. Alkyne 12 then underwent smooth hydrozirconation utilizing the procedure reported by Negishi [2HCl is generated in situ by reduction of ZrCp2Cl2 with DIBALH in THF. Iodinolysis of the zirconium species then gave the diene iodide 4 in good yield. Selectivity for this process was excellent with only a trace of the regioisomer formed.Treatment of the salt tography . The min Negishi  were ZrC5 began with the production of the known bis-TES ether [13 [t-BuOH/H2O to give diol 14 in reasonable yield. The enantioselectivity and absolute configuration of the secondary alcohol was determined by conversion of diol into the bis-(S)-Mosher ester [1H MMR spectrum indicated the e.r. was 93.7:6.3 and Mosher analysis  is shown in 3 and 4 was very efficient giving the alkyne 18 in good yield. Removal of the acetonide was effected by treatment with HCl in MeOH to give the known triol 19 [1 were similar with those previously reported [20 in 76% yield. A large excess of the Zn reagent was required to obtain a good conversion of 19 into 20. The 1H NMR spectrum (CDCl3 solvent) of RvD2 methyl ester (20) compared well to that reported [1).The completion of the synthesis of RvD2  had physical data identical to that reported [D −17.5° ). In our hands, both RvD2 methyl ester (20) and RvD2 (1) itself were highly unstable, especially to acid. Prolonged standing in CDCl3 or CD3CN solution or exposure to light caused rapid decomposition and so NMR spectra were obtained quickly. We found that RvD2 methyl ester (20) was not very soluble in CD3CN so spectra were best run in CDCl3 that was filtered through basic alumina immediately prior to use. Spectra for RvD2 (1) were always measured for CD3CN. Even with short exposure to the solvent, we still observed degradation to unidentified compounds. Samples of RvD2 (1) can be stored in EtOH or frozen in DMSO solution but should be used immediately upon thawing. Alternatively, the triol 19 proved more stable than both RvD2 methyl ester (20) and RvD2 (1) and can be stored for longer periods prior to conversion to 1 which should be used rapidly for biological assessment to avoid degradation.The synthetic RvD2  has been completed using a common linchpin Wittig reaction. Using this approach, we were able to prepare sufficient quantities of this important inflammation resolving compound for further biological evaluation.The total synthesis of RvD2 .


Amelogenesis imperfecta (AI) is a group of inherited conditions featuring isolated enamel malformations. About 5% of AI cases show an X-linked pattern of inheritance, which are caused by mutations in AMELX. In humans there are two, non-allelic amelogenin genes: AMELX (Xp22.3) and AMELY (Yp11.2). About 90% of amelogenin expression is from AMELX, which is nested within intron 1 of the gene encoding Rho GTPase activating protein 6 (ARHGAP6). We recruited two AI families and determined that their disease-causing mutations were partial deletions in ARHGAP6 that completely deleted AMELX. Affected males in both families had a distinctive enamel phenotype resembling “snow-capped” teeth. The 96,240 bp deletion in family 1 was confined to intron 1 of ARHGAP6 (g.302534_398773del96240), but removed alternative ARHGAP6 promoters 1c and 1d. Analyses of developing teeth in mice showed that ARHGAP6 is not expressed from these promoters in ameloblasts. The 52,654 bp deletion in family 2 (g.363924_416577del52654insA) removed ARHGAP6 promoter 1d and exon 2, precluding normal expression of ARHGAP6. The male proband of family 2 had slightly thinner enamel with greater surface roughness, but exhibited the same pattern of enamel malformations characteristic of males in family 1, which themselves showed minor variations in their enamel phenotypes. We conclude that the enamel defects in both families were caused by amelogenin insufficiency, that deletion of AMELX results in males with a characteristic snow-capped enamel phenotype, and failed ARHGAP6 expression did not appreciably alter the severity of enamel defects when AMELX was absent. ENAM, 4q21) FAM83H, 8q24.3) MMP20, 11q22.3-q23) KLK4, 19q13.4) WDR72, 15q21.3) AMELX, Xp22.31-p22.1) AMELX and AMELY), but the gene on the Y-chromosome (Yp11.2) is expressed at low levels AMELX and AMELY do not undergo homologous recombination, have diverged AMELY deletions. DNA repeat sequences on the Y chromosome apparently create a structural instability that leads to recurrent 3–4 Mb deletions inclusive of AMELY, PRKY and TBL1Y, and sometimes PCDH11Y, with no apparent negative selection AMELY deletions varies by population and is generally low, perhaps about 0.6% AMELY deletions have been reported for “normal populations”, with the extrapolation that the tooth enamel must therefore be normal in these people. To our knowledge no oral photos or radiographs have been published of person lacking AMELY, although two individuals with AMELY deletions were specifically reported to have normal teeth Non-syndromic amelogenesis imperfecta (AI) is a heterogeneous collection of inherited defects in dental enamel formation that includes a multiplicity of enamel phenotypes, patterns of inheritance, and causative genes AMEL) belongs to the secretory calcium-binding phosphoprotein (SCPP) family of genes AMEL, all human SCPP genes are clustered on chromosome 4, which include 5 genes encoding acidic proteins and 16 genes encoding proteins enriched proline and glutamine Amel apparently arose in the proline-glutamine-rich gene cluster and later transposed into intron 1 of Arhgap6 (Rho GTPase activating protein 6) in the opposite orientation, resulting in a nested gene structure. This may have occurred in fish, as Amel is already nested in Arhgap6 in African clawed toads, suggesting that such was the localization of Amel in early tetrapods Amel and Arhgap6) translocated to the sex chromosomes Amelogenin (Arhgap6 (inclusive of Amelx) to the 5′end of Mid1 did not cause any detectable phenotypic or behavioral abnormalities beyond dental enamel defects. The enamel malformations were largely the same as those observed in the Amelx null mice  with the possible exception of a “flattened layer” on the enamel surface Arhgap6/Amelx double null mice relative to the Amelx single nulls, two other Arhgap6 knockouts that retained Amelx showed no enamel phenotype. A knockout deleting only exons 6–8 of Arhgap6 resulted in mutant male and female mice “indistinguishable from their wild-type littermates at birth and thereafter” Arhgap6 to Mid1 led to enamel abnormalities but deletion from exon 6 of Arhgap6 to Mid1 did not” Arhgap6 is not essential for normal enamel formation, but might modify the enamel malformation phenotype when Amelx is absent.ARHGAP6 is a GTPase-activating protein of the Rho-GAP family. Mice deleted for a 1.1-Mb genomic region spanning from the first exon of AMELX cause syndromes that include AI as a phenotype HCCS) adjacent to ARHGAP6 and AMELX on Xp. To date, 16 AMELX mutations have been reported that cause non-syndromic amelogenesis imperfecta . A partial AMELX deletion (g.2525_7247del4723) starting in intron 2 and ending in exon 7 retained only the coding region for the amelogenin signal peptide plus 2 amino acids In humans, larger X-chromosome deletions inclusive of ARHGAP6 deletions that remove all of AMELX. In family 1 the deletion is confined to intron 1 and is not predicted to alter ARHGAP6 expression. In family 2 the deletion includes exon 2 of ARHGAP6 and is predicted to eliminate normal ARHGAP6 expression. These naturally occurring gene knockouts are analyzed to see what information can be gained concerning the human enamel phenotype in the absence of AMELX expression and the potential importance of ARHGAP6 in the process of dental enamel formation.In this study we report the characterization of two kindreds with partial The human study protocol and subject consents were reviewed and approved by the Institutional Review Board at the University of Michigan and the Ethics Committee of Istanbul University, Turkey, where one of the families was recruited. Study participants signed appropriate written consents after an explanation of their contents and after their questions about the study were answered. Any minors age 8 or older signed a written assent form after their parent completed a written parental consent for participation of the minor. The animal protocol was reviewed and approved by the University Committee on Use and Care of Animals (UCUCA) at the University of Michigan.The proband (III:4) of Family 1 was a 10-year-old dental patient at the University of Istanbul, Turkey. Oral photographs and radiographs were obtained for the proband and his 11-year-old cousin (III:1). The proband of Family 2 (III:7) was an 11-year-old dental patient at the Children's Clinic, University of Michigan School of Dentistry. His family was Caucasian, of Eastern European decent. Oral photographs and radiographs were obtained for the proband (III:7) and his mother (II:4). Tooth H of the proband was extracted for orthodontic considerations, photographed, and examined by scanning electron microscopy.260 and OD280. Genomic DNA (50 ng) was amplified using the Platinum PCR Supermix , and the amplification products were purified using the QIAquick PCR Purification Kit and protocol . The concentration of purified amplicon was estimated by the intensity of its ethidium bromide-stained band on a 1% agarose gel. The DNA sequencing reactions used 3 ng/µL for each 1000 bp of amplification product size and 1.0 pMol/µL of oligonucleotide primer and were analyzed using an ABI Model 3700 DNA sequencer  at the University of Michigan DNA sequencing core.Peripheral whole blood (5 cc) or buccal swabs were obtained from the 8 members of Family 1 and 5 members of Family 2. Genomic DNA was isolated using the QIAamp DNA Blood Maxi Kit and protocol  and its quality and quantity were determined by spectrophotometry at ODAMELX exons along with adjoining intron sequences were amplified by polymerase chain reaction (PCR); however, amplification products were only observed for male and female controls–not from either proband . We concluded AMELX was deleted in both probands. Because the families reported no notable medical histories and the clinical phenotype was limited to the enamel layer, we expected the deletions to be relatively small. PCR primer pairs were designed to survey upstream and downstream regions. Eight primer pairs sampled intron 1 of ARHGAP6 from nucleotides 50,924 to 291,746 (NCBI genomic reference sequence NG_012494.1) . PCR amplification products were observed from both probands in all reactions. ARHGAP6 exons 2 through 7 were sampled on the other side of AMELX. Only exon 2 of ARHGAP6 in family 2 failed to amplify . Thus the deleted regions were confined to the 5′ region of ARHGAP6. Additional amplifications narrowed down the locations of the deletions in family 1  and family 2 .The seven GCTAATTATTGGTGGAAAAG and Fa1-11R: GAACAGAGGCAGGCTGTGTC, producing a 2200 bp product. The amplification reactions included a 2 min denaturation at 94°C followed by 35 cycles at 94°C for 30 s, annealing at 57°C for 1 min, extension at 72°C for 6 min followed by a final extension for 20 min. The products were characterized by DNA sequencing priming with the primers used for amplification as well as Fa1-BPF: TTGCCAATCTGCTTTTTACAG, and Fa1-BPR: TATCCTCTTTGTGGGACAGC.To sequence across the deletion in Family 1 genomic DNA was amplified with F: TGAAAGCTAGAGGGGAAACC and Fa2-10R: TGCCAAGAGTAGCCATTTGA, producing a 1969 bp product. The amplifcation reactions included a 5 min denaturation at 94°C followed by 35 cycles at 94°C for 90 s, annealing at 58°C for 1 min, extension at 72°C for 3 min, followed by a final extension for 7 min.To sequence across the deletion in family 2, genomic DNA was amplified with Fa2-5F: The primary tooth (H) from the proband of Family 2 was sputter coated with gold for 75 s and then imaged using a Field Emission Gun Scanning Electron Microscope  at the Microscopy and Image Analysis Laboratory at the University of Michigan.Arhgap6 promoters (1A–1D) are used during tooth development, day 5 and day 11 developing first mandibular molars were collected from C57BL6 wild-type mice and processed according to the published protocol GCAAGCATCCTCAGTTCCTC; Ex1bF: GCAGTGAAGTAAGGGGACCA; Ex1cF: GTGACTCCTAGGGGACCACA; Ex1dF: AAGACAGCAAAGACACCGAGA) paired with an exon 4-specific reverse primer (Ex4R: GGGATAAGGGCATTCCAAAT). The PCR amplifications included a 5 min denaturation at 95°C followed by 35 cycles at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 1 min. In the last cycle the extension was for 7 min. The products were analyzed on a 1% agarose gel stained with ethidium bromide.To determine which of the four The proband (III:4) of family 1 was a 10-year-old Caucasian male from Turkey. His mixed dentition displayed thin enamel that was only slightly more radiopaque than dentin and in some locations secondarily affected by dental caries . The occThe proband's affected male cousin (III:1) at age 12 exhibited enamel defects similar to those of the proband , althougThe proband of family 2 (III:7) was an 11-year-old Caucasian male of Eastern European ancestry with an anterior cross-bite. His late, mixed dentition displayed thin, rough enamel that barely contrasted with dentin on radiographs . The incA primary cuspid  extracted from the proband for orthodontic considerations was examined by scanning electron microscopy . The enaThe mother (II:4) of the proband was less severely affected than any of the male subjects . Her enaAMELX exons amplified with the appropriate primers. PCR primer pairs were designed to survey surrounding DNA sequence within ARHGAP6, the results of which narrowly defined the deleted regions in both families . Ultimately, we were able to amplify across the deletions in both families and determine the DNA sequences at the break points (AMELX deletion (X*X). The 96,240 bp deletion in ARHGAP6 in family 1 (g.302534_398773del96240) was confined to intron 1 of that gene, but deleted all of AMELX. The 52,654 bp deletion in ARHGAP6 in family 2 (g.363924_416577del52654insA) deleted the 3′ end of intron 1 including all of AMELX, exon 2, and a small part of the 5′ end of intron 2.Mutation analyses using genomic DNA from the probands of families 1 and 2 were initiated, but none of the seven k points . The delARHGAP6 expression we analyzed the DNA sequences 5′ to exon 2 on human and mouse ARHGAP6 expressed sequence tags (ESTs). This analysis indicated that ARHGAP6 expression is directed by at least 4 different promoters, each associated with a different exon 1 . We identified one human EST and 4 mouse ARHGAP6 ESTs that contained exon 1a; 5 human and 44 mouse ESTs with exon 1b; one human and 9 mouse ESTs with exon 1c; and one human and no mouse ESTs with exon 1d . The structure of the ARHGAP6 gene showing the positions of the alternative promoters, AMELX, and the deletions in families 1 and 2 is shown in To better assess the possible effects of these deletions on Arhgap6 promoters are used during enamel formation, we isolated mRNA from ameloblasts (by laser capture) and enamel organ epithelia (EOE) of mouse first molars at days 5 (secretory stage) and 12 (maturation stage), amplified them with primers specific for exons 1a through 1d, and analyzed the products on agarose gels stained with ethidium bromide , which is entirely coding, would shift the reading frame and likely cause degradation of mRNA expressed from the truncated Arhgap6 gene in family 2. Thus while both of our families have an X-chromosome lacking the amelogenin gene, the shortened X-chromosome in family 1 likely has a functional ARHGAP6 gene, while the one in family 2 does not.AMELX mutations. Multiple amelogenins are expressed due to alternative splicing AMELX mutations produce a diversity of enamel phenotypes that seem to correlate with the position of the mutation Amelx caused extensive cytotoxicity because the ameloblasts failed to secrete the mutant protein, engorging the endoplasmic reticulum/Golgi apparatus. AMELX avoids this ambiguity and gives a clearer picture of how enamel forms when only trace amounts of amelogenin (from AMELY) are secreted. A remarkable observation in both of our families with AMELX deletions is that a thicker layer of enamel forms on the cusp tips and marginal ridges relative to the lateral tooth surfaces. The presence of this enamel cannot readily be explained by expression from AMELY as mice lack an amelogenin gene on the Y-chromosome, and yet Amelx null mice produce a thin enamel layer Enam and Ambn null mice fail to make an enamel layer Amelogenin is a tooth-specific protein that accounts for almost 90% of the protein in secretory stage enamel AMELX is nested within the large (>400 kb) first intron of ARHGAP6ARHGAP6 ESTs (Hs.435291) out of a total of 3,328,058 human ESTs for normal tissues. ARHGAP6 activates the GTPase activity of RhoA AMELX deletions, but the missing parts of ARHGAP6 varied. The deletion in Family 1 removed ARHGAP6 alternative promoters 1c and 1d. Our RT-PCR analyses showed that these promoters are not active in ameloblasts, so it is unlikely that this deletion affects ARHGAP6 expression in developing teeth. The deletion in Family 2 removed exon 2 of ARHGAP6, which should have precluded expression of the ARHGAP6 protein. It seems likely then that Family 1 had an AMELX defect only, while Family 2 had a combined AMELX and ARHGAP6 defect. The enamel phenotypes in both families were similar with a characteristic snow-capped appearance caused by the deposition of a relatively thick layer of enamel on the cusp tips, the buccal-occlusal and lingual-occlusal cusp slopes, and marginal ridges. The enamel defects were severe, but there was not a complete absence of enamel. The proband of Family 2 seemed to have less contrasting enamel on radiographs and greater surface roughness than the affected members of Family 1. The enamel phenotypes in these two families are remarkably similar given the broader phenotypic variation observed among the many persons with defined AMELX mutations . We suspect that the minor variations in enamel phenotype between our two families reflects the natural range of phenotypic variation in persons lacking AMELX, but the suspicion that an absence of ARHGAP6 is the cause of the increased surface roughness cannot be excluded given the minor differences in enamel phenotype observed in Amelx null and Amelx/Arhgap6 double null mice ARHGAP6 could modify the AMELX null phenotype, ARHGAP6 defects alone apparently do not cause enamel malformations. Arhgap6 null mice showed no enamel defects AMELX mutations are consistently found in human kindreds with X-linked AI, suggesting there is no second X-linked gene that causes AI, as this would lead to the accumulation of unsolved X-linked AI cases without AMELX mutations, which is not observed.Both of our AI families had complete Figure S1AMELX disease-causing mutations.(DOC)Click here for additional data file.Figure S2Tooth H from the proband of family 2 before it was processed for SEM analyses.(DOC)Click here for additional data file.Figure S3PCR amplification of the 7 AMELX exons.(DOC)Click here for additional data file.Figure S4PCR amplifications in intron 1 and exons 2 through 7 of ARHGAP6.(DOC)Click here for additional data file.Figure S5PCR amplifications in family 1.(DOC)Click here for additional data file.Figure S6PCR amplifications in family 2.(DOC)Click here for additional data file.Figure S7More PCR amplifications in family 2.(DOC)Click here for additional data file.Figure S8Mouse (A) and Human (B) ARHGAP6 expressed sequence tags.(DOC)Click here for additional data file.


C) into pathogenic conformational isoform (PrPSc). Variations in prions, which cause different disease phenotypes, are referred to as strains. The mechanism of high-fidelity replication of prion strains in the absence of nucleic acid remains unsolved. We investigated the impact of different conformational characteristics of PrPSc on conversion of PrPC in vitro using PrPSc seeds from the most frequent human prion disease worldwide, the Creutzfeldt-Jakob disease (sCJD). The conversion potency of a broad spectrum of distinct sCJD prions was governed by the level, conformation, and stability of small oligomers of the protease-sensitive (s) PrPSc. The smallest most potent prions present in sCJD brains were composed only of∼20 monomers of PrPSc. The tight correlation between conversion potency of small oligomers of human sPrPSc observed in vitro and duration of the disease suggests that sPrPSc conformers are an important determinant of prion strain characteristics that control the progression rate of the disease.The mammalian prions replicate by converting cellular prion protein (PrP Sc), often forming large amyloid deposits and fibrils. However, the apparent absence of protease-resistant PrPSc or amyloid fibrils in growing number of prion diseases raised several fundamental questions; specifically, whether presumably protease-sensitive forms of PrPSc exist as distinct conformers; and whether they comprise the initial steps in prion replication or are related to the alternative misfolding pathway generating noninfectious aggregates. We investigated the conformational characteristics of protease sensitive conformers of PrPSc and their role in the pathogenesis of sporadic Creutzfeldt-Jakob disease (sCJD). Using two different in vitro prion protein (PrPC) conversion techniques in tandem with biophysical methods, we identified small oligomers of protease sensitive PrPSc present in sCJD brains as the most potent initiators of PrPC conversion. Their concentration and conformational stability determine the distinctly different replication potency of PrPSc in individual isolates of sCJD and each of these characteristics correlates tightly with duration of the disease. These features argue for a broad range of distinct prion strains causing the sCJD and imply that small oligomers of protease sensitive conformers of pathogenic prion protein are encoding incubation time and progression rate of the disease.Mammalian prion diseases were originally characterized by accumulation of protease-resistant prion protein (PrP Sc) present in sCJD brains beyond the evidence that it is variably resistant to proteolytic digestion The yeast, fungal, and mammalian prions determine heritable and infectious traits, and thus behave like proteinaceous genes Sc), often forming large amyloid plaques and fibrils CScC) into pathogenic protease resistant (r) conformational isoform (PrPSc) Sc and apparent absence of protease-resistant PrPSc or amyloid fibrils in growing number of prion diseases Sc and prion infectivity ScSc demonstrated a high seeding (replication) potency of small oligomers of the pathogenic prion protein Sc found invariably in sCJD-infected brains might be composed of such highly potent small oligomers.Mammalian prion diseases were originally characterized by deposits of protease-resistant prion protein  Sc generated in PMCA is as infectious as the original brain derived sample is currently debated Sc in sonication-driven or quaking-induced conversion (QuIC), and yielded protease-resistant aggregates with a PK digestion pattern closely related to original brain PrPScSc, these approaches helped to define some key elements of prion structure Sc used as a seed Early important studies with mouse and Syrian hamster PrPde novo” from recombinant proteins and thus prove in principle that mammalian prions are misfolded proteins Sc and translating them into unique phenotypes of the disease in different prion strains is largely unknown. To analyze the mechanistic and structural aspects of the replication of different sCJD prions and specifically the role of sPrPSc, we employed an in-vitro amplification of brain PrPSc with QuIC and sPMCA. Using recombinant human PrP substrate in QuIC, and Tg mice brains expressing human PrPC(129M) in the sonication-driven sPMCA, both reactions demonstrated inverse correlation between conversion efficacy and the conformational stability of small protease sensitive (s) oligomers of PrPSc. The observed link between duration of the disease and conversion potency of small oligomers of sPrPSc in individual sCJD cases suggests that these conformers encode the progression rate of the disease in different prion strains.Although the PMCA and analogous techniques allowed to create prions “6-fold diluted sCJD seeds with matching codon 129 polymorphism and Type 1 or Type 2 PrPSc with levels of rPrPSc (PrP 27-30) easily detectable on WBs . The WB mobility of unglycosylated fragments of rPrPSc from individual cases of sCJD matched the original Type 1 or Type 2 seeds . The results extend previous observations on high fidelity replication of sCJD PrPSc using Tg mouse brain expressing homologous human PrPCC in transgenic mice remains to be established QuIC generated in QuIC resulted in a major PK-resistant fragment with SDS PAGE mobility corresponding to the mass ∼16 kDa . We did not observe differences between masses of peptides generated from rhuPrPQuIC seeded with Type 1 PrPSc(129M) or Type 2 PrPSc(129M). As expected ).After four rounds of sPMCA, the Tg brain homogenate substrate responded to 10Sc present in sCJD brain homogenates of 20 patients homozygous for methionine in codon 129 and containing either Type 1 (n = 10) or Type 2 (n = 10) PrPSc. Both techniques showed statistically significant inverse correlation of the amplification potency and duration of the sCJD . The tight correlation between amplification indexes obtained with sPMCA and QuIC in individual samples  proved that the observed range of values is highly reproducible with two different techniques. When we separated sCJD samples according WB type of PrPSc, we observed overall higher seeding efficacy of MM1 PrPSc over MM2 PrPSc . However, the trends were not statistically significant due to the broad range and overlapping amplification values. We concluded from these data that PrPSc present in cases classified as MM1 and MM2 sCJD display a continuum of conversion rates.Using CDI, we measured the conversion potency of PrP sCJD andFig. 1BSc that were either treated or not with PK before QuIC, we observed up to 10-fold higher seeding potency of the samples that were not treated with PK . We concluded from these experiments that in QuIC reaction, the total PrPSc is more efficient seed than protease-resistant fragment rPrPSc.Using seeds of Type 1 or Type 2 sCJD PrP with PK Fig. 2. , ) suggests conformational heterogeneity beyond that observed with WBs. Therefore, we used the CDI Sc in patients who were homozygous for methionine in codon 129 of the PRNP gene and demonstrate pure Type 1 or 2 PrPSc on WBs Sc as well as rPrPSc is operational and therefore all digestions with proteinase K (PK) were performed at constant protein/enzyme ratio equivalent to 3 IU/ml (∼100 µg/ml) in 10% brain homogenate containing 1% Sarkosyl for one hour at 37°C. The protocol for PrPSc digestion, validated in previously published experiments, was selected according to the following criteria: 1) complete digestion of PrPC determined with CDI in control samples; 2) complete shift of the bands of PrPSc to PrP 27-30 on WBs; 3) unequivocal WB differentiation of Type 1 and Type 2 rPrPSc in all tested samples Sc N-terminus with PK was monitored on WBs in all samples.The broad range of amplification indexes within each Type 1 or Type 2 group . Typical examples of dissociation/unfolding curves before and after PK for MM1 and MM2 PrPSc are shown in 1/2 values, from ∼2.3 to ∼3.0 M  with PK resulted in rPrPSc with invariably increased conformational stability  uniformly, with one exception, produced rPrPSc with decreased stability (Sc(129M) remaining after proteolytic treatment demonstrated higher overall conformational stability than total PrPSc. We observed the opposite effect of PK leading to less stable Type 2 rPrPSc.Comparing ten sCJD Type 2 cases, we found a broad range of Gdn HClo ∼3.5 M Fig. 4ASc, we first evaluated the shift in the [Gdn HCl]1/2 values (Table S1). Alternatively, we subtracted the relative fractional change in stability of rPrPSc after PK treatment from the PrPSc values obtained before PK . In contrast to simple shift in the [Gdn HCl]1/2 values, the ΔFapp take into account the difference in the slopes of the unfolding curves. Using both shifts in the [Gdn HCl]1/2 and ΔFapp values, the overall stability of Type 1 sPrPSc is lower than that of rPrPSc (Sc(129M) and shift in the [Gdn HCl]1/2 values induced by PK both demonstrate that sPrPSc is more stable than rPrPSc in this sCJD group  and MM2 (n = 10) sCJD samples, and in both sPMCA and QuIC  showed higher amplification rates . Similarly, PrPC present in MM1 and MM2 sCJD cases remained in the upper portions of the tubes . On the basis of calibration with standard proteins  and sω2t value distribution in the sucrose gradient, we estimate that the range of S values of CJD prions is from ∼20 to >150. The majority of MM1 PrPSc conformers have an S value in the range 110–130. In contrast, the S value for the majority of MM2 PrPSc conformers is ≥150. We concluded that both MM1 PrPSc and MM2 PrPSc display a broad range of aggregates and that the MM1 PrPSc aggregates are reproducibly smaller that those composed of MM2 PrPSc. The variable low-density flotating fraction of PrPSc in both MM1 and MM2 PrPSc samples suggests that the aggregates are very small, or that some PrPSc exists in a complex with detergent-insoluble low-density lipids Sc have mass 9–11×106 Da and are composed of ∼380–460 monomers of PrPSc. The most frequently occurring aggregates in MM2 sCJD PrPSc have mass ≥14×106 Da and are composed of ≥600 monomers of PrPSc.To investigate the impact of prion particle size on protease sensitivity and amplification, we separated sCJD prion particles according to sedimentation velocity using high-speed centrifugation in sucrose gradient. The sCJD prions present in brain homogenates of twelve sCJD patients with MM1 (n = 6) or MM2 (n = 6) PRNP gene polymorphism and WB pattern of PrPSc were separated in 10–45% sucrose gradient and collected fractions were analyzed by WBs, CDI, and QuIC. The PrPSc with increasing size of the aggregates in MM1 (n = 6) as well as MM2 (n = 6) sCJD cases  trend toward higher PK sensitivity of the fractions containing small oligomers of PrPSc in comparison to the bottom and floating fractions . Cumulatively, the data demonstrate that the conversion potency of PrPSc in individual isolates of sCJD is inversely related to the duration of the disease. Because the length of the duration of a clinically pronounced prion disease is a function of the incubation time, this finding accords with the bioassay data that show a broad spectrum of transmission rates and incubation times of sCJD prions in transgenic mice expressing human PrPC(129M) or human/mouse PrPC chimeras Sc and MM2 PrPSc and broad distribution of values within each group suggests that a continuum of sCJD prions are causing the disease.The distinct conversion efficacies we observed with prion seeds obtained from different cases of MM1 and MM2 sCJD were highly reproducible with both QuIC and sPMCA techniques  in prion infection Sc accumulating in CJD brains consists of sPrPScSc upon inoculation into wild mice Considerable data demonstrate that sPrPSc varied with the incubation time of the disease Previously we found that levels of sPrPSc, rPrPSc, and sPrPSc on the seeding potency of sCJD prions with QuIC and sPMCA. Both methods revealed that the stability of total PrPSc inversely correlated with the amplification index of sCJD PrPSc. Moreover, when sPrPSc proved less stable than rPrPSc, the difference in stability correlated with more efficient amplification. Conversely, when sPrP conformers proved more stable than rPrPSc, we observed the opposite effect-less accumulated PrPSc in both sPMCA and QuIC  of cellular membranes. This association would place both cellular and pathogenic forms of the prion protein into the same compartment and thus support the hypothesis that PrPSc formation occurs within caveolae In this research, we investigated whether small aggregates of PrPSc, in contrast with the lower sedimentation velocity of MM1 PrPSc indicates that MM2 PrPSc forms much larger aggregates and concurs with the pattern of large coarse deposits of Type 2 PrPSc observed with the brain immunohistochemistry in situ, in contrast to the fine  appearance of the immunoreactivity associated with Type 1 PrPScSc may be responsible for the different peptide fragmentation pattern with predominantly 19 kDa fragments of MM2 rPrPSc and 21 kDa in MM1 rPrPSc after PK treatment.The strikingly high sedimentation velocity of MM2 PrPAll procedures were performed under protocols approved by the Institutional Review Board at Case Western Reserve University. In all cases, written informed consent for research was obtained from patients or legal guardians and the material used had appropriate ethical approval for use in this project. All patients' data and samples were coded and handled according to NIH guidelines to protect patients' identities.We selected 20 representative subjects from a group of 340 patients with definitive diagnosis of sCJD. The criteria for inclusion were (1) availability of clinical diagnosis of CJD according to WHO criteria Retrospective charts review was carried out for all subjects, with particular attention to the documented initial cardinal clinical signs of sCJD such as cognitive impairment, ataxia, and myoclonus Sc was classified as (1) Type 1 PrPSc(129M) (n = 10) and (2) Type 2 PrPSc . Patients lacked pathogenic mutations in the PRNP and had no history of familial diseases or known exposure to prion agents. These cases underwent additional detailed WB analyses of the PrPSc so that we could ascertain the accuracy of their original classification and confirm that the same brain homogenate analyzed by CDI contained pure Type 1 PrPSc(129M) or Type 2 PrPSc(129M).All Type 1–2 patients or uncertain cases were excluded from this study. DNA was extracted from frozen brain tissues in all cases, and genotypic analysis of the PRNP coding region was performed as described Coronal sections of human brain tissues were obtained at autopsy and stored at −80°C. Three 200–350 mg cuts of frontal (superior and more posterior middle gyri) cortex were taken from each brain and used for molecular analyses. The other symmetric cerebral hemisphere was fixed in formalin and used for histologic and immunohistochemical purposes.Slices of tissues weighing 200–350 mg were first homogenized to a final 15% (w/v) concentration in calcium-free and magnesium-free PBS, pH 7.4, by 3 75 s cycles with Mini-beadbeater 16 Cell Disrupter . The homogenates were then diluted to a final 5% (w/v) in 1% (v/v) Sarkosyl in PBS, pH 7.4 and rehomogenized. After clarification at 500×g for 5 min, one aliquot of the supernatant was treated with protease inhibitors . The second aliquot was treated with 50 µg/ml of proteinase K  for 1 h at 37°C shaking 600 rpm on Eppendorf Thermomixer  and PK was blocked with PMSF and aprotinin-leupeptin cocktail. Both aliquots were precipitated with final 0.32% (v/v) NaPTA after 1 h incubation at 37°C as described Both PK-treated and untreated samples were diluted 9-fold in 1× Laemmli Buffer  containing 5% (v/v) beta-mercaptoethanol (ME) and final 115 mM Tris-HCl, pH 6.8. Samples were heated for 5 min at 100°C and ∼2 ng of PrP per lane was loaded onto 1 mm 15% Polyacrylamide Tris-HCl, SDS-PAGE gels (Bio-Rad) mounted in Bio-Rad Western Blot apparatus. After electro-transfer to Immobilon-P Transfer Membranes , the membranes were blocked with 2% (w/v) BSA in TBS containing 0.1% of Tween 20 (v/v) and 0.05% (v/v) Kathon CG/ICP . The PVDF membranes were developed with 0.05 ug/ml of biotinylated mAb 3F4  followed by 0.0175 ug/ml Streptavidin-Peroxidase conjugate  or with ascitic fluid containing mAb 3F4 (kindly supplied by Richard Kascsak) diluted 1∶20,000 followed by Peroxidase-labeled sheep anti-mouse IgG Ab  and diluted 1∶3000. The membranes were developed with the ECL Plus detection system (Amersham) and exposed to Kodak BioMax MR Films (Fisher Scientific) or Kodak BioMax XAR Films (Fisher Scientific).2PO4 containing 0.03% (w/v) NaN3, pH 7.5. Second, aliquots of 20 µl from each fraction containing 0.007% (v/v) of Patent Blue V (Sigma) were directly loaded into wells of white strip plates prefilled with 200 µl of Assay Buffer . Finally, the captured PrP was detected by a europium-conjugated −1 cm−1 and 21640 M−1 cm−1, respectively. The purified recombinant proteins were dissolved in 4 M GdnHCl and 50% Stabilcoat , and stored at −80°C. The concentration of PrP was calculated from the CDI signal of denatured samples using calibration cure prepared with either recPrP(23-231) for samples containing full length PrPSc or recPrP(90-231) for samples containing truncated rPrPSc (PrP 27-30) after proteinase-K treatment. This separate calibration was necessary due to the ∼3.5-fold lower affinity of mAb 3F4 with full-length human PrP compared to PrP The CDI for human PrP was performed as described previously Sc was performed as described previously Sc were thawed, sonicated 3×5 s at 60% power with Sonicator 4000 , and the concentration was adjusted to constant ∼50 ng/ml of PrPSc. The 15 µl aliquots in 15 tubes were treated with increasing concentrations of 8 M GdnHCl containing 0.007% (v/v) Patent Blue V  in 0.25 M or 0.5 M increments. After 30 min incubation at room temperature, individual samples were rapidly diluted with Assay Buffer  containing diminishing concentrations of 8 M GdnHCl, so that the final concentration in all samples was 0.411 M. Each individual aliquot was immediately loaded in triplicate to dry white Lumitrac 600, High Binding Plates , coated with mAb 8H4, and developed in accordance with CDI protocol using europium-labeled mAb 3F4 for detection The denaturation of human PrPOBS−TRFN)/(TRFU−TRFN) where TRFOBS is the observed TRF value, and TRFN and TRFU are the TRF values for native and unfolded forms, respectively, at the given Gdn HCl concentration Sc is unfolded ([Gdn HCl]1/2), the data were fitted by least square method with a sigmoidal transition model (Equation 1):The raw TRF signal was converted into the apparent fractional change of unfolding (Fapp) as follows app) in the TRF signal is the function of Gdn HCl concentration(c); c1/2 is the concentration of Gdn HCl at which 50% of PrPSc is dissociated/unfolded and r is the slope constant. To determine the impact of protease treatment on the conformational stability of PrPSc, the values of fractional change after PK were subtracted from Fapp values obtained before PK (ΔFapp = F0−FPK) and then fitted with a Gaussian model to estimate the proportion and average stability of sPrPSc conformers (Equation 2):The apparent fractional change  in 10 mM Sodium Acetate buffer, pH 4.0, was pretreated with 12 mM HCl at 1∶3.9  ratio for 7.5 min and immediately diluted to final 0.1 mg/ml into the reaction buffer composed of 20 mM NaH2PO4, 130 mM NaCl, pH 6.9, and containing 0.1% SDS, 0.1% Triton X-100, and 1∶5000 (v/v) N2 . The QuIC was performed with final volume 100 µl per well in sterile V-bottom, low-binding polypropylene 96-well plate  equipped with a 3 mm diameter PTFE bead  in each well. The aliquots of sCJD brain homogenates were diluted into the complete QuIC reaction buffer to obtain final 10−4 dilution of sCJD prions, and the plates were sealed with sterile AxyMat Silicone Sealing Mat . The QuIC was performed in samples seeded with sCJD PrPSc at 55°C for 20 hrs in Eppendorf Thermomixer  set for 1 min shaking at 1400 rpm followed by 1 min incubation.The QuIC was performed as described To each well containing 100 µl of QuIC reaction buffer was added 50 µl of PBS, pH 6.9, containing 3% (w/v) Sarkosyl and Proteinase K  to obtain the final Sarkosyl concentration 1% (w/v) and PrP/enzyme ratio 10∶1 (w/w). The plates was incubated for 1 h at 37°C at 1200 rpm on the Eppendorf Thermomixer with 1 min interval. The PK was blocked in each well with protease inhibitors .Sc was replicated using brains of transgenic mice overexpressing human PrP with methionine at position 129 6-fold dilution of the initial sCJD brain homogenate.Sonication-driven PMCA of sCJD samples was performed as described obs were calculated from formula sobs = k/ tubes . The ultracentrifugation was performed at 50,000 rpm for 73 min at 5°C in Optima TL ultracentrifuge  equipped with Beckman SW 55 Ti rotor. These conditions correspond to the adjusted proportionality constant k = 58.7 and angular velocity ω = 5236 rad/s. Observed sedimentation coefficients sSc in Gdn HCl before and after PK treatment We investigated the effect of the following demographic and laboratory variables on survival: sex; age at onset; duration of the disease; electrophoretic Type of PrP 27-30; and the concentration and stability of PrPFigure S1C(129M) and with QuIC using recombinant human PrP substrate. (A) Sonication-driven sPMCA with brain PrPC substrate preserves the differences in mobility of unglycosylated PrPSc in three MM1 and three MM2 sCJD PrPSc after four rounds of amplification and final dilution of brain sCJD prions 106-fold. Asterisk signifies residual full length PrP after PK treatment. (B) 16 kDa protease-resistant fragments of rhuPrPQuIC detected after QuIC by WBs developed with monoclonal antibody 12B2 (epitope residues 89–93) (21) or 3F4 (epitope residues 107–112) (9). The sCJD seeds were in QuIC reaction diluted 104-fold.A typical amplification of sCJD prions in sPMCA using brain homogenate of Tg mice expressing human PrP(TIF)Click here for additional data file.Figure S2A) The typical results of QuIC seeded with different sCJD prions and low background levels of de novo PrP. (B) The amplification of sCJD PrPSc after four rounds of sPMCA (n = 20) correlates to a highly significant degree with QuIC (n = 20). The amplification index is the ratio between the concentration of PrPSc before and after PMCA measured with CDI. The data points are averages of three PMCA experiments, each measured in triplicate with CDI.The time course of QuIC reaction and comparison with sPMCA. ((TIF)Click here for additional data file.Figure S3Sc recorded with both sPMCA and QuIC in MM1 and MM2 sCJD. (A) Amplification index obtained with sPMCA for (red circles) MM1 (n = 10) and (blue squares) MM2 (n = 10) sCJD cases. (B) Amplification index obtained with QuIC for (red circles) MM1 (n = 10) and MM2 (n = 10) sCJD. In both sPMCA and QuIC, the differences observed between MM1 and MM2 samples are not statistically significant. The amplification index was determined as described in legend for Continuum of amplification indexes of PrP(TIF)Click here for additional data file.Figure S4The CDI demonstrated the same end point concentrations of recombinant PrP substrate in samples with PK treated or untreated seeds at the end of the QuIC reaction. The total (substrate+seed) PrP concentration was measured with CDI in duplicate in six samples at the end of QuIC reaction.(TIF)Click here for additional data file.Figure S5A) elution profile and (B) calibration plot for estimating S20,w and mass. The calibrants were bovine serum albumin , alcohol dehydrogenase , thyroglobulin monomer , and apoferitin  (20). The protein content in each fraction was determined with BCA protein assay .Calibration of sucrose gradient ultracentrifugation with standard proteins. The ((TIF)Click here for additional data file.Figure S6C. The (A) PrPC in the human platelets isolated from blood of healthy donors and (B) in a control brain of the patient with other than prion disease (OND). The distribution of PrPC (green bars), total PrPSc (blue bars), and rPrPSc (red bars) in sucrose fractions was determined with and without PK treatment by CDI. The PrPSc and rPrPSc values oscillating around zero were used to establish the cutoff and baseline sensitivity limit of CDI in each fraction. The bars represent average ± SEM from CDI performed in triplicate.Sucrose gradient ultracentrifugation of control samples containing only PrP(TIF)Click here for additional data file.Figure S7C (green bars), total PrPSc (blue bars), and rPrPSc (red bars) in sucrose fractions was determined with and without PK treatment by CDI. The bars represent average ± SEM from CDI performed in triplicate.Fractionation by ultracentrifugation in sucrose gradient and protease sensitivity of samples taken from frontal cortex of individual (left column) MM1 (n = 6) and (right column) MM2 sCJD (n = 6) sCJD cases. The distribution of PrP(TIF)Click here for additional data file.Table S1Descriptive statistics of the data and demographics of sCJD cases.(DOC)Click here for additional data file.


FLC expression by vernalization (extended cold) has become a model for understanding polycomb-associated epigenetic regulation in plants. Antisense and sense non-coding RNAs have been respectively implicated in initiation and maintenance of FLC repression by vernalization. We show that the promoter and first exon of the FLC gene are sufficient to initiate repression during vernalization; this initial repression of FLC does not require antisense transcription. Long-term maintenance of FLC repression requires additional regions of the gene body, including those encoding sense non-coding transcripts.The repression of Arabidopsis  FLC gene is a repressor of flowering that confers a requirement for vernalization  to promote flowering in spring FLC encodes a MADS box protein that binds to and represses expression of the floral promoting genes FT and SOC1FLC by a plant homeodomain-polycomb repressive complex 2 (PHD-PRC2) mechanism FLC locus. Detailed studies of the timing of changes in FLC mRNA expression and H3K27me3 levels showed that transcription of FLC is repressed and H3K27me3 increases at a region around the transcription start site during the cold FLC transcription FLC expression and reduced repression of FLC after vernalization FLC locus is important in down-regulating its expression pre- and post-vernalization. Experiments to define the parts of the FLC gene mediating the different phases of the vernalization response showed that the promoter and first exon are sufficient to confer repression of a reporter gene during vernalization, but maintenance of repression after return to warm conditions requires the first intron in addition to the promoter and exon 1 FLC gene fragment including approximately 1.8 kb of FLC intron 1 and the remainder of the 3′ end of the gene recruits PRC2 and H3K27me3 in the absence of transcription FLC repression after vernalization.The Arabidopsis HOTAIR, which targets PRC2 to the HoxD locus, and is associated with HoxD silencing in humans FLC locus have been identified. The COOLAIR antisense transcripts originate from a promoter adjacent to the FLC 3′ untranslated region and consist of two classes, terminating at proximal or distal sites  originating from a region within the first intron of FLCCOLDAIR transcript has been shown to interact with PRC2 and its abundance also increases during vernalization. Reduction of COLDAIR transcript levels by RNAi showed that it is not required for the initial repression of FLC but is required for subsequent maintenance of repression.A complex array of non-coding RNAs is transcribed from eukaryotic genomes, the majority of which are of unknown function. Some long non-coding RNAs (lncRNAs) associate with and target protein complexes to regulate gene expression al sites Figure 1COOLAIR antisense transcripts in the initial vernalization-induced repression of FLC and the results of reporter gene studies we used FLC insertion mutants to test the role of these transcripts in FLC repression.As there are apparent contradictions in the proposed role of the FLC promoter and first intron are required for the stable vernalization-mediated down-regulation of a reporter gene we tested the effect of a Ds insertion of approximately 6 kb (flc-20) that separates these regions in the endogenous FLC gene FLC 5′ unspliced transcript as an approximation of transcription rate flc-20 mutant that was not maintained after plants were returned to warm conditions  in both C24 and flc-20 at the end of cold, but this increase is not maintained in flc-20 after return to warm temperatures .As the flc-20 mutant together with previous data from FLC reporter constructs raised the question of whether antisense transcription from the COOLAIR promoter is a requirement for vernalization-induced repression of FLC. To test this we carried out further experiments with T-DNA insertions close to the 3′ end of the FLC gene that disrupt the distal (SALK_092716) or both proximal and distal (SALK_140021) antisense transcripts , with the exception of regions 3′ of the SALK_092716 insertion which had high H3K27me3 under all conditions as seen previously for non-transcribed parts of FLCThe data from the COOLAIR transcripts is not an essential component of vernalization-induced repression of FLC. The observations presented here for these SALK lines are in agreement with previous results showing that sequences consisting of the promoter, exon 1 and intron 1 but not the COOLAIR promoter, are sufficient to confer stable repression on a reporter gene COOLAIR lncRNAs may play a redundant role in regulating FLC expression, our data shows that they are not required for the vernalization response.These data show that the production of COLDAIR lncRNA acting to maintain repression of FLC by recruiting the PRC2 machinery COLDAIR transcript and all show maintenance of repression after vernalization.Our data are consistent with the FLC, we measured 5′ unspliced FLC transcripts in the swn7clf28 double mutant and in the vin3-4 mutant. CLF and SWN encode histone methyl transferases components of PRC2; loss of function of these genes leads to a genome-wide loss of H3K27me3 swn7clf28 plants have increased 5′ unspliced FLC transcript in non-vernalized plants and show a similar fold-repression as ColFRI during 4 weeks of cold exposure  and crossed with ColFRI, a Col line with an active FRI allele FRI allele activates expression of FLC. PCR was used to identify F2 plants in which the active FRI allele and the T-DNA insertions were homozygous. The flc-20 mutant contains a modified Ds element inserted in the first intron of FLCSalk insertion mutants were obtained from the Arabidopsis Stock Center . Primers used are listed in Table S1. PCR were reactions carried out in quadruplicate, quantified using a standard curve of diluted cDNA and normalized to At4g26410 RNA was extracted using Qiagen Plant RNeasy Mini columns with an on-column DNase treatment according to the manufacturer's protocol. cDNA was synthesized using Superscript III (Invitrogen), primed with oligo dT  or with gene specific primers for the Table S1.Chromatin immunoprecipitation was carried out and amplicons for ChIP-qPCR are as described Figure S1H3K27me3 ChIP qPCR across FLC in C24 and flc-20. ChIP qPCR for amplicons 1–12 flc-20 plants grown for 12 long days (12LD), 12 long days followed by 4 weeks at 4°C (12+4V) or 12 long days, 4 weeks at 4°C followed by 2 long days (12LD+4V+2LD).(TIF)Click here for additional data file.Figure S2H3K27me3 ChIP qPCR across FLC in ColFRI, SALK_092716, SALK_140021 and SALK_131491. ChIP qPCR for amplicons 1–12  in ColFRI, SALK_092716, SALK_140021 and SALK_131491 plants grown for 12 long days (12LD), 12 long days followed by 4 weeks at 4°C (12+4V), 12 long days, 4 weeks at 4°C followed by 2 long days (12LD+4V+2LD) or 12 long days, 4 weeks at 4°C followed by 5 long days (12LD+4V+5LD).(TIF)Click here for additional data file.Table S1Oligonucleotide sequences.(XLS)Click here for additional data file.


Transmembrane roundabout receptor family members (ROBO1–ROBO4) principally orchestrate the neuronal guidance mechanism of the nervous system. Secreted glycoprotein SLITs are the most appreciated ligands for ROBOs. Recently identified ROBO4 is the key mediator of SLIT-ROBO mediated developmental and pathological angiogenesis. Although SLIT2 has been shown to interact with ROBO4 as ligand, it remains an open question whether this protein is the physiologic partner of ROBO4. The purpose of this review is to summarise how reliable SLIT2 as ligand for ROBO4 is, if not what the other possible mechanisms demonstrated till date for ROBO4 mediated developmental and pathological angiogenesis are. We conclude that ROBO4 is expressed specially in vascular endothelial cells and maintains the vascular integrity via either SLIT2 dependent or SLIT2 independent manner. On the contrary, it promotes the pathological angiogenesis by involving different signalling arm(s)/unknown ligand(s). This review explores the interactions SLIT2/ROBO1, SLIT2/ROBO1–ROBO4, ROBO1/ROBO4, and ROBO4/UNC5B which can be promising and potential therapeutic targets for developmental angiogenesis defects and pathological angiogenesis. Finally we have reviewed the ROBO4 signalling pathways and made an effort to elaborate the insight of this signalling as therapeutic target of pathological angiogenesis. Hence, the current available information is not sufficient to delineate the SLIT2/ROBO1/ROBO4 mediated mechanism of pathological angiogenesis. In this review we have summarized the molecular mechanism of developmental and pathological angiogenesis mediated by magic roundabout (ROBO4) as one of the major processes supporting the tumor growth.The neural and vascular networks often undergo the same routes and similar mechanisms of signalling. Many classes of guidance molecules have been characterized to play critical roles during angiogenesis , 2 such Structural organization of magic roundabout shows significant difference with other ROBO members, most notably in the extracellular region . ROBO4 hROBOs, the neuronal guidance receptors, are newly introduced in pathological vascular development. While ROBO1 is expressed in both endothelial cells and other cell types, ROBO4 is expressed specially in vascular endothelial cells including the tumor vasculature , 11, 42.in vitro, and vascular leak in vivo. This happens by blocking the activation of nonreceptor tyrosine kinases  in ROBO4+/+ endothelial cells but not in ROBO4−/− cells [Since ROBOs are extensively involved in guidance mechanism, they should be expressed in the leading end cells. Contrary to this hypothesis, ROBO4 was found to be transcribed in stalk cells (mature vascular cells) of retinal blood vessels and absent from many of the tip cells that sense and respond to extracellular cues. This suggests that ROBO4 may have a biological role that is unrelated to the guidance mechanisms regulating vascular patterning . Park et/− cells . These r/− cells , 46. SLI/− cells  indicati/− cells . ROBO4-p/− cells  and lead/− cells , 46. TheAnother mechanism, in this context, is the interaction of ROBO4 with UNC5B, a vascular netrin receptor that also counteracts the VEGF signalling . This rein vitro [The N-terminal Ig like domains of ROBO1 and ROBO4 are 42% identical but the residues identified for SLIT2 binding are not conserved in ROBO4 and are replaced by residues that are not compatible with binding of SLIT2. This suggests that ROBO1 has more efficient binding with SLIT2 compared to ROBO4. Park et al.  have demin vitro , 32 suggin vitro , 32, 55.in vitro . Therefoin vitro  which enHence, there are three complexes that can be exploited to target the pathological angiogenesis: SLIT2/ROBO1, ROBO4/UNC5B, and ROBO1/ROBO4. By promoting or inhibiting the affinity and/or association of these axes according to the mechanism involved in a particular system, inhibition of pathological angiogenesis and vascular high permeability can be appreciated as therapeutic target.Danio rerio) demonstrating that ROBO4 plays an essential role during angiogenesis and guides endothelial cells to their target analogous to ROBO1, ROBO2, and ROBO3 during neuronal guidance [We discussed that ROBO4 has endothelial specific expression and SLIT2/ROBO1–ROBO4 signalling inhibits the endothelial cell migration and pathological angiogenesis by counteracting the VEGF signalling. Contrary to this antiangiogenic nature of ROBO4 signalling, there is also evidence supporting that ROBO4 promotes the pathological angiogenesis , 57. Binguidance . Collectguidance  demonstrguidance .α (HIF-1α) leading to VEGF production and angiogenesis [Evidence shows that SLIT2 is not the ligand for ROBO4 because (1) N-terminal ligand binding domain of ROBO4 is not fit for SLIT2 binding; (2) dimerization of cytoplasmic region of ROBO4 is independent of SLIT2 binding ; (3) ROBogenesis . Also, iogenesis . It can In neurons interaction of SLIT2 with ROBO1 induces the recruitment of Rho GTPase activating proteins (srGAPs) to its CC3 domain which increases the intrinsic GTPase activity of CDC42 leading to its inactivation  Figure . LocalizHow does ROBO4 mediate the proangiogenic attraction guidance signalling in endothelial cell? Again as discussed earlier there could be two possibilities: (1) an unknown ligand for ROBO4 and (2) SLIT2 may signal by interacting with ROBO1–ROBO4 heterodimer. There should be a protein interacting with cytoplasmic region of ROBOs, to determine the antiangiogenic or proangiogenic nature of this signalling. One such appreciable protein is srGAP (SLIT-ROBO GTPase activating protein). These proteins are particularly abundant in SLIT responsive regions. Binding of srGAP to CC3 domain of ROBO1 increases the intrinsic GTPase activity of CDC42, which converts the GTP-CDC42 into GDP-CDC42 inactivating CDC42. Reduction of active CDC42 eventually decreases actin polymerization affecting the cell movement . Repulsi(WIP) and syndapin [To understand the mechanism of ROBO4 to promote or inhibit the fundamental events of angiogenesis, it is important to explore the other interacting partner protein(s) with intracellular region of ROBO4, especially proteins involved in actin regulatory machinery. The novel proteins interacting with ROBO4 involved in actin regulatory machinery include the Wiskott-Aldrich syndrome protein (WASP) and neuronal WASP proteins (NWASP) and their regulatory proteins: WASP-interacting protein syndapin . WASP hasyndapin . Glutathsyndapin . The idesyndapin . Thus, RIt is obvious that interaction of activated CDC42 (CDC42-GTP) presumably with other proteins is needed for the recruitment of MENA, WASP, NWASP, and WIP to ROBO4 for the formation of filopodia and migration. One such protein previously implicated in CDC42-GTP mediated filopodia formation is an SH3 domain-containing insulin receptor substrate protein 53 (IRSp53) . IRSp53 Our discussion converges to the point that ROBO4 is a key mediator of pathological angiogenesis and unravelling its mechanism of action may prove it as double edged sword. To unravel its mechanism there are some key questions to be addressed such as the following: (1) What are the physiological partners of ROBO4? (2) Does the choice of partner proteins of ROBO4 depend on microenvironment of cell that determines the proangiogenic or antiangiogenic nature of ROBO4 signalling? (3) What are the factors that regulate the homodimerization or heterodimerization of ROBOs? (4) Syndecans, a conserved family of heparan- and chondroitin-sulfate, are emerging as central players in cell surface interactions. It has been demonstrated that heparan sulfate serves as essential co-receptor in Slit-Robo signalling , 69. DoeConverging points of evidence discussed in this review indicate that ROBO4 has prominent role in regulation of angiogenesis. ROBO4 inhibits the pathological angiogenesis by counteracting the VEGF signalling either via SLIT2 or by interacting with UNC5B. This inhibition mechanism may be dependent on SLIT2-ROBO1 and ROBO1–ROBO4 interactions. ROBO4 signalling also promotes the pathological angiogenesis either via SLIT2/ROBO1–ROBO4 axis or via interaction of ROBO4 with an unknown ligand other than SLIT2. SLIT2 signalling via ROBO1–ROBO4 heterodimerization and srGAP interaction with cytoplasmic region of ROBO1 may be responsible for proangiogenic attraction guidance signalling in endothelial cells. Most of the literature considering therapeutic aspect of ROBO4 is focused on interaction of SLIT2 and ROBO4. In this review we have attempted to explore whether ROBO1–ROBO4 interaction can be targeted for antitumorigenic and antiangiogenic therapy. Since ROBO4 has dual behaviour of promoting and inhibiting angiogenesis depending on cell/tissue type, the therapeutic agents having properties of promoting and inhibiting ROBO4 signalling, respectively, can be used to target tumor angiogenesis according to the cell/tissue type under consideration. Because of its specific expression at the site of neoangiogenesis, ROBO4 can be used as angiogenesis marker. Although many questions still remain to be addressed regarding ROBO4 signalling, its understanding may be a milestone in targeting the pathological angiogenesis.