Investigation of the mechanism in primary cell culture studies demonstrated that resveratrol treatment significantly protected endothelial cells against an in vitro ‘ischemia’ resulting in improved viability against oxygen and glucose deprivation . An inhibition of nitric oxide synthesis did not prevent the improved cell viability following oxygen glucose deprivation but SIRT-1 inhibition with sirtinol partially blocked the protection (P<0.001) suggesting endothelial protection is to some extent SIRT-1 dependent. Collectively, the results support that oral resveratrol treatment provides a low risk strategy to protect the brain from enhanced damage produced by recurrent stroke which is mediated in part by a protective effect of resveratrol on the endothelium of the cerebrovasculature.Despite increased risk of a recurrent stroke following a minor stroke, information is minimal regarding the interaction between injurious mild cerebral ischemic episodes and the possible treatments which might be effective. The aim of the current study was to investigate recurrent ischemic stroke and whether resveratrol, a nutritive polyphenol with promising cardio- and neuro- protective properties, could ameliorate the associated brain damage. Experiments in adult rats demonstrated that a mild ischemic stroke followed by a second mild cerebral ischemia exacerbated brain damage, and, daily oral resveratrol treatment after the first ischemic insult reduced ischemic cell death with the recurrent insult (P<0.002). Further investigation demonstrated reduction of both inflammatory changes and markers of oxidative stress in resveratrol treated animals. The protection observed with resveratrol treatment could not be explained by systemic effects of resveratrol treatment including effects either on blood pressure or body temperature measured telemetrically. Investigation of resveratrol effects on the blood-brain barrier  Thrombo/embolic ischemic stroke is a leading cause of death and long-term neurological disability. An important population of patients at risk of stroke is one that has had a transient ischemic attack (TIA) or minor stroke. In these patients cerebral vessel occlusion is of short duration and its recannalization is followed by near or complete functional recuperation. Despite functional recovery, there is limited knowledge concerning the extent of full tissue recovery. There is increasing evidence that there can be ischemic damage not always detectable with standard diagnostic imaging techniques. Health status of the brain is likely crucial in these patients considering they are at higher risk of a recurrent stroke, with stroke recurrence being reported in 6–15% of patients within 90 days and in up to 21% of patients by one year post initial insult Thus, in the present study we used a novel model of recurrent stroke in the rat to investigate the interaction between multiple mild ischemic insults and the effects of resveratrol treatment. The results demonstrate that brain damage is enhanced when a mild recurrent ischemic stroke follows an initial mild ischemic insult. Oral administration of resveratrol between insults was found to reduce ischemic cell death. The beneficial effects of resveratrol treatment were not related to altered regulation of systemic blood pressure, body temperature or regional cerebral blood flow (CBF) but are consistent with it acting, at least partially, via beneficial effects on endothelial function.Experiments were performed to investigate the concentrations of resveratrol in blood and tissues following oral resveratrol administration. Significant levels of resveratrol were detected using high performance liquid chromatography tandem mass spectrometry (LC/MS/MS) in the plasma and tissues of resveratrol treated animals; levels reaching a peak that fell quickly soon after feeding  A. ResveOf the 42 animals randomized to MCAO, six died as a result of surgical complications. During MCAO all animals had >90% reductions in cortical perfusion as measured in the core region using a single laser Doppler probe. A mild ischemic stroke produced cortical injury and scattered cell necrosis evident seven days post-insult  C. The dOral treatment with resveratrol for three days prior to a single mild stroke, reduced the extent of damage observed compared with vehicle treated animals  C, D. SiThe effects of resveratrol on physiological parameters potentially affecting ischemic stroke outcome were investigated carefully. During MCAO, blood glucose, mean arterial blood pressure, PO2, PCO2, PH, and tympanic temperature were similar between groups . All ratDespite a lack of evidence for acute physiological differences during MCAO in the anesthetized animals, it was possible that resveratrol had systemic effects which could be more readily detected in conscious animals. There has been evidence suggesting that resveratrol can affect blood pressure Next we considered whether resveratrol had beneficial effects on signs of inflammation or oxidative stress following mild or recurrent ischemic stroke. Using immunohistochemistry, we assessed alterations in staining for reactive astrocytes using anti-glial fibrillary acidic protein (GFAP), alterations in staining for microglia/macrophage reactivity using anti-ED1, and altered oxidative stress using immunostaining with anti-nitrotyrosine (NT), a product of free radical oxidation of nitric oxide Following a mild ischemic stroke, sections immunostained with GFAP demonstrated areas of increased astrogliosis in peri-infarct/penumbral regions- an effect which was enhanced with recurrent stroke  A. The gFollowing a mild ischemic stroke, there was increased ED1 staining of microglia/macrophages evident within the ischemic core and peri-infarct/penumbral regions  B. QuantSimilar to ED1, NT staining was increased ipsilaterally in the ischemic region following either a mild or recurrent ischemic stroke  C. In reThe mechanisms causing stroke injury are complex and maintenance or restoration of tissue perfusion and vascular integrity may be key for mitigating complex neuroinflammatory processes at the blood brain barrier precluding neuronal cell death In contrast to a lack of effect of resveratrol on CBF, there were differences in BBB function in these same animals. Recurrent mild stroke resulted in an increase in Evans Blue extravasation in the cortex ipsilateral to the injury that was more marked in vehicle compared to resveratrol treated rats  C. Resvein vitro. Resveratrol treatment of endothelial cells at low doses (0.1–1 µM) for 3 days prior to exposure to oxygen glucose deprivation increased cell viability , was investigated and found to not prevent the improved cell viability following oxygen glucose deprivation. However, the SIRT-1 blocker, sirtinol, partially blocked the protection at a dose of 50 µM (P<0.001)   B. This The major original finding of the current study is that resveratrol treatment protects against brain injury in our new model of mild recurrent stroke. In addition to a reduction in brain injury, resveratrol treatment lessens oxidative stress and inflammation, prevents edema, protects endothelial cells and preserves BBB function following recurrent stroke, but does not alter regional CBF or systemic variables such as blood pressure and core temperature. The results support that a treatment such as resveratrol is promising for its ability to reduce the severity of damage due to stroke recurrence and could have a significant impact on quality of life and associated costs of caring for stroke survivors.We show for the first time protection with resveratrol against ischemic brain injury due to recurrent mild stroke. We also observed protection with pre-treatment prior to a single mild stroke which is consistent with what has been found with resveratrol pre-treatment in other stroke models Resveratrol has been widely studied but its mechanisms of action are still controversial and poorly defined, including identification of the potential cardiovascular or systemic effects that may modify cerebral ischemic injury or recovery. Determining cardiovascular impact is essential for a better understanding of resveratrol's mode of protection because even minor stroke may lead to effects such as impaired cerebral autoregulation to blood pressure changes In addition to the parenchymal protection observed, resveratrol was investigated for its potential to improve the health of the cerebral microvasculature during and after stroke. Microvessel health is essential to neuronal survival and requires preservation of vessel reactivity, adequate cerebral perfusion and optimal BBB function and its associated neurovascular exchange. We found no evidence for resveratrol-mediated augmentation of vascular responsiveness and flow in the ischemic area, during or after ischemic stroke using focal measures of laser Doppler flowmetry or measures of regional CBF with laser speckle imaging. However, we did demonstrate a substantial reduction in BBB disruption and a suppression of edema following recurrent stroke in resveratrol-treated rats that was independent of flow changes.Several mechanisms may be involved in the protection of the BBB. BBB dysfunction following ischemia is associated with changes in tight junction proteins such as occludin and claudin involving mediators such as matrix metalloproteinases (MMPs) that cause degradation of these tight junction proteinsTo conclude, the current results indicate that a recurrent stroke consisting of two mild transient ischemic episodes produces damage exceeding that of a single mild stroke. Resveratrol administered at moderate doses and within a clinically relevant acute time frame of three days reduces parenchymal brain damage and inflammation produced by either a single or a recurrent mild stroke. These beneficial effects of resveratrol treatment appear related at least partially to a reduced BBB extravasation and cerebral edema associated with improved viability of endothelial cells against deprivation of substrate and oxygen delivery. The results support further investigation of the protective role of resveratrol treatment as a dietary supplement that could potentially provide a low risk and effective option for protective therapy in patients following their first TIA or minor stroke.For experiments conducted in Calgary, animals were used and handled in accordance with the Canadian Council on Animal Care and University of Calgary Health Sciences Animal Care Committee (Approved Protocol MO7073). For laser speckle flow and blood-brain barrier (BBB) experiments conducted in Szeged, Hungary, all experimental procedures were approved by the Ethical Committee for Animal Care of the University of Szeged, Szeged, Hungary. All animals were randomized to their treatment groups and the surgeon and investigators performing analysis were blinded to the treatments.13C6, Sigma-Aldrich Co. Oakville, Canada). The supernatant was dried and then reconstituted in 200 µL of 50∶50 acetonitril:1 mM ammonium formate. The LC/MS/MS analysis was carried out with an Agilent 1100 HPLC  coupled to an API 4000 triple quadrupole Mass Spectrometer equipped with a Turbo-Ionspray Interface . Resveratrol was separated using a 2.1×100 mm, 5 µ Hypersil ODS column . The mobile phase composed of 1 mM ammonium formate (A) and acetonitrile (B) was delivered at a flow rate of 0.3 mL/min. A binary gradient started with 85% of A and 15% of B, which was then increased to 50% of B at 6 min. and subsequently to 70% of B at 8 min. Then, the column was equilibrated with 15% of B for 10 min before the next 5 µL sample injection. The mass spectrometer was operated in negative, multiple reaction monitoring mode with the following settings: source temperature 350°C, curtain gas 10, collision gas 6, gas1 50, gas2 50, and ion spray voltage −4200 V. Quantitation of trans-resveratrol (Sigma-Aldrich Co) was achieved using the 227/185 transition with the 227/143 transition monitored as confirmation ion. Transitions monitored for internal standard (resveratrol-phenyl-13C6) were 233/149 and 233/191.Resveratrol levels in blood and tissue were first assessed using LC/MS/MS in 200–300 g male Wistar rats  treated with either trans-resveratrol  or vehicle (n = 3). Blood samples were obtained in anesthetized rats at various time points  following daily treatments and the plasma was frozen for analysis. Two hours after the last (fourth) treatment, samples of brain and liver were dissected on ice, homogenized in lysis buffer, centrifuged at 5000 g for 15 min, and the supernatant was frozen. The sample preparation and analysis was as described previously with some modifications Thirty rats were randomized to receive either single mild  or recurrent mild stroke . Animals receiving a single stroke were further randomized to receive, daily for 3 days prior to the stroke, either vehicle  or trans-resveratrol in vehicle , the dose being within ranges used previously Transient focal ischemia was achieved by placing a microaneurysm clip  on the middle cerebral artery through a small burr hole in the skull, as previously described, with some modifications To simultaneously monitor core temperature, heart rate and mean arterial blood pressure in awake and freely moving rats we used telemetry probes . These were implanted abdominally using aseptic techniques under isoflurane anesthesia. Two leads from the probe, tunneled under the skin, measured heart rate and a catheter inserted into the descending aorta without obstructing blood flow measured arterial blood pressure. After 1 week of recovery, animals were placed in separate cages, allowing measurements to be recorded every 30 sec via RPC-1 receivers . Following 24 h of baseline measurements, rats were fed vehicle (n = 3) or 25 mg/kg RSV (n = 3) daily for 4 doses. One hour following the final dose, rats were subjected to a single mild stroke as described above. After recovery animals were further monitored using telemetry for 48 h following stroke.nd insult of recurrent stroke, animals were perfused with 10% formalin under anesthesia. Brains were embedded in paraffin and 6 µm sections were stained with hematoxylin and eosin to determine cell death. Standard infarct volume assessment did not allow us to accurately evaluate the scattered cell death that we observed following mild stroke; therefore, a cumulative score for brain injury at each level was assessed by an investigator blinded to the animal identity. A scoring system similar to that used previously One week after the either the single stroke or the 2Immunohistochemical staining was performed in adjacent sections that were dewaxed and incubated first with 10% goat serum, then primary antibody overnight, and then visualized using the appropriate biotin-conjugated IgG (Jackson ImmunoResearch Lab followed by incubation with peroxidase-congugated streptavidin (HRP)  and diaminobenzidine . Reactive astrocytes were assessed using anti-glial fibrillary acidic protein . Inflammatory signs of activated microglia/macrophages were assessed using anti-ED1 . Signs of oxidative stress were assessed using rabbit anti-Nitrotyrosine ) to detect the accumulation of peroxynitrite metabolites indicative of damage related to reactions with nitroxyl radicals. Antigen retrieval was performed prior to incubation with primary antibodies by immersing slides in boiling citrate buffer pH = 6 for 10 mins for ED1 and NT.Areas of altered staining were measured in GFAP stained sections that were scanned digitally (Nikon COOLSCAN 5.0). Areas of altered stain were corrected for edema by measuring the contralateral area and the unaltered area of staining ipsilaterally and presenting the difference as a percentage of the contralateral area. The ED1 and NT sections were scanned and assigned scores similar to the hematoxylin and eosin stained sections above, except that increased levels of staining were scored in the four different cortical areas as follows: 0 - no increase in staining, 1- modest increase in <25% of the area, 2 - modest increase in 25–75 of the area and/or intense increase in <25% of the area, 3 – modest increase in >75% and/or intense increase in 25–75% and 4 – intense increase in >75% of the area.nd 30 minutes transient MCAO was performed as described above. Images were taken (2 per sec) during the entire ischemic period and for 1 hour of reperfusion. Analysis of laser speckle images was performed using PIMsoft; a PeriCam PSI dedicated software . Maps of the speckle contrast value at each pixel of the image show the pattern of blood flow on the cortical surface during imaging. The mean perfusion of the parenchymal microcirculation was calculated by measuring the average perfusion values in four regions of interest of equal size and shape placed in four conserved locations  or vehicle (n = 6), for the 3 days between the first and second stroke. After carotid artery isolation, rats were placed into a standard stereotaxic frame and the skull was exposed with a midline scalp incision. The ipsilateral parietal bone was carefully thinned with a water-cooled drill  and covered with a UV-light cured doming  to render the skull transparent. A lateral window was also prepared to expose the distal MCA for clipping. Rats were placed under a PeriCam PSI HD System , which utilizes a built-in 70 mW laser diode to illuminate the cortical surface. Blood perfusion in the scanned area was monitored using a 1388×1038 pixel CCD camera at a speed of 2 Hz. Ten minutes of baseline was recorded, after which the 2BBB integrity and water content were investigated in the same animals as those used for measuring CBF changes. Blood brain barrier permeability was assessed by measuring extravasated Evans Blue dye in animals following exposure to recurrent stroke and treated with either resveratrol (n = 7) or vehicle (n = 6). For treatment, 4 ml/kg of 2% Evans Blue  in saline was injected through the tail artery immediately prior to reperfusion with the recurrent stroke. Rats were sacrificed 48 hours after reperfusion by transcardial infusion of saline. Wet and dry weights (dried at 60°C for 24 h) of the ipsilateral and contralateral cerebral cortices were measured and the corresponding water content was calculated by the formula: water content = 100% (wet weight–dry weight)/wet weight.Dry cortices were incubated overnight in formamide (5 ml/g dry weight) and the amount of Evans Blue released into the formamide was quantified by florescence spectrophotometry . Raw data were compared with a standardized curve of known quantities of Evans Blue in formamide. Results are presented in ng/g tissue.ueous One Solution Assay was acquired from Promega .Plastics used for endothelial cultures were obtained from Becton-Dickinson . Dulbecco's modified Eagle medium (DMEM), Neurobasal medium, B27 Supplement, 2-mercaptoethanol, and fetal bovine serum were obtained from Invitrogen  and fetal bovine plasma-derived serum was purchased from Animal Technologies . Dispase I was purchased from Roche  and Collagenase type II was obtained from Worthington . CellTiter 96 AQA total of 3 Wistar dams and 22 pups were used for endothelial cultures. Rat brain endothelial cells were isolated from two week old Wistar rat pups , as described previously An ischemic type insult sufficient to evoke ∼50% cell death was produced using oxyygen and glucose deprivation by washing the cells and replacing the medium with glucose-free Dulbecco's modified Eagle medium (DMEM). This was followed by exposure of the cells to a humidified anaerobic environment for 3.5 hours. Control cell cultures were incubated under normal conditions in glucose-containing DMEM.sample−absorbancebackground×100/(absorbancecontrol−absorbancebackground).Cell viability in the cultures was determined 24 hours after oxygen glucose deprivation using the tetrazolium-based CellTiter 96 AQueous One Solution assay. Absorbance at 490 nm was measured with a microplate reader . Comparisons were made between cultures treated in a similar way, with the exception of omitting the noxious stimulus. Cell viability (%) was expressed as:  inhibitor  or the non-specific nitric oxide synthase blocker .Resveratrol's effects were investigated in cell cultures treated with various doses of resveratrol (100 nM-10 µM) dissolved in culture medium containing up to 1.5% DMSO. Control cultures were treated with 1.5% DMSO alone. Cells were treated once a day (a new resveratrol solution was made each day) for three consecutive days to match the Differences between treatments were assessed with t-test or a one-way analysis of variance (ANOVA) and a Student–Newman–Keuls test. Non-parametric data were analyzed using a Kruskal Wallis ANOVA on ranks and a Mann–Whitney rank sum test. A value of P<0.05 was considered significant.


Multiple HLA class I alleles have been shown to influence both HIV-1 transmission and viral load. In transmission pairs, viral loads of acutely infected partners correlate with viral loads (VL) of their chronically infected donors. This correlation becomes highly significant after adjustment for host factors known to modulate viral load. In addition, we have previously demonstrated that transmission of a virus containing multiple HLA-I associated polymorphisms resulted in a lower set point VL in Zambian linked recipients. These studies imply that transmitted viral characteristics play a role in defining early HIV-1 pathogenesis, and it will be important for vaccine development to understand which viral characteristics are responsible for this.We investigated the role that the in vitro replicative capacity (RC) of the transmitted Gag plays in defining HIV-1 clinical parameters, by cloning gag genes from the founder virus in newly infected partners of 149 epidemiologically linked transmission pairs into the subtype C, R5 tropic MJ4 provirus.We observed a statistically significant positive correlation between the RC of Gag-MJ4 chimeras and set point VL in seroconverters (P=0.013). The RC of the transmitted Gag also correlated (P=0.025) to the viral load in the chronically infected donor, pointing to RC as the major viral characteristic responsible for the link between donor and linked recipient viral loads. The long term clinical benefit associated with the transmission of attenuated viruses was investigated by performing a Kaplan Meier analysis of time to CD4+ count less than 350. Individuals infected with attenuated gag sequences (RC< 1) were delayed in their progression to CD4+ counts < 350 compared to high (RC >2) replicating viruses (P = 0.034).Interestingly, this phenomenon seemed to be independent of viral load perhaps highlighting the role that early viral replication may play in defining HIV-1 pathogenesis.


Drosophila heart as an assay to identify interacting CHD candidate genes by expressing them alone and in all possible pairwise combinations and testing for effects on rhythmicity or heart failure following stress. This comprehensive analysis identified DSCAM and COL6A2 as the most strongly interacting pair of genes. We then over-expressed these two genes alone or in combination in the mouse heart. While over-expression of either gene alone did not affect viability and had little or no effect on heart physiology or morphology, co-expression of the two genes resulted in ≈50% mortality and severe physiological and morphological defects, including atrial septal defects and cardiac hypertrophy. Cooperative interactions between DSCAM and COL6A2 were also observed in the H9C2 cardiac cell line and transcriptional analysis of this interaction points to genes involved in adhesion and cardiac hypertrophy. Our success in defining a cooperative interaction between DSCAM and COL6A2 suggests that the multi-tiered genetic approach we have taken involving human mapping data, comprehensive combinatorial screening in Drosophila, and validation in vivo in mice and in mammalian cells lines should be applicable to identifying specific loci mediating a broad variety of other polygenic disorders.A significant current challenge in human genetics is the identification of interacting genetic loci mediating complex polygenic disorders. One of the best characterized polygenic diseases is Down syndrome (DS), which results from an extra copy of part or all of chromosome 21. A short interval near the distal tip of chromosome 21 contributes to congenital heart defects (CHD), and a variety of indirect genetic evidence suggests that multiple candidate genes in this region may contribute to this phenotype. We devised a tiered genetic approach to identify interacting CHD candidate genes. We first used the well vetted  Drosophila heart, where they disrupted several indices of heart function. We then over-expressed these genes in the mouse heart alone or in combination and found that while expression of either gene alone had little or no effect, co-expression of the genes, as in flies, lead to severe cooperative defects in heart physiology and morphology. The strategy we have followed in this study is broadly applicable to identifying genes involved in other polygenic disorders, such as obesity, autism, and schizophrenia, which have been linked to altered copy number of multiple genes.A large fraction of human genes may contribute to polygenic disorders, yet few experimental methods for identifying such genes are currently available. For example, with regard to congenital heart defects (CHD) caused by extra copies of genes on chromosome 21 in Down syndrome patients, it is not known which genes contribute to this complex phenotype. In this paper, we identify two genes, DSCAM and COL6A2 that interact strongly to produce CHD when over-expressed at modest levels in the mouse heart. These two genes were identified as the most strongly interacting pair of CHD candidate genes when over-expressed in the  The Online Inheritance in Man (OMIM) lists over 3500 loci which when mutated give rise to heritable human disease. Approximately one third of these disorders are dominant, most of these cases being due to dosage sensitive requirements for gene function  and a minority resulting from the production of an aberrant interfering protein product as has been extensively studied for peripheral neuropathies Drosophila as a model to systematically examine the effect of over-expressing these candidate genes individually or in pairwise combinations in the pumping heart tube of adult flies as well as in a neurologically relevant tissue (the eye). Although the fly heart is a much less complex structure than its vertebrate counterparts, it has several important basic properties common to all hearts including developmental genes involved in specifying the heart primordium Given the large numbers of potentially interacting loci that could underlie polygenic disorders, systematic approaches to identify such loci are urgently needed. In the current study, we present a multi-tiered genetic approach that could be generalized to a broader range of disorders, to identify genes that interact to cause congenital heart defects (CHD). Based on human genetic data delimiting a short interval on the distal tip of chromosome 21 containing a small set of candidate genes which may contribute to CHD in human DS patients Drosophila identified DSCAM and COL6A2 as the most strongly interacting pair of genes. The effect of modestly over-expressing these genes in the mouse heart was then examined and, as in flies, these two genes interacted synergistically to cause defects in heart morphology and physiology. Over-expression of DSCAM and COL6A2 also resulted in a transcriptional response in cardiac H9C2 cells. Consistent with the known cell biological roles of these two genes in mediating cell-matrix adhesion, we found clear transcriptional signals for genes involved in cell adhesion as well as genes involved in cardiac disease. We discuss the prospects of using similar multi-tiered genetic approaches to identifying genes involved in other polygenic disorders.Our comprehensive screening of CHD candidate genes in Drosophila  altered two indices of heart function . Using tIn parallel to testing for defects in heart performance, we examined the effect of expressing CHD candidate genes in the fly eye, which is another widely used assay system for genetic interactions and a well established model for defining mechanisms underlying neurological disorders. As in the case of the heart, we expressed each CHD candidate gene alone and in all pair wise combinations using the eye specific GMR-GAL4 driver and observed varying degrees of roughened eyes  ranging In flies, we next examined the basis for the interaction between DSCAM and COL6A2 by taking movies of individual semi-intact fly heart preparations using high speed digital video imaging. We analyzed autonomous heart function using a semi-automated heartbeat analysis that provides several quantitative measures of the dynamic contractile properties of the beating heart Having demonstrated a cooperative interaction between DSCAM and COL6A2 in flies, we next asked whether over-expression of this same pair of genes would similarly result in a synergistic disruption of heart function and/or morphology in mice. We over-expressed each of these genes separately and in combination under the control of the murine heart specific alpha-MHC promoter, which is active in myocardial cells both during early development and in the adult Since we targeted expression of the DSCAM and COL6A2 transgenes specifically to the heart, the reduced proportion of viable double transgenic mice suggested that some of these individuals may have succumbed to severe heart defects and that surviving adults might have observable abnormalities in heart morphology or function. Consistent with this possibility, dissected hearts from double transgenic 3-month-old adult mice weighed more than those from wild type controls . We searin vivo imaging methods. The first method, digital subtraction angiography (DSA), can detect abnormal shunting of blood between the heart chambers by injecting a radio-opaque dye into the right jugular vein and following it through the heart cycle in real-time by radiography We also examined hearts in greater detail using the full resolution of Micro-CT reconstructive imaging of fixed whole mount hearts as well as standard dissection procedures . Both tyThe second assay we employed, saline contrast echocardiography, provides a yet more sensitive test for shunting in which agitated saline infused with highly reflective micro-bubbles is injected into the right jugular vein and then followed by 2-dimensional echocardiography. During the period when the pressure in the right atrium exceeds that in the left atrium, even minute amounts of right-to-left blood flow shunts can be detected DSCAM is a cell adhesion molecule and its genetic interaction with COL6A2, an extracellular matrix component, suggested a possible direct or indirect interaction involving cell-substrate adhesion. We investigated this possibility by transfecting the rat cardiac myoblast cell line H9C2 with DSCAM to generate a stable cell line constitutively expressing DSCAM (H9C2-DSCAM). These cells moderately over-express DSCAM and exhibit a subcellular distribution that is similar to the endogenous protein . We platSince H9C2-DSCAM cells exhibited elevated adhesion in a COL6-dependent fashion and because adhesive interactions are known to induce transcriptional responses, we asked whether expression of DSCAM in H9C2 cells might alter the transcriptional profile of these cells. We purified RNA from H9C2-DSCAM and control H9C2 cells plated on COL6 and performed RNA deep sequencing (RNA-Seq) and compared the transcriptomes of the DSCAM-expressing and parental cell lines. Our analysis identified 1251 genes that were differentially expressed in the two cell lines . Gene onin vivo in double transgenic DSCAM+COL6A2 mice. We therefore examined the relative expression levels of a select set of genes in hearts dissected from wild-type and double transgenic embryos. mRNA was isolated from individual hearts and tested for quantitative changes in gene expression by qRT-PCR. This analysis confirmed that several gene transcripts altered in the cellular cardiomyocyte model were also affected in the hearts of the double transgenic mice by qRT-PCR (Tnn), hypertrophy associated cardiac troponin gene (Tnnt2), calcium binding protein S100A4, which is involved in fibrosis and tissue remodeling in several diseases Cxcr7, which is associated with various cardiac defects including septal defects Gata2, which is associated with familial early-onset coronary artery disease The altered expression levels of genes mediating cell-substrate interactions or genes misregulated in cardiac myopathy and hypertrophy in H9C2-DSCAM/COL6 cardiomyocyte cells suggested that these genes might also be misregulated  qRT-PCR , includiIn this study, we began with information provided by decades of mapping in human DS patients that delimited a small region of chromosome 21 responsible for causing CHD Because DSCAM and COL6A2 cooperatively altered heart function in flies when expressed in myocardial cells, we selected this combination of genes to over-express at modest levels in the murine heart. We chose the myosin-alpha chain gene promoter to drive over-expression of these genes in the myocardium since it is heart specific and drives modest levels of expression both during heart development and in adult hearts. While this mode of expression obviously does not precisely recapitulate the endogenous pattern of DSCAM and COL6A2 expression, it does restrict expression of these genes to the heart throughout a protracted period. Since we were using this driver to express molecules targeted to the extracellular space, we reasoned that these proteins might similarly accumulate extracellularly whether they were expressed by cardiomyocytes or other cardiac cells such as fibroblasts that are known to produce these proteins as well . Additional studies with endogenous promoters or promoters driving expression of these genes in other cell populations in the heart  are clearly warranted. The choice of gene expression vehicle notwithstanding, we observed a very strong synergistic interaction between DSCAM and COL6A2 when over-expressed in the mouse heart including, highly penetrant and prominent cardiac hypertrophy, atrial septal defects associated with physiological shunting, and a mortality rate of 50%. Most importantly, these dramatic phenotypes were not observed in transgenic mice expressing either transgene alone, but only in mice co-expressing both transgenes.An obvious possible mechanism by which DSCAM and COL6A2 might disrupt heart development and/or function is by altering cell-substrate adhesion given that DSCAM is a transmembrane adhesion molecule and COL6A2 is constituent of the ECM. In line with this possibility, a rat cardiomyocyte cell line expressing DSCAM exhibits a time-dependent increased adhesion to COL6 coated plates. Although it is known that adhesion of cells has transcriptional effects, we were surprised that much of this transcriptional response seems to be focused on regulation of genes involved in the adhesion network, suggesting that positive feedback mechanisms further stabilizing adhesive interactions may be a prominent element of this interaction. These pervasive changes in gene expression could be mediated either by DSCAM itself, which is known to transduce a variety of signaling events during axonal pathfinding and axon branching including self-adhesive signaling and a response to Netrins, or by other adhesion dependent effectors such as the focal adhesion pathway components of which are regulated in the transcriptional response of H9C2-DSCAM grown on COL6. The reciprocal regulation of several components promoting adhesion versus cell migration may also contribute to the CHD phenotypes that are observed in DSCAM+COL6A2 double transgenic mice, particularly since a number of the responsive genes identified in the cell culture experiments were also misregulated in hearts of double transgenic mice. While further studies will be needed to assess the importance of altered cell-substrate adhesion and cell migratory processes in mediating the morphological and physiological effects of DSCAM+COL6A2 over-expression, we speculate that such defects could lead to a developmental delay in closing the atrial septum and may also contribute, either developmentally or as part of a physiological feedback loop, to hypertrophic phenotypes that we observed in affected mice.Since we set out to identify genes contributing to DS CHD, a natural question is whether the phenotypes we observe recapitulate those associated with DS in humans. It is certainly noteworthy that we observed ASDs with high penetrance in double transgenic mice, since this is one of several salient features of DS CHD. However, other typical DS CHD phenotypes were not observed such as atrial ventricular septal defects, perimembranous and muscular ventricular septal defects, Tetralogy of Fallot, or persistent ductus arteriosus. Also, since atrial septal defects can have many etiologies, in order to determine whether this phenotype is similar to that in human DS patients, it will be important to examine in greater detail the origin of these defects in mice  and compare them to the occurrence of these defects in DS patients There are several possible reasons for DSCAM+COL6A2 double transgenic mice exhibit only a subset of DS CHD phenotypes. Perhaps most obviously, as noted above, endogenous promoters may drive expression of these genes at different levels or in distinct spatial and temporal patterns than we achieved using a heterologous promoter. With regard to expression level, however, we note that DSCAM+COL6A2 double transgenic mice express only modestly elevated levels of the transgenes. Whether these match precisely with the altered dose in DS patients is unknown. It is noteworthy in this context that the level of altered gene expression in DS patients is not always elevated by precisely 50%, and in some cases can be as much as 2-3 times the normal level The most prominent phenotype we observed in DSCAM+COL6A2 double transgenic mice was pronounced left ventricular hypertrophic cardiomyopathy, which is not typical of DS CHD a priori assumptions including genetic analysis in human DS patients, expression in the developing heart, and the potential to physically interact in the extracellular environment. While these criteria naturally limit our analysis to a subset of possible contributing genes, they nonetheless lay the groundwork for future studies to analyze contributions of additional genes from the CHD candidate region.Success in identifying DSCAM and COL6A2 as mediators of CHD phenotypes in these studies resulted from the combined use of three systems: comprehensive candidate testing in flies, validation of synergistic genetics interactions in mice and cell culture, and high resolution genetic mapping in humans. In addition to these genetic studies, we then employed cell based assays to investigate the underlying molecular mechanisms regulated by these gene products. For this initial study, we restricted our screen to genes that we suspected had a high chance of causing heart defects based on We suggest that application of this multi-tiered genetic strategy should be broadly applicable to other multigenic genetic diseases in which sorting though many genetic combinations is necessary to identify promising candidate loci underlying disease phenotypes. Examples of such multigenic disorders include the identification of interacting genes within loci defined by the HapMap initiative Drosophila full-length cDNA of SH3β (CG8582) and COL18A1 (CG33171) were obtained from Open Biosystems. The mammalian and Drosophila cDNAs were cloned into pUAS vector and injected to w1118 embryos. Several independent transgenic lines were generated by P-element-mediated germ line transformation technique for each pUAS construct. GMR-GAL4 Stock was obtained from Bloomington, Indiana. The GMH5-GAL4 line All fly stocks were raised at 25 °C. Full-length cDNAs were mis expressed in the heart or developing eye using the GAL4-UAS transactivation system Heart rate, electrical pacing, semi intact heart preparation and movie analysis were conducted as described previously 250 µL of nonionic contrast was injected into the jugular vein over a period of 1 to 2 seconds and video images were acquired on half-inch super-VHS videotape at 30 frames per second under constant fluoroscopy with the XiScan 1000 C-arm x-ray system . Later, the interlaced video images were edited and digitally processed off-line  with a resolution matrix of 512x512 pixels, 256 shades of gray, 60 fields per second Mice were sedated with isoflurane. The jugular vein was dissected and cannulated for the intravenous saline administration. Imaging of the heart was performed using an Echo ultrasound system. Image acquisition in the axial four-chamber views was begun just before injection of contrast and continued until contrast effect in the myocardium had dissipated 3 isometric voxel resolution using an eXplore Locus SP MicroCT specimen scanner . Visualization of sections was performed with MicroView Software  and volume rendering was performed with OsiriX Medical Image software.High-resolution volumetric Computed Tomography (CT) of hearts was performed by Numira Biosciences  at 10-µmHearts were cut at the horizontal short-axis plane, fixed in 4% paraformaldehyde, embedded in OCT and sectioned. Frozen cryosections wild-type and double transgenic mice were stained with anti DSCAM (N-16) (Santa Cruz biotechnology) or anti COL6A2 (D20) (Santa Cruz biotechnology), and anti Rabbit Alexa594 secondary , counter stained with Hoechst 33342 (Invitrogen) imaged with a Perkin Elmer UltraView Vox spinning disk confocal microscope. For myocytes size determination, 14 micron frozen cryosections were stained with Alexa 594 –conjugated wheat germ agglutinin (Invitrogen), and the myocyte cross-sectional area was measured for assessment of cell size using NIH image J software. For staining of H9C2 and H9C2-DSCAM cells, cells were grown on 35 mm plates, fixed in 4% paraformaldehyde, permeabilized in PBS/Triton X100 0.1%, and stained with rabbit anti DSCAM , or mouse anti Myc  primary antibodies, Alexa594 secondary antibodies , counter stained with Hoechst 33342 (Invitrogen) and imaged with a Zeiss Axioplan 2 fluorescence microscope.DSCAM was cloned in to an expression vector expressing a C-terminal myc tag and a puromycin resistance gene. The promoter used was a modified chicken beta actin promoter which can be expressed in a wide variety of tissues and cell types (pCAGGS promoter). H9C2 cells were transfected and selection resistant clones were isolated. We chose a cell line that over-expressed DSCAM at the lowest level that we could still detect tagged DSCAM by western blotting.++, -Mg++) overnight, washed twice with PBS , and then incubated for 2 hours with 1% BSA/PBS  to block non-specific binding. In control (BSA) plates addition of collagen was omitted and plates were processed in parallel. 2.5×105 cells/ml H9C2 or H9C2-DSCAM cells were plated in serum free media on blocked 96 well plates in triplicate. At the indicated time points, wells were washed twice gently in serum free media, and attached cells were fixed in 10% neutral buffered formalin for 5 minutes. Cell number was determined by crystal violet staining, read at OD540. The percent of cells bound was calculated relative to cells plated in non-blocked wells which displayed 100% adherence at the end point of the experiment.Cell adhesion was performed as described Poly-T capture beads were used to isolate mRNA from 5 µg of total RNA from either H9C2 or H9C2-DSCAM cells grown on COL6 coated plates. First-strand cDNA was generated using random hexamer-primed reverse transcription, and subsequently used to generate second-strand cDNA using RNase H and DNA polymerase. Sequencing adaptors were ligated using the Illumina Genomic DNA sample prep kit. Fragments ∼200 bp long were isolated by gel electrophoresis, amplified by 16 cycles of PCR, and sequenced on the Illumina Genome Analyzer II.http://david.abcc.ncifcrf.gov/) Sequence reads were filtered for quality, failing tags with more than 8Ns or 8 bases with quality values lower than 3 standard deviations from the mean. Tags were then aligned using the top hat splice read mapper Dataset S1H9C2-DSCAM mRNA-seq data.(XLS)Click here for additional data file.Figure S1DSCAM and COL6A2 over-expression cause contractility defects in the fly heart. a) Representative 10 second M mode traces extracted from high speed movies of semi intact 1 week old flies. The genotypes tested were the heart specific GAL4 driver GMH5 alone (GMH5/+), flies carrying one or both the DSCAM and COL6A2 transgenes without GAL4 driver , or flies carrying both the GMH5-GAL4 driver and one or both transgenes . b-d) Histograms of Heart beat parameters distribution at 1 week old flies (N = 20), of heart period (b), diastolic interval (c) and systolic interval (d).(TIF)Click here for additional data file.Figure S2Documenting atrial shunting and atrial septal defects in the same heart of a DSCAM/COL6A2 double transgenic mouse. a) Saline contrast echocardiography showing abnormal shunting detected as bubbles that appear within 1 beat in the LV. Panels depict echocardiograms of the cardiac chambers in the axial plane of the heart, both before and after injection of saline. Blue arrows indicate time of injection, red arrows indicate the time bubbles are detected in LV, and yellow arrows indicate bubbles. Each panel consists of 10 frames merged for visualization purposes. b) Unstained serial cryosections of the same DSCAM/COL6A2 double transgenic heart that exhibited abnormal shunting as shown in (a) sectioned at 14 µm. The sections reveal frank holes in the atrial septum within the foramen ovale (red arrows) as well as dysmorphology of the atrial septum (green arrows), which may contribute to a fenestrated septum. Consecutive sections are shown at the atrial septum level, from the apex towards the base of the heart . Atrial septum (AS), right atrium (RA), left atrium (LA) right ventricle (RV), left ventricle (LV), tricuspid valve (TV), mitral valve (MV).(TIF)Click here for additional data file.Figure S3Expression of DSCAM in H9C2-DSCAM cells. a) Western blot of H9C2 and H9C2-DSCAM cells showing the relative expression level of DSCAM using a rabbit anti-DSCAM antibody, showing moderate increased level of expression in the H9C2-DSCAM cells relative to control H9C2 cells. b) Immunostaining using rabbit anti-DSCAM antibody showing increased expression in H9C2-DSCAM cells, mainly in perinuclear (Golgi/ER) (arrow) and membrane regions (arrowhead) . c) Immunostaining against the Myc tagged DSCAM identifies both perinuclear (arrow) as well as membrane staining, especially in areas between adjacent cells (arrowhead) .(TIF)Click here for additional data file.Table S1Drosophila homologs. Asterisk (*) indicates DS CHD fly and mammalian genes used to generate transgenic lines).Human DS CHD candidate genes and their (DOC)Click here for additional data file.Table S2Summary of heart performance parameter changes in flies expressing all candidate CHD genes in single and pairwise combinations. The three asterisks (***) indicate statistically significant differences from the corresponding control  and a minus (-) indicates no significant difference from the control.(DOC)Click here for additional data file.Table S3Transmission frequencies of DSCAM and COL6A2 transgenes in mice. For double transgenic mice, the frequency of transmission is reduced to 58% of the expected rate .(DOC)Click here for additional data file.Table S4List of Gene Ontology KEGG pathway genes.(DOC)Click here for additional data file.Table S5Genes associated with cardiomyopathy.(DOC)Click here for additional data file.Video S1Saline contrast echocardiography movie of 3 month old wild-type mouse.(AVI)Click here for additional data file.Video S2Saline contrast echocardiography movie of 3 month old DSCAM and COL6A2 double transgenic mouse.(AVI)Click here for additional data file.


We hypothesized that newer techniques would perform as well as solid media culture to quantify mycobacterial burden in laboratory specimens.Accurate quantification of mycobacterial load is important for the evaluation of patient infectiousness, disease severity and monitoring treatment response in human and 3H]-uracil incorporation assays, luciferase-reporter construct bioluminescence, and quantitative PCR(Xpert -MTB/RIF) using serial dilutions of Mycobacterium bovis and Mycobacterium tuberculosis H37RV. Mycobacterial colony-forming-units(CFU) using 7H10-Middlebrook solid media served as the reference standard.We compared the turn-around-time, detection-threshold, dynamic range, reproducibility, relative discriminative ability, of 4 mycobacterial load determination techniques: automated liquid culture (BACTEC-MGIT-960), [3 organisms. By contrast, BACTEC-MGIT-960 liquid culture, although only providing results in days, was user-friendly, had the lowest detection threshold (<10 organisms), the greatest discriminative ability , and the best reproducibility . Xpert-MTB/RIF correlated well with mycobacterial load, had a rapid turn-around-time (<2 hours), was user friendly, but had a detection limit of ∼100 organisms.All 4 assays correlated well with the reference standard, however, bioluminescence and uracil assays had a detection threshold ≥1×10Choosing a technique to quantify mycobacterial burden for laboratory or clinical research depends on availability of resources and the question being addressed. Automated liquid culture has good discriminative ability and low detection threshold but results are only obtained in days. Xpert MTB/RIF provides rapid quantification of mycobacterial burden, but has a poorer discrimination and detection threshold. M. tuberculosis burden is fundamental to the study of biologically meaningful immunological pathways, and drug and vaccine development in both human and murine models of disease. The latter include early bacteriocidal activity (EBA) studies related to drug development and evaluating vaccine efficacy in murine models Determining mycobacterial burden is a basic requirement for many laboratory and clinical studies, including those assessing disease severity and examining the efficacy of new therapies and interventions for tuberculosis (TB) control Culture on solid media using colony-forming units (CFU), is widely considered to be the gold standard for determining the number of viable organisms in a specimen or experimental condition, but is labor-intensive and has a long turn-around-time More recently, newer technologies such as the Xpert MTB/RIF system  have been developed for the rapid detection of TB using clinical samples. However PCR methods have been limited by their inability to distinguish viable from degraded organisms. Whilst detecting TB-specific mRNA from viable organisms is a potential solution, like PCR We hypothesized that newer automated mycobacterial load determination techniques perform as well as traditional measures. We therefore compared the performance characteristics of 4 quantitative techniques by evaluating turn-around-time, detection threshold, dynamic range, reproducibility and quantitative discriminative ability, with CFU on solid media as the reference standard.6 CFU per 500 ul) in sterile phosphate buffer solution (PBS) from 3 ml of frozen stock for each strain. Three vials of each dilution were provided for the five predetermined assays. In addition, all dilutions were inoculated onto solid media to confirm the number of CFUs at each dilution (Both BCG and H37RV luciferase reporter constructs (pSMT1 luciferase) dilution .Aliquots of 1, 10 and 100 CFU were plated in 6 replicates of 10 µl on 7h10 Middlebrook agar enriched with OADC. Plates were sealed in airtight bags and incubated at 37°C. Colonies were counted daily between days 7 and 14 using an inverted light microscope. The colony counts were over two days by two readers.A 500 µl aliquot of PBS containing the specified number of organisms was injected into pre-prepared mycobacterial growth indicator tubes (MGITs) , and the MGITs then incubated using the BACTEC-MGIT-960 automated culture system, which monitors the tubes for the presence of growth on a constant basis. Each dilution was prepared in triplicate and blinded to the MGIT operator. Time to positivity was recorded by instrument as the time between start of incubation and detection of growth.Luminescence was measured using a Modulus™ Microplate Multimode Luminometer (Turner). 25 µl of 1% (v/v) n-decyl aldehyde (Sigma), was injected into each well containing serial dilutions (1∶10 with 0.25% Tween/PBS) to a final volume of 125 µl. The results were read using a 0.5 s delay and a 0.5 s integration time and were expressed as relative light units (RLU).T values of probes were obtained using the software (Xpert MTB/RIF version 2.0).A 500 ul aliquot of H37RV and BCG was treated with sample buffer at a ratio of 1∶3 supplied by the manufacturer as recommended. The 2 ml mixture was later transferred into an Xpert MTB/RIF cartridge. The cartridge was loaded into the Gene Xpert IV instrument and the automated procedure started. 3H uracil  was added to each well. The plates were incubated for 24 hours at 37°C in a 5% CO2 humidified chamber and harvested onto fiberglass filter mats. The discs were placed into scintillation bottles containing 1 ml of Quicksafe  and the amount of incorporated tritiated uridine determined using a liquid scintillation counter and reported as counts per minute (CPM).200 µl of each dilution was pipetted into 6 wells of 4 separate U-bottomed 96 well plates and 50 ul of The turnaround time was defined as the time taken from the start of each assay after preparation of the aliquots until a bacterial load determination was possible (either automated output or colony counting).The detection threshold was defined as the lowest colony number detected by the assay: The result from 2 out of 3 MGIT bottles, 2 out of 3 Xpert MTB/RIF cartridges or >50% of CFU/RLU/CPM replicates for a particular dilution were as the reliable lower limit of detection for comparative purposes.www.graphpad.com).The ability of the assay to detect a difference between serial dilutions was assessed using a 1-way ANOVA with correction for multiple comparisons (Tukey). GraphPad Prism software  across all dilutions. To compare reproducibility between assays the mean coefficient of variance for BCG dilutions was calculated for each assay.A comparative overview of all the mycobacterial load assays is summarised in 1 and 1×102 CFU were used for plating on 7H10 agar. The coefficient of variance across the three dilutions ranges was 22%.Although technically simple to perform, plating for CFU was time consuming and accuracy and reproducibility are affected by pipetting skills as well visual counting of colonies. Counting of CFUs and determination of mycobacterial load was possible approximately 10–14 days after plating . Determi6 to 1×100 CFU; 6 CFU. BACTEC-MGIT-960 was able to detect differences in mycobacterial load as small as 1×102 organisms at low concentrations and 1×105 at higher concentrations.The turnaround time for the BACTEC-MGIT-960 system was defined as time to positivity (TTP) and ranged from 117 hours (∼5 days) to 467 hours (∼19 days) for BCG and 123 hours (∼5 days) to 528 hours (∼22 days) for H37RV  values as compared to BCG (coefficient of variance 6%) and did not detect less than 100 CFU. For the BCG experiments mean CT values were statistically significant across all ranges thus providing excellent discriminative ability. Although the mean Ct values across the range of loads tested were statistically different for the H37RV experiments (1way ANOVA p<0.001), when correcting for multiple comparisons (Tukey), statistical significance was only present at a 2 log change in CFU i.e. 1×104 vs. 1×106.The automated real time MTB/RIF assay using the Gene Xpert IV system was user friendly and minimal technical steps were required prior to inserting the cartridge into the machine. Xpert MTB/RIF results were available within 2 hours of the experiment using the Gene Xpert IV machine with 4 cartridge bays (only 4 samples could be run at once). BCG reproducibility was excellent (coefficient of variance 3%) and the automated PCR reliably detected 100 organisms . At the Accurate determination of mycobacterial numbers is essential for evaluating infectivity, the efficacy of treatments and for many projects in translational research. However, there are limited reports comparing the performance of different mycobacterial load determination techniques. We evaluated several techniques and to our knowledge head-to-head comparison of several technologies has not been previously undertaken.Our study has demonstrated that (i) Xpert MTB/RIF provides a rapid measure of mycobacterial burden above a threshold of ∼100 organisms/sample; (ii) TTP using MGIT 960, whilst accurate, with better discriminative ability of mycobacterial load (than Xpert MTB/RIF) and a detection threshold down to 1 CFU, has a substantially longer turn-around-time; (iii) bioluminescence and uracil incorporation assays are limited by lack of discrimination below 1000 organisms. Thus, none of these assays has ‘ideal’ performance characteristics and the selection of an assay has to based on the experimental question under study and details of the study design including the likely range of bacterial burden which is anticipated, need for rapid results, desired reproducibility, and available laboratory and financial resources. In resource-limited settings determination of CFU using solid media is likely to remain the method of choice as it is likely to be associated with the least cost.Although PCR for mycobacterial load is not a novel technique, the Xpert MTB/RIF assay may overcome several of the known drawbacks of real-time PCR. Most notably, apart from the semi automation of the methods, DNA from degraded organisms is thought to be removed in the wash step and only intact organisms are retained in the cartridge mesh for the PCR step Rapid turn-around-time is a further advantage of the Xpert MTB/RIF assay, which might be of value in drug development. For example, for providing ‘real time’ serial quantification of bacterial burden during early bacteriocidal activity studies. Its ability to detect quantitative changes in viable organisms will need to be evaluated as all PCR techniques will detect DNA from both live and dead organisms Liquid culture using the automated BACTEC 960 MGIT system is an attractive quantification technology for both clinical and laboratory studies. Already incorporated into EBA studies Bioluminescence and uracil incorporation assays both provide rapid turnaround time and are have been extensively used in laboratory studies in vitro laboratory work done with a specific strain and thus may not be generalizable to clinical strains or clinical samples where sample quality and inhibitors etc. may impact on findings. Smear microscopy was not used as a reference standard in this study as the primary focus was on laboratory settings where organism load below 100 000 organisms/ml are frequently used and smear looses sensitivity below this threshold. Moreover, we required a highly discriminative technique from a quantitation point of view . Therefore we chose not to evaluate smear microscopy. Thus, because a single strain was used for all experiments the MGIT findings may also not be generalizable to clinical samples or different strains. Whilst Ct correlated well with mycobacterial burden, as shown in recent studies There are several limitations to this study. The findings here pertain to In summary, no single mycobacterial quantitative technique has ideal performance characteristics. Thus, the choice of assay will largely depend on the research context, study question, and the relative tradeoffs of cost versus turnaround time. Automated systems like MGIT are sensitive and have high discriminatory value but a long turnaround time. Xpert MTB/RIF is a good quantitative tool with rapid turn-around-time but its detection threshold was not as good as automated liquid culture. Although solid culture is the most labor-intensive it likely remains the cheapest option for highly discriminative quantification of mycobacterial load over a wide dynamic range. Cost effectiveness and clinical treatment follow up studies are still required for Xpert and MGIT.


To clarify their phylogenetic position, we amplified and sequenced the SSU rRNA gene obtained from four independent DNA extractions of A. flavum and three independent DNA extractions of A. wrightianum. Our molecular data demonstrate that genera Archerella and Amphitrema form a fully supported deep-branching clade within the Labyrinthulomycetes (Stramenopiles), together with Diplophrys sp. (ATCC50360) and several environmental clones obtained from a wide range of environments. This newly described clade we named Amphitremida is diverse genetically, ecologically and physiologically. Our phylogenetic analysis suggests that osmotrophic species evolved most likely from phagotrophic ancestors and that the bothrosome, an organelle that produces cytoplasmic networks used for attachment to the substratum and to absorb nutrients from the environments, appeared lately in labyrithulomycete evolution.Micro-eukaryotic diversity is poorly documented at all taxonomic levels and the phylogenetic affiliation of many taxa – including many well-known and common organisms - remains unknown. Among these  It includes the genera Amphitrema and Archerella has always been problematic. Penard Archerella within Amphitrema and described Amphitrema flavum as a “Thecamoebidae” with a filamentous pseudopodia and rich with zoochlorellae endosymbiont. Later, Wailes Amphitrema and DiplophrysAmphitrema ( =  Archerella) flavum and its tubulocristate mitochondria; such structures are repeatedly found within “core Cercozoa” Amphitrema and Archerella are considered as forming part of a single family, the Amphitremidae, together perhaps with the enigmatic Paramphitrema. They have been placed as testate amoebae with filopodia incertae sedisAmphitrema remained amongst the protist genera with uncertain affiliation.Taxonomical placement of genera Archerella and Amphitrema. In a second step, we performed a search in GenBank to assess the environmental diversity of this clade and the variety of environments colonized.In order clarify their phylogenetic position within the tree of eukaryote; we performed the first molecular study based on SSU rRNA gene sequences in the two most common genera of Amphitremidae, Archerella flavum and Amphitrema wrightianum  and Praz-Rodet bog in the Jura Mountains of Switzerland (46°34′ N 6°10′ E). An authorization (No 1449) was delivered by the “Service forêts de la faune et de la nature du canton de Vaud” (state office for nature conservation) for sampling in Praz-Rodet for 2011 and 2012 (January 2011 to end of summer 2012). The Duffey Lake Sphagnum sample was not collected within the Provincial Park and therefore, no permits were required. Cells were extracted from Sphagnum mosses through serial of filtrations, and then were washed 3 to 4 times with distilled water Archerella flavum and three from Amphitrema wrightianum, each of these extractions contained between 50 to 70 cells. Both species were documented using light microscopy (We sampled ghtianum  from wetcroscopy .Archerella flavum and three from Amphitrema wrightianum) were obtained by two amplifications. The first amplification was performed using universal eukaryotic primers, 1EKF (CTGGTTGATCCTGCCAG) and 1498R (CACCTA CGGAAACCTTGTTA) or 1520R (CYGCAGGTTCACCTA), in a total volume of 30 µl with amplification profile consisting of . The positive products were cloned into pCR2.1 Topo TA cloning vector (Invitrogen) and transformed into E. coli TOP10’ One Shot cells (Invitrogen) according to the manufacturer’s instructions. Clone inserts were amplified with vector T7 and SP6 primers. The expected size clones from PCR amplifications were purified with the NucleoFasts 96 PCR Clean Up kit from Macherey-Nagel  and sequenced with an ABI PRISM 3700 DNA Analyzer  using a BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit (PE Biosystems). We also designed the following primers for internal sequencing: Archer1F (GTAAATTACCCAATCCYAAMTCG), Archer1R (AAACATTTTGCTTTCGC), and Archer2R (TTTGTCCTGCCCTGCT). The positive products were cloned; and two to five clones from each extraction of Archerella flavum and Amphitrema wrightianum were sequenced. Sequences are deposited in GenBank with the Accession Numbers: Amphitrema wrightianum PR-1 (KC245091); Amphitrema wrightianum PR-2 (KC245092); Amphitrema wrightianum PR-2 (KC245093); Archerella flavum BC-1 (KC245094); Archerella flavum BC-2 (KC245095); Archerella flavum BC-3 (KC245096) and Archerella flavum BC-4 (KC245097).DNA was extracted using a guanidine thiocyanate-based protocol All SSU rRNA gene sequences were submitted to BLAST http://phylobench.vital-it.ch/raxml-bb/) using the GTR+Г+I model. Model parameters were estimated in RAxML over the duration of the tree search. The obtained tree was compared to the one that built by Bayesian analysis using the software MrBayes v. 3.1.2 th generation, the tree with the best likelihood score was saved, resulting in 10,000 trees. The burn-in value was set to 25%. Trees were viewed using FigTree (a program distributed as part of the BEAST package). In addition, we performed approximately unbiased (AU) tests The maximum likelihood tree was built using RAxML version 7.2.8 algorithm Archerella flavum and three from Amphitrema wrightianum with SSU rRNA gene length of 1287 bp and 1351 bp, respectively. The most similar SSU rRNA gene sequences to ours as revealed by BLAST were members of the labyrinthulida and thraustochytrida , plus the amoeboid Diplophrys and some environmental sequences. Therefore, we built an alignment that included some of the available SSU rRNA gene sequences of Labyrinthulomycetes/Labyrinthulea and other related taxa. Our constructed phylogenetic trees inferred from both RAxML and Bayesian analyses had the same topology  and (3) the group formed by Archerella flavum and Amphitrema wrightianum together with other related taxa: Diplophrys sp. ATCC50360(AF304465), and several environmental sequences, including PR3_4E_52 (GQ330589) from a peat bog, 528-O7 (EF586082) from freshwater, plus fourteen sequences from anoxic/micro-oxic deep-sea environment. As the position of Paramphitrema remains dubious Amphitrema and Archerella. As members of this group have traditionally been treated under the ICZN , this code will continue to being applied to it regardless of current or future phylogenetic placement Amphitrema. Amphifilidae and Amphitremida both received maximal support values  (Amphitrema wrightianum and Archerella flavum plus clone PR3_4E_52 (i.e. Amphitremidae) formed together a moderately supported clade . All the four obtained A. flavum sequences were exactly identical, while the obtained sequences of A. wrightianum PR-2, showed two nucleotides substitution at position 595 bp and 854 bp in comparison of both A. wrightianum PR-1 and PR-3 sequences. Our results have been confirmed by cloning the SSU rRNA fragments from each taxa.We obtained seven SSU rRNA gene sequences, four from topology . Labyrin = 1.00) . AmphitrA. wrightianum sequences. The freshwater environmental sequence 528-O7 (EF586082) had a basal position with respect to Archerella flavum and Amphitrema wrightianum. Fourteen SSU rRNA sequences from marine anoxic and micro-oxic water column branched as a sister clade to the peat bog+freshwater clade.The environmental peat bog clone PR3_4E_52 was also very closely related to these sequences and showed 99% similarity to p-values = 0.49 and 0.48 respectively).Following up on these results, we performed approximately unbiased (AU) test Archerella and Amphitrema belong to the Labyrinthulomycetes/Labyrinthulea (Stramenopiles) (Pseudodifflugia (Cercozoa), or the Foraminifera. Filose pseudopodia therefore appeared several times in eukaryotic evolution, not only within Rhizaria but also in some Opisthokonts  , rather Labyrinthula and Aplanochytrium also use these cytoplasmic networks for gliding Archerella, Amphitrema, Diplophrys and Amphifila move also by filose ectoplasmic extensions they do not possess a true bothrosome, and biflagellated stage have not yet been observed The labyrinthulida and thraustochytrida are characterized by the presence of a bothrosome or a ‘sagenetosome’ , an organelle that produces cytoplasmic networks (extensions of the plasma membrane) to absorb the nutrients from the surrounding environments, similar in that to fungi Amphitrema, Archerella, Diplophrys and Amphifila marina might be ancestral to both Amphitremida and Amphifilidae. The AU test does not reject the existence of one clade grouping both Amphitremida and Amphifilidae and a second clade uniting the thraustochytrida together with labyrinthulida, an evolutionary pathway that appears the most parsimonious because it implies a single appearance of the bothrosome, a unique feature of thraustochytrida and labyrinthulida , in contrast to earlier suppositions Our phylogenetic analysis suggests that the typical organisation with two symmetrical pseudopodial tufts found in nthulida , and a sSorodiplophrys stercorea, an organism isolated from cow and horse dung with bilateral symmetry and filose pseudopodia, and devoid of bothrosome; interestingly, it relies entirely on osmotrophy Another possible candidate for the assignment to both Amphitremida and Amphifilidae is Archerella flavum sequences, and also, surprisingly, fourteen sequences obtained from anoxic and micro-oxic water column from the Cariaco Basin in the Caribbean Sea Archerella and Amphitrema respectively, but were not included in our analysis due to their short length. Nevertheless, these sequences further illustrate the diversity of habitats colonized by the hitherto unrecognised clade of Amphitremida.Our tree analysis including environmental clones revealed an unexpected diversity of organisms branching within Amphitremida that derived from a very wide range of environments . These iAmphitrema wrightianum sequences, from which it differs by three nucleotides at most. The different extractions of A. wrightianum also showed small differences in their sequences (up to two nucleotides). This diversity suggests the presence of several genotypes within the morpho-species A. wrightianum and/or the existence of several closely related taxa (cryptic species). Indeed, confusion with the very similar-looking peat bog species A. stenostomaAmphitrema species  obtained by Lara et al., The Amphitremida represents a novel major clade within the Labyrinthulomycetes. This clade has colonised environments as divergent as peat bogs, freshwater and the oceans, ranging from nitrogen-depleted environments to eutrophic (sludge); their metabolisms vary from aerobic to anaerobic/micro-aerophilic, they can be phagotrophic or mixotrophic and have a planktonic or a benthic lifestyle. This illustrates the immense versatility of this group that certainly encompasses an even larger environmental genetic diversity than currently known. The true magnitude of this diversity will most probably be revealed by future massive environmental sequencing studies.


In past, numerous methods have been developed for predicting efficacy of short interfering RNA (siRNA). However these methods have been developed for predicting efficacy of fully complementary siRNA against a gene. Best of author's knowledge no method has been developed for predicting efficacy of mismatch siRNA against a gene. In this study, a systematic attempt has been made to identify highly effective complementary as well as mismatch siRNAs for silencing a gene.http://www.imtech.res.in/raghava/desirm/) for designing highly effective siRNA for silencing a gene. This tool will be helpful to design siRNA to degrade disease isoform of heterozygous single nucleotide polymorphism gene without depleting the wild type protein.Support vector machine (SVM) based models have been developed for predicting efficacy of siRNAs using composition, binary and hybrid pattern siRNAs. We achieved maximum correlation 0.67 between predicted and actual efficacy of siRNAs using hybrid model. All models were trained and tested on a dataset of 2182 siRNAs and performance was evaluated using five-fold cross validation techniques. The performance of our method desiRm is comparable to other well-known methods. In this study, first time attempt has been made to design mutant siRNAs (mismatch siRNAs). In this approach we mutated a given siRNA on all possible sites/positions with all possible nucleotides. Efficacy of each mutated siRNA is predicted using our method desiRm. It is well known from literature that mismatches between siRNA and target affects the silencing efficacy. Thus we have incorporated the rules derived from base mismatches experimental data to find out over all efficacy of mutated or mismatch siRNAs. Finally we developed a webserver, desiRm ( RNA interference (RNAi) is a natural mechanism evolved in complex organisms to regulate the gene expression. This mechanism also provide defense against viruses and transposable material to maintain the genome integrity et al. analyzed experimental data to understand relationship between the siRNA sequence and its silencing effect on 34 mRNA species siRNAs have become an important tool for silencing gene of interest and have emerging as potential therapeutics. The beauty of the system that makes it a powerful tool lies in sequence specificity towards particular gene, its quick effect, and cost effectiveness. Importantly, it makes feasible for large-scale functional genomics studies. It has been shown that knockdown effect (efficacy) of siRNA is varying according to target site on mRNA and hence, very limited set of siRNAs show high efficacy et al. reveals position of the mismatch generated in the target influence silencing and categorized them as; (a) High tolerance: mismatch at position 1, 2, 18, or 19, which does not affect the efficacy. (b) Low tolerance: mismatch at position 5-11 which results into abolishing the RNAi activity and remain position is (c) of moderate tolerance e.g. at position 17 of siRNA both A:C and C:A mismatched are well tolerated while at position 12 only A:C mismatch is tolerated not C:A. However, study also demonstrated the importance of creating mismatch between sense and antisense strand of siRNA in order to make more asymmetric siRNA which leads to improve silencing efficacy Initially it was believed that full complementary siRNA is needed to silence a target gene. However, studies have shown that siRNA behaves like miRNA and suppress protein synthesis when it is not fully complementary to the target, indicating mismatches are allowed during target selection by siRNA Investigation indicates that a large portion in mRNA could not be targeted for siRNA because of having low efficacy http://www.imtech.res.in/raghava/desirm/.This study having two sections: (1) The development of a model for predicting siRNA efficacy, and (2) The creation of mutation in the siRNA sequence to enhance its efficacy. This facility is accessible to scientific community through web based portal at et al.et al.The main dataset used in this study contains 2182 siRNAs. All models trained, tested and evaluated using five-fold cross-validation techniques on main dataset. This dataset was obtained from Huesken Nucleotide composition: The nucleotide composition determines the occurrences of different types of nucleotides, dinucleotides, trinucleotide etc. We compute mono-, di,- tri-, and tetra-nucleotide composition of siRNAs that generate vector of 4 , 16 , 64 , and 256  respectively.Split nucleotide composition: In this case whole sequence was divided into two equal parts and nucleotide composition of each part is calculated separately. Composition of both part is used to develop our models, in this case dimension of input vector was doubled st order) where interaction between ith and (i + 1)th nucleotide is taken into account. In case of second order dinucleotide composition, interaction of 1st with 3rd nucleotide is considered i.e. ith and (i + 2)th. Similarly in case of third order dinucleotide composition interaction of 1st with 4th nucleotide is considered.Higher order nucleotide composition: In simple dinucleotide composition we considered local order . Thus, a sequence of 21 nucleotides of miRNA was represented by a vector of dimensions 84 (4×21).Binary pattern of dinucleotides: Instead of considering one nucleotide as in binary pattern, occurrence of two consecutive nucleotides at particular position was considered.Binary of condense: Sequence was divided into two equal parts and binary pattern of both part were calculated and merged into each other (like hairpin structure) so that 5′-end and 3′-end of a sequence are at same position.Hydrogen bond: The hydrogen bonding properties were depicted as “3” for G and C while “2” was assigned in case of A and U.Thermodynamic: The value of thermodynamic propertied at each position were taken from Target site accessibility: Target site accessibility in terms of probability of being unpaired is calculated using RNAplfold http://www.csie.ntu.edu.tw/~cjlin/papers/guide/guide.pdf).Scaling of feature: During hybrid approach various different features were considered at a time creating a large range of feature values that resulted into the poor performance of models lightIn order to develop models for siRNA efficacy prediction, various features of siRNAs were used. SVM2), III) mean absolute error (MAE), root mean squared error (RMSE). All models were evaluated using five-fold cross validation technique.In order to evaluate performance of our models, we used following standard parameters; 1) correlation coefficient (R), II) coefficient of determination . First SVM based model was developed using binary pattern of nucleotide composition and achieve correlation coefficient of 0.637 . This mo21 in this study  for model development. The best SVM model (desiRm) achieved correlation coefficient of 0.647 and 0.654 on training dataset (2182-sequences) and independent dataset (249-sequences) respectively. The marginal improvement in the performance was observed due to incorporating target site accessibility information. This supports earlier finding about the importance of this feature in designing functional siRNA In past, several investigations reported the importance of target site accessibility in mRNA to design effective siRNA. Hence, we also integrated target site accessibility feature along with nucleotide frequency and binary pattern feature (desiRmMismatch Efficacy (ME) of mutated siRNA.In order to get more potent siRNA, we generated mutation on every position of 19 nt antisense with all four nucleotides of an siRNA. Efficacies of these mutated siRNAs were predicted using our SVM model desiRm. However, the mutant siRNA when bound with target sequence caused mismatch and hence affected the silencing efficiency. Therefore, based on the experimental data a scoring method has been developed, which deduced effect of position and/or identity of mismatch from predicted efficacy to find out overall ME =  predicted efficacy- Σ reduced efficacy (due to mismatch)Initially we generated the single mutation that makes 57 different permutation of single siRNA. The repression changes affected by position of mismatch and identity of mismatch between siRNA:target is taken from experimental data th position (uAGUGGAAAGUACAUCAGA) its predicted efficacy will be 0.776, when it is fully complementary with target sequence. But as we noticed that first base of siRNA causes mismatch of U:G and causes decrease in efficacy by 0.066  is made against a target region (UCUGAUGUACUUUCCACUG) in a mRNA NM015213. The siRNA is fully complementary with target having actual and predicted efficacy of 0.479 066 from . Thus miet al. only used single siRNA against mutated targets The experimental studies carried out by Dahlgren (1) submit mRNA: effective siRNA can be detected against the mRNA ((2) submit siRNA, where user can put its 19 nt long antisense siRNA generated from other software and its target sequence. However, this field did not consider the target site accessibility feature during efficacy calculation. Further mutations in siRNA can be generated by clicking the antisense sequence. This strategy can also be used to generate siRNA with very low efficacy against an off-target.A user-friendly webserver has been developed on SUN server under Solaris environment using HTML, PERL, and CGI-PERL. There are two input fields; the mRNA . The outthe mRNA . If one the mRNA . The outet al.i.e. targeting same site with different siRNAs (each siRNA having one-base substitution at different position) to manipulate the efficacy of siRNA and to get those siRNAs which can better discriminate between mutant and wild type target. Thus siRNA give rise to single-nucleotide mismatch with mutant-target while two-nucleotide mismatch with wild-type. They reported that introducing base-substitution at specific position in siRNA depleted the mutant transcript while least affected on wild-type. When we predict the efficacy of siPrnp 102 (T9) by desiRm a correlation coefficient of 0.725 was achieved between actual and predicted efficacy Click here for additional data file.Figure S2Snapshot of desiRm input field where mRNA can be submitted to get siRNAs.(JPG)Click here for additional data file.Figure S3Snapshot of desiRm output result with fully complementary siRNAs. Each row contains sequence of siRNA, target position, target sequence and accessibility with predicted efficacy. To improve the efficacy of 197th siRNA targeting on 164th position (highlighted), click this sequence.(JPG)Click here for additional data file.Figure S4Snapshot of desiRm output result with single-mutated siRNAs. Each row contains mutated siRNA, position of mutation, type of mutation, target sequence and accessibility, with predicted efficacy. First sequence (WT) is original, mutation at 1st position in siRNA increase their efficacy to 0.710. Further improvement could be achieved by click on siRNA.(JPG)Click here for additional data file.Figure S5Complete CDS of Homo sapiens prion protein (PRNP) gene (wild type). The nucleotides in bold and red color indicate the position of nucleotide variation in mutant genes reported. Mutant PRNP-P102L has mutation at position 377(C→U); mutant PRNP-P105L has mutation at position 386(C→U); mutant PRNP-D178N has mutation at position 564(G→A). Highlighted regions are targeted by siRNAs in both wild type and mutants by Ohnishi et al.(PDF)Click here for additional data file.Table S1Performance of SVM-based model for siRNA efficacy prediction developed using hybrid of features.(DOCX)Click here for additional data file.Table S2Assessment of desiRm on experimentally verified mismatched siRNAs of siPrnp105(T10). siPrnp105(T10) and its various mutant siRNAs were targeted against prion protein genes (PRNP) and its mutant allele (PRNP-P105L). Mutated base in siRNA is denoted by small letter while mismatch base between siRNA and target are denoted by bold letter. Data of actual efficacy of siRNAs were taken from experimental work reported by Ohnishi et al. Predicted efficacy denotes efficacy of desiRm. All sequences are in 5′ to 3′ direction. Correlation coefficient between actual and predicted efficacy is R = 0.586.(DOCX)Click here for additional data file.Table S3Assessment of desiRm on experimentally verified mismatched siRNAs of siPrnp102(T10). siPrnp102(T10) and its various mutant siRNAs were targeted against prion protein genes (PRNP) and its mutant allele (PRNP-P102L). Mutated base in siRNA is denoted by small letter while mismatch base between siRNA and target are denoted by bold letter. Data of actual efficacy of siRNAs were taken from experimental work reported by Ohnishi et al. Predicted efficacy denotes efficacy of desiRm. All sequences are in 5′ to 3′ direction. Correlation coefficient between actual and predicted efficacy is R = 0.607.(DOCX)Click here for additional data file.Table S4Assessment of desiRm on experimentally verified mismatched siRNAs of siPrnp178(A9). siPrnp178(A9) and its various mutant siRNAs were targeted against prion protein genes (PRNP) and its mutant allele (PRNP-D178N). Mutated base in siRNA is denoted by small letter while mismatch base between siRNA and target are denoted by bold letter. Data of actual efficacy of siRNAs were taken from experimental work reported by Ohnishi et al. Predicted efficacy denotes efficacy of desiRm. All sequences are in 5′ to 3′ direction. Correlation coefficient between actual and predicted efficacy is R = 0.666.(DOCX)Click here for additional data file.


Significant overexpression of CD133 (p<0.02) was observed by immunohistochemical staining in tissues from patients with recurrent disease versus those without disease recurrence. Relative risk analysis between these two groups suggested that the patients with recurrence or metastatic lesion had a greater than 2-fold overexpression of CD133. In addition, immunodetectable CD133 corroborated with upregulation of CD133 RNA levels (14- to 30-fold) as assessed by quantitative real-time reverse transcription-PCR (qRT-PCR) comparison of melanoma cell lines derived from patients with poor clinical outcomes and short overall survival (<10 months), vs. those derived from patients with good clinical outcomes and longer overall survival (>24 months). Further, cells derived from patients, and MACS-sorted according to their CD133 status retained their CD133-positivity (>95%) or CD133-negativity (>95%) for more than 8 passages in culture. CD133+ cells could repopulate and form tumors (p<0.03) in athymic NCr-nu/nu mice within 8 weeks while no tumors were observed with CD133− phenotype . Taken together, the study demonstrates, for the first time, that there exists a clonal dominance of a CD133+ population within the hierarchy of cells in cutaneous tissues from patients that have undergone successive progressive stages of melanoma, from primary to metastatic lesions. CD133, thus, provides a predictive marker of disease as well as a potential therapeutic target of high-risk melanoma.Chemotherapeutic refractoriness of advanced cutaneous melanoma may be linked with melanoma-initiating cells, also known as melanoma stem cells. This study aimed to determine relative risk of clonal dominance of the CD133 High-risk cutaneous melanoma, a highly aggressive form of skin cancer, is minimally susceptible to current treatment strategies for systemic disease and thus these strategies have not yielded any appreciable survival benefit. Chemotherapeutic refractoriness of advanced cutaneous melanoma is attributable to several resistance mechanisms resulting from the outgrowth of resistant clones, subsequently promoting tumor recurrence and metastasis. Recently, therapy-resistant clones are implicated as melanoma initiating cells (MICs) also known as melanoma stem cells (MSCs). CD133 is now recognized as an important cancer stem cell-associated marker in cutaneous melanoma.The existence of cancer cells with stem cell-like characteristics has been reported in a variety of human malignancies –5 includSeveral reports have implicated the expression of different markers of melanoma stem cells, including the self-renewal transcriptional factor, Bmi-1, in primary and metastatic melanoma. Four subpopulations of cells have been identified in melanoma tissues, including CD20-positive, CD133-positive, label-retaining cells and side-population (SP) cells. These cell populations possess stem-cell characteristics with defined self-renewal capacity, differentiation potential, and high tumorigenicity , CD166 a+ MSCs in tissues that could be applied to early diagnosis, prognosis, or to the development of more effective strategies have or disease monitoring and treatment modalities. The expression levels of CD133 were thus assessed in tissues from patients with or without recurrent disease and compared with melanoma thickness as a prognostic factor. In addition, CD133 expression in tissues from patients with primary melanoma successively progressed to lymph node metastasis and distant metastasis were compared, utilizing immunohistochemistry (IHC) and quantitative real-time reverse transcription-PCR (qRT-PCR).The present study examines relative risk analysis on clonal dominance of CD133μm were subjected to immunohistochemical staining using monoclonal antibodies to a distinct subset of MSCs including CD133/prominin-1 (Abcam), a CSC and neural stem cell marker; ABCB5 (Genway), a chemoresistance mediator; CD166 (BD Biosciences), an adhesion molecule; and Nestin (Abcam); a lineage marker. The tissue sections were immunostained using a Bond-Max automated staining system (Leica). The reactions were developed using a biotin-free bond polymer refine detection kit (Leica) and visualized with 3′3-diaminobenzidine (DAB) substrate. Immunoexpression of stem cell markers was evaluated with a Zeiss Axioscope microscope using a semi-quantitative scoring system for intensity of staining of stem cell markers on tumor cells. Fisher’s exact test, Odds ratio analysis and relative risk analysis were performed to determine statistical significance.Formalin-fixed, paraffin-embedded (FFPE) tissues were selected from 3 groups of consenting patients, after Institutional Review Board protocol approval. Group I consisted of melanoma patients who did not develop disease recurrence or metastases (n=18). Group II was comprised of melanoma patients who developed metastatic lesions (n=18). Group III included patients with disease progression from primary melanoma to lymph node (LN) metastasis and subsequently to distant metastasis (n=8). Tissue sections measuring 5 Human melanoma cell lines were established from fresh metastatic tumor tissues of consenting patients comprising two groups. Group A (n=4) consisted of patients  with poor clinical outcomes and short overall survival (<10 months), and group B (n=4) was comprised of melanoma patients  with good clinical outcomes and longer overall survival (24 months). The majority of cell lines were derived from lymph node metastases.2, floating debris was discarded and fresh complete medium was added. Cultures were fed 2–3 times per week, replacing about half of the spent medium. Melanoma cell lines were split when near confluence and sub-cultured at 4×104 viable cells per cm2 surface area in polystyrene culture flasks. Cultures were shown to be free of mycoplasma contamination using the MycoProbe™ mycoplasma detection kit . To insure that each cultured cell line consisted of melanoma cells, each cell line was stained and analyzed by flow cytometry for melanoma-specific antigens MART-1, gp100, TRP75, or melanoma-associated chondroitin sulfate proteoglycan (MCSP). At least one of these four melanoma antigens was observed in order to include the cell line in the study. All cell lines were early passages of less than 20.Single cell suspensions were prepared from freshly resected tumor tissue specimens by mechanical mincing; no enzymatic dissociation was used. Viable tumor cells were cultured in Iscove’s medium supplemented with 10% fetal bovine serum (FBS) and antibiotics. After overnight incubation at 37°C with 5% CO+ or CD133− cells were isolated from cell culture suspensions per kit instructions using magnetically labeled CD133 Micro Beads and a MACS® Column . To increase the purity, the positively selected cell fraction containing the CD133+ cells were further separated over a second MACS® column.CD133+ or CD133− cells were cultured in DMEM/F-12 (1:1) plus 1% penicillin/streptomycin, 20 μg/ml each of epidermal growth factor (EGF) and fibroblast growth factor (FGF), and 1X B-27 supplement (Invitrogen). Culture plates were coated in 10 mg/ml 2-hydroxyethly methacrylate  in order to ensure a spheroid suspension. Flow cytometry was used to confirm the populations as CD133+ or CD133− using the monospecific mouse mAb CD133/2 (Miltenyi Biotech). Athymic NCr-nu/nu mice were purchased from Harlan Laboratories (IN). Purified CD133 cells were counted using a Coulter Counter  and then serially diluted. Cells were re-suspended in a mixture of growth media containing Matrigel , and injected subcutaneously into each hind flank. Animals were each injected with 2×105 or 1×103 of either CD133+ or CD133− cells; each group of cells was injected in replicates of four. Animals were allowed to recover from anesthesia and monitored daily to check for the presence of palpable tumors. Tumor growth was measured using a digital micrometer and calculated using the modified ellipsoid formula (1/2 length x width2) for tumor volume as described previously  according to the manufacturer’s instructions. Absolute quantitation of CD133 gene transcripts was performed using qRT-PCR analysis on a 7500 real-time PCR system using the SYBR Green method (Applied BioSystems). The following human-specific intron spanning primer pairs for CD133 were used: forward, CATCCACAGATGCTCCTAAGGC; reverse, GCTTTATGGGAGTCTTGGGTC. Cycle conditions were as follows: 1 cycle at 95°C for 10 min, 40 cycles at 95°C for 15 sec, and 60°C for 1 min. The specificity of the PCR product was verified by melting curve analysis. Threshold cycles of primer probes were normalized to those of 18S rRNA. Absolute values of transcripts were calculated using the standard curve method.Total RNA was prepared from 8 melanoma cell lines established as described above using Trizol (Invitrogen) and an RNeasy Mini Kit (Qiagen). From 1 As shown in Significantly greater expression of CD133 in tissues was seen in melanoma patients who developed metastatic lesions/recurrence of the disease, when compared with the group of melanoma patients who did not develop any metastatic lesions or recurrence  and distant metastasis .To investigate the potential of CD133 as a molecular biomarker of melanoma progression and disease recurrence, we assessed CD133 mRNA transcript levels by qRT-PCR in low passage (<20) primary cell lines established from fresh tissues of patients with either poor outcome and short overall survival, or from patients with good outcome and long overall survival status. Using 18S rRNA as a control, CD133 mRNA expression levels in each individual cell line derived from patients with poor clinical outcome and short overall survival  were compared with four cell lines derived from melanoma patients with good clinical outcome and longer overall survival  in athymic NCr-nu/nu mice within a short span of 8 weeks. However, tumor formation could not be observed in the mice injected with matched cell density of CD133− phenotype, nor with up to 200,000 cells  and CD133 gene transcript levels in cell lines from metastatic patients with poor clinical outcome and short overall survival status. These findings suggest that CD133 represents a hyperpolarized cell population with tumorigenic potential at metastatic sites and is linked to survival status of the patient.in vitro and in vivo, these studies have not been able to define a phenotypically distinct cell population with tumorigenic potential in target tissues with different clinical outcome or associated with the progression of the disease.Although most CSCs in metastatic colon cancer , non-smain vivo assessment of a CD133+ subset population further confirmed that CD133+ MSCs contain self-renewal and repopulating capabilities.It has been widely reported that CSC represent rare tumor cell populations that possess unique properties making them important for tumor initiation, growth and metastasis. As per the CSC hypothesis, tumor initiation is regarded as an exclusive characteristic of CSCs . HoweverAlthough the CD133 cell population co-expresses other stem cell markers or may represent overlapping markers, recent reports on metastatic melanoma containing CD133 and ABCG2-positive cells with enhanced tumorigenic potential further support our present findings on consistent expression of stemness marker CD133 in conjunction with ABCB5 in various progressive stages of cutaneous melanoma ,26. FurtOur gene quantitation analysis of CD133 in cell lines from metastatic melanoma patients with poor clinical outcomes and short overall survival strongly supports our hypothesis that CD133 is a reliable marker of disease recurrence and an abundance of CD133 transcripts may play a pivotal role in progression of human cutaneous melanoma.+ phenotypes in tumor metastasis and tumor recurrence as determined by immunohistochemical expression in tissues from patients with short overall survival and poor clinical outcome, as well as gene expression analysis by qRT-PCR, we further validated our hypothesis by examining their in vivo tumorigenicity by utilizing xenotransplantation experiments in immunodeficient mice.Based on our encouraging leads on clonal dominance of tumorigenic CD133+ cells in nude mice clearly demonstrated that as few as 1,000 CD133+ cells are capable of generating rapidly growing tumors in nude mice within a short period of 8 weeks thus demonstrate positive correlation between enrichment of more aggressive CD133+ phenotypes and tumor growth. This further reminds us that even CSCs are susceptible to selection pressure in aggressive tumors. This observation is a reflection of its pre-existence in the original metastatic melanoma prior to its resection from the patient. We believe it is likely that the metastatic phenotype existed in unrecognized form in the pre-metastatic lesions in the patients. Our findings sufficiently justify the basis of identifying patients whose cancers are at high-risk for metastases even in the absence of clinical evidence of metastases.Our xenotransplantation studies with CD133It is still not clear whether melanoma stem cells are derived from melanocytic stem cells, melanocytic progenitors, or mature melanocytes. It is assumed that primary CSCs originate through cell fusion with the cell population and that they have altered intrinsic/epigenetically unstable mechanisms with an enhanced metastatic potential and drug resistance. Further, the pathways and signaling molecules involved in stem cell homeostasis, de-differentiation, and transformation to melanoma stem cells remain to be determined. These molecules or pathways associated with melanoma stem cells would serve as potential candidates for targeted therapies.Substantial variation exists on the precise definition of melanoma stem cells based on the traditional stochastic model. However, our results uniquely provided a rationale for the definition of a predictive factor of disease progression and represent a significant step towards identifying a more promising target in the clinical management of high-risk cutaneous melanoma.