Agrobacterium-mediated transformation were optimized for three east African farmer-preferred cultivars . Our results demonstrated transformation efficiencies of about 14–22 independent transgenic lines per 100 mg of FEC for farmer-preferred cultivars in comparison to 28 lines per 100 mg of the model cultivar 60444. The presence, integration and expression of the transgenes were confirmed by PCR, Southern blot analysis and histochemical GUS assay. This study reports the establishment of a cassava transformation platform at International Institute of Tropical Agriculture (IITA) hosted by Biosciences eastern and central Africa (BecA) hub in Kenya and provides the basis for transferring important traits such as virus resistance and prolonged shelf-life to farmer-preferred cultivars in east Africa. We anticipate that such platform will also be instrumental to transfer technologies to national agricultural research systems (NARS) in sub-Saharan Africa.Cassava genetic transformation capacity is still mostly restricted to advanced laboratories in the USA, Europe and China; and its implementation and maintenance in African laboratories has remained scarce. The impact of transgenic technologies for genetic improvement of cassava will depend largely on the transfer of such capabilities to researchers in Africa, where cassava has an important socioeconomic niche. A major constraint to the development of genetic transformation technologies for cassava improvement has been the lack of an efficient and robust transformation and regeneration system. Despite the success achieved in genetic modification of few cassava cultivars, including the model cultivar 60444, transgenic cassava production remains difficult for farmer-preferred cultivars. In this study, a protocol for cultivar 60444 developed at ETH Zurich was successfully implemented and optimized to establish transformation of farmer-preferred cassava cultivars popular in east Africa. The conditions for production and proliferation of friable embryogenic calli (FEC) and  Manihot esculenta Crantz) is the third most important source of calories in the tropics, after rice and maize  were obtained from Kenya Agricultural Research Institute (KARI), Kenya and cultivar 60444 was obtained from ETH, Zurich. The cultivars Mkombozi, Kibandameno, Albert, Kibaha and TME14 were obtained from germplasm collection at IITA. All the cultivars were maintained as shoot cultures on cassava basic medium  were induced from axillary buds (AB) and immature leaf lobe (ILL) explants, according to procedure previously described  of OES production for each cultivar.Nodal explants were cut and placed horizontally on cassava axillary bud medium  contains hygromycin phosphotransferase gene (hpt) as selection marker and β-glucuronidase (gusA) reporter gene with a plant intron, both driven by the constitutive Cauliflower mosaic virus promoter (CAMV 35S) , rifampicin (50 mg l−1) and streptomycin (100 mg l−1) were used to initiate 2 ml LB medium as starter cultures. After 48 h shaking at 150 rpm at 28°C, this suspension was used to inoculate a 20 ml YEP medium containing the same antibiotics, and grown overnight on a shaking platform at 150 rpm to reach an OD600 of 0.75–1.0. Bacteria were spun down and washed twice with liquid GD medium. The pellet of bacteria was re-suspended in liquid GD medium supplemented with 200 μM acetosyringone  to an OD600 of 0.5.Agrobacterium tumefaciens strain harboring pCAMBIA1301 in sterile 50 ml falcon tubes, mixed vigorously to disaggregate the callus tissues and incubated for 30 min with gentle shaking (40 rpm). Agro-inoculated FEC tissues were transferred onto a 100 μm mesh placed on sterilized paper towel for 5 min to remove excess bacteria. The FEC were co-cultivated on GD medium for 3 days with Agrobacterium under light (16/8 h photoperiod) at 22°C. Following the co-cultivation step, Agro-inoculated FEC were washed 3 times with liquid GD medium containing 500 mg l−1carbenicillin and transferred to mesh. The mesh was placed on GD media supplemented with 250 mg l−1carbenicillin and incubated for 4 days of recovery at 28°C 16/8 h photoperiod. After 4 days of incubation, the mesh was transferred to fresh GD medium supplemented with 250 mg l−1 carbenicillin and 5 mg l−1 hygromycin and kept under 16/8 h photoperiod at 28°C for 7 days. This step was repeated twice with gradually increasing the hygromycin selection to 8 and 15 mg l−1. Following the FEC selection on GD media, the mesh with FEC was transferred to MSN medium supplemented with 250 mg l−1 carbenicillin and 15 mg l−1 hygromycin and kept under 16/8 h photoperiod at 28°C with fortnightly subculturing on fresh MSN medium.Three month old FECs (100 mg) were co-cultivated with 10 ml of the −1 carbenicillin with fortnightly subculturing onto fresh CEM medium for cotyledon development and shoot elongation. The elongated shoots were transferred to CBM for rooting. Rooted plantlets were screened for escapes by transferring stem cuttings to CBM supplemented with carbenicilin 50 mg l−1 and hygromycin 10 mg l−1 as previously described  containing sterile coconut peat. Pots were placed in trays and covered with a transparent cover and placed in a glasshouse at 28°C. The cover was opened partially after a week and removed after 2 weeks. The hardened plantlets (10 cm in height) were transferred into bigger plastic pots (30 × 40 cm) for further development of tuberous roots and growth monitoring under glasshouse conditions.Histochemical GUS assays for transient gene expression was performed 3 days after co-cultivation according to the modified procedure of Jefferson  as descrin vitro grown shoots using the cetyltrimethylammonium bromide (CTAB) method  overnight at 37°C and resolved on 0.8% agarose gels. DNA was blotted onto Hybond-N+ nylon membrane (Roche) and fixed by cross-linking in a STRATA-LINK™ UV cross-linker. The blots were hybridized with digoxigenin (DIG)-labeled gusA-specific probe generated using a PCR DIG Probe Synthesis Kit . Hybridization and detection were carried out using a DIG Luminescent Detection Kit for Nucleic Acids , according to the manufacturer's instructions.For Southern blot hybridization 15 μg of DNA was digested with 3 U μgAll the experiments were repeated three times and data for all parameters were analyzed by analysis of variance (ANOVA) using PROC ANOVA of SAS 9.2 , and Duncan's new multiple range test was used to detect significant differences between means.The identification of cassava cultivars that will be readily adopted by significant numbers of farmers in sub-Saharan Africa is essential for the development of genetically modified (GM) product. Resource poor farmers prefer cultivars that offer several important traits like cooking quality, time to maturity, yield and disease resistance. The local cultivars preferred in east Africa  were selected, based on consultation with cassava breeders, for establishment of genetic transformation system. These cultivars were selected due to their economic importance and wide cultivation across several countries in east Africa Table . HoweverSuccessful genetic transformation of cassava using FECs depends initially on the production of OES, and the ability of OES to induce and proliferate into pure and homogeneous FEC  in addition to model cultivar 60444 Figure , whereasThe genotypic effect on FEC production of cassava genotypes tested in this study was also observed in the FEC production of a range of Asian cassava genotypes  in germination of cotyledon-stage embryos of Serere in comparison to cultivar 60444, whereas germination of cotyledon-stage embryos of Ebwanatereka and Kibandameno was significantly (p ≤ 0.05) lower. The differences in regeneration efficiencies would be due to genotypic differences. The relatively high germination frequencies of FECs from Serere, Ebwanatereka, Kibandameno and cultivar 60444 suggested that FEC tissues produced from these cultivars could be suitable for genetic transformation. It was, however, noted that not all somatic embryos produced plantlets showing poor correlation between capacity to form somatic embryos and plant regeneration.Since genetic transformation system depend on regeneration of transformed FEC tissue and each cassava cultivar may not respond in those reported condition, we evaluated the regenerative potential of FEC of the three farmer-preferred cultivars  and compared with cultivar 60444. The regeneration potential of FEC from these cultivars is presented in Table In this study, we demonstrated successful and reproducible transformation and regeneration system for three local farmer-preferred cassava cultivars  commonly grown in east Africa, using the modified protocol of Bull et al. . TransfoAgro-inoculation was done by dropping Agrobacterium liquid suspension directly to FEC clusters on the propagation solid medium  better in comparison to model cultivar 60444 at this stage of regeneration of transformants, whereas Ebwanatereka and Kibandameno had a significantly (p ≤ 0.05) lower efficiency of somatic embryo germination . This disease is present in all regions where cassava is grown and production losses can reach up to 80 or 100%  in cassava, could provide a broad and durable resistance to CBB, as has been demonstrated for other plants  and plant ferredoxin-like protein (Pflp) from sweet pepper (Capsicum annuum) , caused by gram-negative bacteria Although cassava yield is high, and its storage root is rich in starch, the PPD of cassava roots seriously affects its storage and utilization  in institutions in Africa will promote research capabilities and allow scientists autonomy to adapt cassava to suit local agro-ecosystems, ultimately serving to develop a sustainable biotechnology infrastructure in African countries.Agrobacterium-mediated transformation of African farmer-preferred cassava cultivars in a laboratory based in sub-Saharan Africa. It is a result of collaborative process involving IITA, Nairobi and ETH, Zurich. The scientific approach followed in this report will serve as model to implement genetic transformation of other cassava cultivars that are agronomically important in Africa. In sub-Saharan Africa, capacities for the production of transgenic crop plants have only recently been developed and remain limited to few crops like banana and maize (Tripathi et al., To our knowledge this is the first report on successful In conclusion, a platform for the high throughput production of transgenic cassava has been developed in farmer-preferred cultivars in east Africa at IITA which will serve as a potential platform for training NARS in sub-Saharan Africa for developing improved cassava varieties with important traits like disease resistance.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


VInv) expression was suppressed in cultivars Russet Burbank and Ranger Russet using RNA interference to determine if this approach could control sugar-end defect formation. Acid invertase activity and reducing sugar content decreased at both ends of tubers. Sugar-end defects and acrylamide in fried potato strips were strongly reduced in multiple transgenic potato lines. Thus vacuolar invertase silencing can minimize a long-standing French fry quality problem while providing consumers with attractive products that reduce health concerns related to dietary acrylamide.Sugar-end defect is a tuber quality disorder and persistent problem for the French fry processing industry that causes unacceptable darkening of one end of French fries. This defect appears when environmental stress during tuber growth increases post-harvest vacuolar acid invertase activity at one end of the tuber. Reducing sugars produced by invertase form dark-colored Maillard reaction products during frying. Acrylamide is another Maillard reaction product formed from reducing sugars and acrylamide consumption has raised health concerns worldwide. Vacuolar invertase gene ( Potato plants are subjected to a variety of biotic and abiotic stresses that impact plant health, marketable tuber yields and final tuber quality. Poor tuber quality due to the combined effect of environmental and cultural practices in the field can be visualized in finished processed products—the French fry or potato chip Sugar-end defect is an internal tuber quality disorder observed primarily in elongate tubers of fry processing potatoes such as Russet Burbank and Ranger Russet Sugar-end defects are typically associated with plants that have had to endure transient periods of high air and soil temperatures during tuber initiation and early tuber bulking VInv and a subsequent increase in invertase activity are the immediate causes of sugar-end defects The molecular and biochemical changes that cause sugar-end defects to develop in tubers from stressed plants have not been described completely. Changes in tuber sugar and starch content have been observed during early season heat and water stress, but these differences often do not persist after the stress is removed, and are not observed in tubers at harvest. Increased transcription of VInv/Pain-1, InvGE, InvGF, InvCD111 and InvCD141, were found in potato VInv/Pain-1 gene located on chromosome III encodes a single copy vacuolar invertase. The other four genes are located on chromosome IX or X and encode apoplastic invertases Three types of invertase isoenzymes are found in plants Acrylamide is an undesirable by-product of the Maillard reaction that results when reducing sugars react with free asparagine during high temperature cooking VInv to decrease sugar-end defect formation. This method has been shown previously to control CIS in fry and chip processing potato tubers VInv-silencing to lower the frequency of sugar-end defects has not been shown previously. Data presented here on multiple independently transformed lines of Russet Burbank and Ranger Russet with reduced expression of VInv demonstrate the potential for using VInv-silencing to reduce sugar-end defect development in fry processing potatoes.The present study details a method that utilizes the forced down-regulated expression of VInv expression relative to that of the reference gene Actin97 was quantified by real-time quantitative PCR using the SYBR Advantage qPCR Premix (Clontech) on MJ Research Opticon 2 (Bio-Rad Laboratories). PCR Primers for the Actin97 were fp:5′-AGTATGACGAATCTGGTCCTTCTATTG-3′ and rp:5′-ACCCAACAATCAACTCTGCCCTCTC-3′ , and primers for the VInv were fp:5′- CATCAAAGACATTTTATGACCCGAA-3′ and rp:5′-TGTGTCCCTGTCTTCTTGTCGTAA-3′ . Twenty-five independent transformants were selected from the 53 original lines for further study based on VInv expression in leaves of clones relative to expression in leaves of wild-type Russet Burbank. Multiple clones with a high percentage of VInv silencing were selected in order to assess the potential effectiveness of the approach to reduce sugar-end defects. Additional clones with relatively low amounts of VInv silencing were included for comparison. Overall, 22 lines showed 80–98% reduction of VInv expression and three lines showed 0–80% reduction of VInv expression in leaf tissues. Transgenic plants were grown under a 16 h photoperiod using natural light supplemented with sodium vapor lamps as needed to maintain a minimum of 500 μmol m−2 s−1 photosynthetically active radiation. Tubers were harvested from plants that had senesced naturally and were stored at 4°C for 14 days to stimulate CIS and strong expression of VInv in wild-type Russet Burbank. Equal amounts of tissue from positions corresponding to tuber stem end, bud end and middle of the tuber were frozen in liquid nitrogen and ground together. RNA was extracted from tuber tissues using Plant RNA Isolation Mini Kit (Agilent) and quantitative real-time PCR was used to confirm that the VInv RNAi-silencing construct was functioning in tubers of the selected lines. Based on relative VInv expression in leaves and in cold-stored tubers, five RNAi lines were selected for further analysis because they covered a wide range of VInv silencing. These included RBK1, RBK22, RBK25, RBK27, and RBK46 .The previously developed RNAi construct InvBP2 VInv-silenced lines in the Russet Ranger background were described previously The vector, which contains a potato tuber specific ADP glucose pyrophosphorylase promoter, and transformation methods used to create the VInv-silencing lines and of Russet Burbank controls were grown in Metromix 360  in a greenhouse for four weeks and then eight plants of each line were transplanted to research plots at the University of Wisconsin Agricultural Research Station in Hancock, Wisconsin on June 4th, 2012. Production practices used were standard for irrigated potato at the Hancock station. Vines were desiccated with two applications of diquat  and tubers were harvested by hand on September 14th, 2012. For each line, the 20 largest tubers were subsampled with five tubers randomly assigned to each of four sample periods for analysis of gene expression, acid invertase enzyme activity, tuber sugar contents and color uniformity after frying at harvest and after one, three, and five months of cold storage. Harvested tubers were stored in the Hancock Storage Research Facility at 13°C for 47 days for wound healing and preconditioning. The storage temperature was decreased from 13°C to 9°C at a rate of 0.1°C per 8 hours, which took a total of 24 days. Tubers were then held at 9°C, a temperature typical for commercial storage of Russet Burbank.Tissue culture plantlets of Russet Burbank To produce tubers for molecular and biochemical analyses and seed tubers for field experiments, Ranger Russet transformants were grown for 3 months in Sunshine Mix-1 in 8 liter pots in a greenhouse that was controlled for temperature (18°C minimum and 27°C maximum) and light with a 16 h day and 8 h night photoperiod).VInv-silenced lines of Ranger Russet were conducted at the University of Idaho Parma Research and Extension Center in Parma, Idaho. Applications of macro and micronutrients followed management recommendations suggested by the University of Idaho. Plots were irrigated using a solid set system with moisture maintained above 65% throughout the growing season. In 2011, each control and transgenic line was represented by 1 plot of 5 hills. In 2012, each control and transgenic line was represented by 5 plots of 20 hills. In both years, in-row spacing was 10 inches with 36 inches between rows. Tubers were harvested 130 to 140 days from planting and stored at 13°C to precondition and encourage wound healing. The final storage temperature of 8°C for tubers of Ranger Russet was achieved by stepping down 0.1°C every 8 hours. Sugar end determinations were made on Ranger Russet tubers stored at 8°C for 1 month. Five randomly selected tubers of lines with the lowest percentages of sugar-end defects in 2012, 1632-1, 1632-3 and 1632-5, and Ranger Russet controls were used for analysis of gene expression and acid invertase enzyme activity using the methods described for Russet Burbank. Tubers (2013 field year) were held at 13°C for 45 days and ramped to 8°C at a rate of 0.33°C per 24 hours. Tubers were held at 8°C for an additional month prior to taking tissue samples for VInv quantification and acid invertase activity assays.Field trials using untransformed controls, empty vector controls and VInv-silencing lines. These tissue samples were cut into subsamples of approximately 1 cm length, frozen immediately in liquid nitrogen and used to quantify VInv expression, tuber sugar content and acid invertase activity. Gene expression was quantified by qPCR as described above. Acid invertase assays and sugar quantification for Russet Burbank lines used the methods described previously At the time of harvest and after 1, 3, and 5 months of storage, 1-cm diameter cylinders of tuber tissues were removed from the apical (bud) and basal (stem) ends of five tubers of Russet Burbank and the five Russet Burbank VInv-silencing lines grown in 2011, a fry sample consisted of a minimum of twelve pounds of tubers taken from a pooled sample of the 5 hills. In 2012, 20 tubers from a pooled conglomeration from each replicate of 20 hills were used and all 5 replicates were measured. All tubers were cut lengthwise on a 0.95-cm×0.95-cm grid fry knife and the four center strips were fried at 191°C for 3 minutes. Fried strips were laid on a white tray and compared to the USDA Munsell Color Chart for French Fried Potatoes. A sugar-end defect fry had a dark end 0.64 cm long or longer on the darkest two sides of the strip, for the full width of the strip, testing number 3 or darker when compared to the USDA Munsell Color Chart.Fried strips of Russet Burbank lines were prepared by slicing longitudinal sections 0.95-cm thick and 3-cm wide from the center of individual tubers and cooking in cottonseed oil at 191°C for 3 min 30 s. Acrylamide content of fried strips was assayed as described previously VInv-silencing lines, relative expression was calculated using Gene Expression Macro software version 1.1 (Bio-Rad Laboratories). All the data for qPCR, tuber sugar contents and acid invertase activity were calculated using the Statistical Analysis System version 9.1 (SAS v9.1) . Analyses of variance (ANOVA) were carried out using PROC GLM. Differences in sugar-end defect percentage between the Ranger Russet and transformed lines of Ranger Russet were determined using one-way ANOVA followed by Holm-Sidak multiple comparisons test using GraphPad Prism version 6.0b for Mac .For the Russet Burbank VInv silencing were selected for in-depth analyses . The expression of VInv in lines RBK1, RBK22, RBK25 and RBK27 was consistently less than the expression in control Russet Burbank from harvest through five months of storage . VInv expression in bud end tissue was lowest and reduced 85–96% in line RBK1 at harvest, and one month and three months after harvest. Strongly reduced VInv expression was also observed in line RBK22 at harvest and one month and 5 months after harvest. Surprisingly, the constitutive VInv-silencing construct was less effective at the tuber stem end than at the tuber bud end  with a wide range of  storage Table 1.VInv expression was greater at the tuber stem end than at the bud end for the silencing lines and the Russet Burbank control  and remained low, with activity between 0.11 and 0.93 units mg−1 protein at one, three and five months after harvest . Tuber fructose content in the silencing lines and Russet Burbank controls was similar to tuber glucose content, although tuber fructose content tended to be less than tuber glucose content . Acrylamide in bud end tissues was also much less in these two silencing lines than in Russet Burbank and RBK46 (p<0.0001). Thus, differences in acrylamide content mirrored observed differences in tuber reducing sugar contents.The acrylamide contents at the tuber stem end of fried strips prepared from Russet Burbank controls and RBK46 were 814 and 762 μg kgectively Table 5.VInv was silenced using the potato tuber specific ADP glucose pyrophosphorylase promoter were evaluated over two field years for susceptibility to sugar-end defect formation. In 2011, a small sample size due to limited seed supply revealed trends toward all silencing lines having reduced sugar-end defects  to basal (stem) differences in post-transcriptional mRNA stability and context-dependent efficiency of the silencing construct. Tuber bud end and stem end tissues also differed in the correspondence, or lack thereof, between changes in VInv mRNA abundance and changes in acid invertase activity. In stem end tissues of control Russet Burbank and line RBK46, changes in VInv mRNA accumulation through 5 months of storage were mirrored by changes in enzyme activity. This was not observed in bud end tissues of Russet Burbank and RBK46 tubers. Pronounced increases in VInv mRNA occurred between 1 and 5 months of storage, but acid invertase activity did not change in either of those lines during that period. It may be that relatively small increases in VInv mRNA, i.e. expression relative to actin97 of less than approximately 5, do not result in an increase in acid invertase activity at either end of the tuber. This suggestion is supported by the data from RBK1, RBK22, RBK25 and RBK27 at the tuber stem end and from all VInv-silencing lines and the Russet Burbank control at the tuber bud end.VInv expression was decreased by more than 50% is consistent with this idea.Differences in tuber bud and stem end sucrose content are often observed in Russet Burbank and this difference has been attributed to a higher rate of vacuolar invertase activity at the tuber stem end compared with the tuber bud end −1 greater on the stem end than on the bud end of fried strips for Russet Burbank controls and RBK46, respectively. Since each strip was cut in half prior to acrylamide analysis, average acrylamide content for intact strips would be approximately the mean of bud and stem end samples. The mean value for Russet Burbank was approximately 600 μg kg−1, which equals the indicative value for acrylamide in Europe as established by the European Commission VInv mRNA, acid invertase activity, and reducing sugar content in both tuber ends compared to those of Russet Burbank and RBK46.The dark color observed after frying was associated with a substantial increase in overall acrylamide content of the fried strips Table 5.VInv and asparagine synthetase-1 genes are silenced.Silencing of the asparagine synthetase-1 gene provides an alternative approach to reduce the acrylamide content in processed potato products VInv-silencing lines demonstrated that tuber specific gene silencing can be used to decrease sugar-end defect formation. For all of the Ranger Russet lines described, tubers harvested in 2011 and 2012 and stored at 8°C produced fries with acceptable color in tissue corresponding to the tuber bud end. However, a very stressful growing season in 2012 demonstrated a requirement for very effective silencing if sugar-end defects are to be managed routinely. Of the lines described, only 1632-1 had very low incidence of sugar-end defects in 2012, although 1632-3 and 1632-5 had fewer sugar-end defects than Ranger Russet controls.Ranger Russet VInv, but further evaluation of the VInv-silenced lines described in this research is needed in order to show this conclusively.Dark fry color was not directly addressed in this research on sugar ends. A number of stresses that occur in the field can cause an increase in reducing sugars throughout the tuber. Tuber sugar contents are influenced by many factors including disease stress, temperature and water stress, management practices and mechanical handling. In cases where such stresses occur, the consequence is usually an increase in fry color distributed over the length of the French fry strip. Defects such as these are also likely to be reduced by silencing of VInv can be used to reduce the incidence of sugar-end defects in fry processing potatoes. Success of this approach depends on the extent of VInv-silencing, with more effective silencing at the tuber stem end producing fewer defects. Decreasing sugar-end defect frequency could benefit potato growers and processors by reducing rejections at the processing plant, and providing a more reliable supply of raw product. Consumers also benefit, in that low vacuolar invertase lines produce attractive end products that are potentially much lower in acrylamide than comparable products made from control varieties.In summary, the data presented here show that silencing the Figure S1VInv in potato leaf and tuber tissues varied between Russet Burbank VInv-silencing lines and Russet Burbank controls. VInv expression was determined using actin97 as a reference gene and presented as a percentage of the level in Russet Burbank controls. Potato leaf tissues were collected from transgenic plants at the four-leaf stage and tuber tissues were collected from cold-stored tubers (14 d of storage at 4°C). Data are presented for five lines with different degrees of VInv silencing. Bars represent mean ± standard error of two independent leaf samples and three independent tuber samples.Expression of (TIF)Click here for additional data file.


We differentiated Olig2-GFP-mESCs into purified Olig2+-GFP+-OPCs and transplanted them into the rats’ cervical 4–5 dorsal spinal cord level at 4 months after irradiation injury. Eight weeks after transplantation, the Olig2+-GFP+-OPCs survived and integrated into the injured spinal cord. Immunofluorescence analysis showed that the grafted Olig2+-GFP+-OPCs primarily differentiated into adenomatous polyposis coli (APC+) oligodendrocytes (54.6±10.5%). The staining with luxol fast blue, hematoxylin & eosin (LFB/H&E) and electron microscopy demonstrated that the engrafted Olig2+-GFP+-OPCs attenuated the demyelination resulted from the irradiation. More importantly, the recovery of forelimb locomotor function was enhanced in animals receiving grafts of Olig2+-GFP+-OPCs. We concluded that OPC transplantation is a feasible therapy to repair the irradiated lesions in the central nervous system (CNS).Demyelination contributes to the functional impairment of irradiation injured spinal cord. One potential therapeutic strategy involves replacing the myelin-forming cells. Here, we asked whether transplantation of Olig2 The spinal cord is one of the important dose-limiting normal tissues in clinical radiotherapy. Excessive doses of radiation to the spinal cord result in radiation injury, which is a rare but serious complication of radiotherapy for cancer in vitro before transplantation.Cell therapy offers an attractive option as grafted cells may replace the lost ones as well as provide neurotrophic benefits to surrounding tissue +-GFP+-OPCs into the spinal cord of rat that underwent irradiation injury, the grafted OPCs survived, differentiated to myelinating oligodendrocytes and improved locomotor function of the injured rats.In the present study, we employed a mESCs line that carries a GFP reporter in the locus of Olig2, a transcription factor that is critical for OPCs development This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of Fudan University (Permit Number: SCXK 2009-0019). All efforts were made to minimize suffering.Female Wistar adult rats  were anesthetized by an intraperitoneal injection of 7.5 mg/kg ketamine, 60 mg/kg xylazine and immobilized during irradiation. The rats’ cervical spinal cords were irradiated using a 6 Mev Electron Beam Linear Accelerator . A 22 Gy radiation dose was delivered to a 2 cm×2 cm radiation field, maintaining cervical 4–5 as the center of the electron-irradiated zone. Radiographs were taken before and after X-irradiation to confirm that all the animals were in the same position. After irradiation, the animals were given subcutaneous saline (5 ml) and housed in a 25°C warm room.Olig2-GFP-mESCs were the gift of Dr. Su-Chun Zhang  and have been previously described 4 cells/µl) were plated on coverslips coated with poly-L-lysine and human laminin (Sigma).For immunofluorescence, dissociated spheres (2×10For fluorescence activated cell sorting (FACS), cells were harvested at day 12, gently dissociated to single cells and washed with a FACS buffer . Cells were analyzed by a Becton Dickinson FACSCaliber with CellQuest Pro .+-GFP+-OPCs were resuspended in a fresh culture medium and cultured for 2 days. At day 14, cells were dissociated with accutase (Invitrogen) and prepared at approximately 100,000 cells/µl in artificial cerebrospinal fluid (aCSF). Trypan blue exclusion testing indicated that this preparation consisted of 95% viable cells at time of transplantation.For transplantation, FACS-sorted Olig2Transplantation was performed as described For the histological analysis, the animals were deeply anesthetized with an overdose of 3% pentobarbitone and perfused with 0.9% saline following by 4% ice-cold paraformaldehyde prepared with phosphate buffer . The spinal cord was divided into eight 1 mm blocks that extended 4 mm cranial to and 4 mm caudal to the injury epicenter. Alternate blocks were processed to produce cryostat sections. Some of the cryostat sections were stained with LFB/H&E to examine general morphology and the extent of demyelination. Some of the cryostat sections were used for immunofluorescent analysis of grafted cells.http://rsbweb.nih.gov/ij/index.html) to measure the relative optical density of the dorsal funiculus from all groups. A pre-defined region of interest was selected to encompass the dorsal funiculus. Color images were acquired at the same exposure level, coverted to 8-bit gray scale, and the mean density calculated from the threshold pixels excluding the background. Density was analyzed in four regions of the dorsal funiculus per section. Eight sections per spinal cord were analyzed. There were six animals in each group.To quantify the demyelination of injured spinal cord, we used Image J  and pure acetone, the sections were embedded in Epon 812. Ultrathin sections (80 nm) obtained using an ultramicrotome  were stained with urany1 acetate and lead citrate, examined and photographed under a transmission electron microscope .For electron microscopic processing, the animals were sacrificed by aortic perfusion with isotonic, heparinized saline followed with 4% paraformaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer. The fixed spinal cords were washed in 0.1 M phosphate buffer and embedded in 3% agar. Transverse sections (40 µm thick) were obtained on a vibrotome  and mounted onto slides. The sections were washed in PB and subjected to an additional fixation in 2% OsOThe clinical degree of weakness was graded according to a scale developed by Ushio et al. p<0.05.All data are reported as Mean±SEM. Repeated measures ANOVA followed by the Tukey-Kramer test was used for comparison of clinical grade scores at each time point. The difference between the two was compared using Student’s tests. Statistical analysis was performed using SPSS 16.0. Differences were considered to be statistically significant at Non-irradiated animals (n = 6) did not show any evidence of demyelination . Even thThe clinical degree of weakness was consistent with the histological changes. The mean interval between irradiation and manifestation of the first neurological symptoms  was 112±14 days. Four months after irradiation, the animals (n = 12) that did not manifest neurological symptoms  were omitted from the experiment.+ spheres began to appear and by day 12 almost all the spheres were green (We used an established mESCs line (Olig2-GFP-mESCs) in which GFP was knocked into the locus of Olig2, a transcription factor expressed by ventral spinal cord progenitors re green . FACS anre green . To furtre green , PDGFRα re green  and O4  without cell transplantation did not exhibit such labeling. The GFP-expressing cells were mainly distributed in the dorsal funiculus with a small number of cells migrating into the gray matter . The behavioral results showed that transplantation of Olig2+-GFP+-OPCs significantly improved forelimb functions.To determine whether transplantation of OPCs improved recovery of function after injury, locomotion was assessed by using the clinical scoring. Six to eight weeks after transplantation, Olig2ol group  (all p<0in vitro and in vivo. More importantly, the oligodendrocytes derived from the Olig2+-GFP+-OPCs express MBP, an essential component of myelin sheath, which corresponds to correction of locomotion deficits in rats with irradiation injury of spinal cord. These results raise the prospect of cell-based therapy as a potential treatment for irradiation spinal cord injury.In this study, we have directed mESCs to a purified population of OPCs based on the GFP expression in the Olig2 locus, which differentiate into oligodendrocytes Cell transplantation has been attempted for the radiation injured spinal cord. Adult rat neural stem cells differentiate predominantly into Schwann-like cells and generated peripheral myelin after transplantation into the irradiation injured spinal cord Most irradiation injury models are made using 40 Gy of X-irradiation which creates acute demyelination in the spinal cord +-GFP+-OPCs transplantation, suggesting the protection of axonal integrity by transplanted cells. Further study is needed by immunoelectron microscopy to prove remyelination occurred by transplanted cells.Promoting remyelination is an important strategy to treat spinal cord injury. Chronic demyelination predisposes axons to degeneration The increased recovery in the transplantation group may also be explained by mechanisms other than remyelination. CNS neurons require multiple signals for optical survival and maturation, and continued oligodendrocyte-derived signals are necessary to maintain neuronal integrity


Categorizing protein-encoding transcriptomes of normal tissues into housekeeping genes and tissue-selective genes is a fundamental step toward studies of genetic functions and genetic associations to tissue-specific diseases. Previous studies have been mainly based on a few data sets with limited samples in each tissue, which restrained the representativeness of their identified genes, and resulted in low consensus among them.This study compiled 1,431 samples in 43 normal human tissues from 104 microarray data sets. We developed a new method to improve gene expression assessment, and showed that more than ten samples are needed to robustly identify the protein-encoding transcriptome of a tissue. We identified 2,064 housekeeping genes and 2,293 tissue-selective genes, and analyzed gene lists by functional enrichment analysis. The housekeeping genes are mainly involved in fundamental cellular functions, and the tissue-selective genes are strikingly related to functions and diseases corresponding to tissue-origin. We also compared agreements and related functions among our housekeeping genes and those of previous studies, and pointed out some reasons for the low consensuses.The results indicate that sufficient samples have improved the identification of protein-encoding transcriptome of a tissue. Comprehensive meta-analysis has proved the high quality of our identified HK and TS genes. These results could offer a useful resource for future research on functional and genomic features of HK and TS genes. In the last decade, massive gene expression microarray data has opened an avenue toward transcriptomic study. Benefitting from this progress, the ideas of housekeeping (HK) genes and tissue-selective (TS) genes can now be investigated on a large-scale manner.Housekeeping genes were first described as those genes always expressed in the cell Tissue-selective genes are predominantly expressed in one or a few biologically relevant tissue types These two transcriptomic extremities, HK genes and TS genes, have prompted research interest in their expressional and functional characteristics 2DB, the microarray meta-analysis database In this study, we compiled 1,431 quality controlled samples in 43 normal human tissue types from Mfraction of Present detection calls (fraction Present) of samples in a group has shown to preserve reliably detected and biologically informative probe sets Most previous HK gene studies identified expressed genes in each tissue type by applying a threshold to expression intensity. Recently, filtering by To evaluate the three indicators and the sample size in assessing gene expression in a tissue, our study used muscle samples as an example to perform the receiver operative characteristic (ROC) analysis . For allAdapting FPEI for searching representative PE transcriptomes, we identified 2,064 genes universally expressed across all analyzed 43 tissues as HK genes  were excluded to ensure reliable annotation matches; and, 2) probe sets with multiple Entrez Gene IDs were excluded. Finally, we obtained 19,106 reliable probe sets, which corresponded to 12,559 unique Entrez Gene IDs. For each tissue, expression intensities and present calls from probe sets of a gene were summarized into expression intensity and fraction Present for the gene, respectively.We used the annotation build 30 of NetAffx ijFPEI is the fraction Present weighted expression intensity (FPEI) for gene i in tissue type j, which combines the expression abundance ijEI and the confidence of detection ijFP. We considered a gene constitutively expressed in a tissue type if its ijFPEI exceeded 100, the product of commonly applied expression intensity of 200, and a relatively fair fraction Present of 50%. By applying the threshold to ijFPEI, housekeeping genes were defined as with ijFPEI above 100 in all 43 studied tissues.In addition to expression intensity and fraction Present, we weighted expression intensity with fraction Present for each gene in each tissue type to simply integrate the two indicators:ijFPEI according toijb is the normalized ijFPEIiw is the gene-specific weight defined as tissue-specificity index N tissuesijS above the cutoff and with ijFPEI above 100 in at least one tissue  were considered as tissue-selective genes.We computed a tissue-selectivity score P-value) to select over-represented functions. We applied the biological process category of GO and the KEGG pathway for functional enrichment analysis of HK genes. Functions were considered as significant with FDR-adjusted EASE scores smaller than 0.05, and were consolidated into eight functional clusters according to DAVID functional annotation clustering and GO term lineage. For TS genes, in addition to GO and KEGG pathway, we applied Genetic Associated Database to find the associated diseases, by which functions or diseases with EASE scores smaller than 0.01 were considered as significant.Functional enrichment analysis were accessed through DAVID Figure S1Expression intensity and fraction Present of four example genes. Two HK genes ACTB and GAPDH, and CD8+ T lymphocyte selective gene CD8B and liver selective gene CYP4A11 are shown as examples. Weighting expression intensity with fraction Present would emphasize the selective expression of CD8B in T cell related tissues, especially in CD8+ T lymphocyte, as well as the selective expression of CYP4A11 in liver and kidney.(PDF)Click here for additional data file.Figure S2Comparison of enriched functions common to HK gene lists. This graph shows enriched functions common to most HK gene lists presented in (PDF)Click here for additional data file.Figure S3Comparison of enriched functions unique to each HK gene list. This graph shows enriched functions unique to each HK gene list presented in (PDF)Click here for additional data file.Figure S4Clustering of 43 tissues with expression pattern of TS genes. Tissues were hierarchically clustered with average linkage according to gene expression patterns (shown in shown in . Tissues(PDF)Click here for additional data file.Figure S5Percentage of expressed genes in each tissue with three different indicators. Percentages of expressed genes range from 36% (thyroid gland) to 55% (epididymis) by applying a cutoff of 100 to FPEI (green bars). These numbers are proportional to that identified by cutoff of 50% to fraction Present (red bars) and the mean percent presents of each tissue. Applying a cutoff of 200  to expression intensity tends to set 50% of genes as expressed (blue bars). Mean percent presents are not shown for clarity.(PDF)Click here for additional data file.Figure S6Comparison of percent present of samples in Gene Expression Atlas II to other experiments. Samples of Gene Expression Atlas II show a significant lower percent present when compared to other samples used in this study (P = 1.8×10−64 by two-tailed t-test). Bars are means from different sample origins; error bars show s.d..(PDF)Click here for additional data file.Figure S7Expression of seven both housekeeping and tissue-selective genes.FDX1 is selectively expressed in adrenal gland (2), and TUBA3C is selectively expressed in testis (36). Other five genes, BNIP3L, RNF10, MKRN1, ADIPOR1, and MARCH8, are selectively expressed in umbilical cord blood (40).(PDF)Click here for additional data file.Table S1The list of 2064 HK genes and their FPEI in 43 tissues.(XLS)Click here for additional data file.Table S2Functional enrichment analysis results of HK genes.(XLS)Click here for additional data file.Table S3The list of 2293 TS genes and their FPEI in 43 tissues.(XLS)Click here for additional data file.Table S4Functional enrichment analysis results of TS genes.(XLS)Click here for additional data file.Table S5The list of all 1431 samples.(XLS)Click here for additional data file.Text S1Results for Receiver Operating Characteristic (ROC) analysis with 93 muscle samples.(PDF)Click here for additional data file.


The role of the endothelium in the pathogenesis of cardiovascular disease is an emerging field of study, necessitating the development of appropriate model systems and methodologies to investigate the multifaceted nature of endothelial dysfunction including disturbed barrier function and impaired vascular reactivity.We aimed to develop and test an optimized high-speed imaging platform to obtain quantitative real-time measures of blood flow, vessel diameter and endothelial barrier function in order to assess vascular function in live vertebrate models.2 (TXA2) analogue, U-46619 (1 µM), in transgenic zebrafish larvae. Data obtained in 3 and 5 day post-fertilization larvae show that these methods are capable of imaging blood flow in a large (1 mm) segment of the vessel of interest over many cardiac cycles, with sufficient speed and sensitivity such that the trajectories of individual erythrocytes can be resolved in real time. Further, we are able to map changes in the three dimensional sizes of vessels and assess barrier function by visualizing the continuity of the endothelial layer combined with measurements of extravasation of fluorescent microspheres.We used a combination of cutting-edge optical imaging techniques, including high-speed, camera-based imaging (up to 1000 frames/second), and 3D confocal methods to collect real time metrics of vascular performance and assess the dynamic response to the thromboxane AWe propose that this system-based microscopic approach can be used to combine measures of physiologic function with molecular behavior in zebrafish models of human vascular disease. To this end, we focused on the application of intravital imaging of vascular dynamics in 3–5 days post fertilization (dpf) embryos to investigate the multifaceted nature of endothelial dysfunction PTU)) and size of the zebrafish embryo are ideally suited to modern microscopy such that complex biologic processes can be visualized in toto and in vivo. Importantly, the vascular is anatomically and functionally comparable to other vertebrate species Our goal in these studies was to develop and test a system-based, high speed microscopic approach that can be used to combine measures of physiologic function with molecular behavior in zebrafish Accurate real-time measurement of vascular resistance (vessel diameter) and regional blood flow demands high speed detection methods such that individual erythrocytes can be tracked and flow velocity calculated over a defined segment of vessel. Until very recently, conventional camera technologies were not able to collect images at sufficient speed or resolution to make dynamic measures of flow velocity in living organisms, including zebrafish. High speed, charge coupled devices (CCD) have low pixel counts  which compromises resolution. Conventional CCD and electron multiplying CCDs (EMCCD) have higher pixel counts (between 512×512 and 1300×1000), but have total pixel read speeds of 10–20 mHz . Strategies have been developed to circumvent these limitations and obtain gross estimates of blood flow, including time-lapse imaging of endogenous  or tracer particles to obtain displacement information between consecutive frames of data Danio rerio) were maintained according to standard protocols b107/+;Tg(kdrl:GFP)la116;Tg(gata1.dsRed)sd2chrna1 incross. Transgenic lines la116Tg(kdrl:GFP) and sd2Tg(gata1:dsRed) have been previously described chrna1 mutants lack functional nicotinic muscle acetylcholine receptors (AChRs) b107chrna1 mutation were imaged, thereby circumventing the need for anesthesia or agar embedment. Embryo medium was supplemented with 0.003% phenylthiourea (PTU)  at 24 hours post-fertilization (hpf) to prevent melanin formation Zebrafish ((Embryos were placed in a coverslip glass bottomed (#1.5) petri dish  and mounted in a humidified chamber  atop the motorized stage of a Nikon TiE inverted fluorescent microscope equipped with a 10X, 0.5NA plan apochromat lens , a fluorescent illuminator , DsRed and EGFP filter cubes  and a Hamamatsu Flash 2.8 CMOS camera (Hamamatsu Corporation). The sensor in this camera has 3.63 micron pixels in a 1920 by 1440 array; the full chip readout is 45 frames/second, each pixel row (1920 pixels) is readout 15 microseconds such that a block of pixel rows consisting of 133 rows can be read at 500 frames/second. The use of a relatively high numeric aperture (NA), low magnification objective allowed large areas to be imaged with a high photon recovery, and a relatively large depth of field. The EGFP labeled vasculature Fig. 1A kdrl-driven EGFP were collected using a Nikon Ti equipped with a Nikon sweptfield scan head, a Mad City Labs  piezo driven Z-drive, and a 40X high NA (1.15 NA) long working distance water immersion objective. The sweptfield confocal pinhole used a 22 µm slit, and 25% laser power . 60 Z-positions were collected for each stack allowing a full stack to be collected in less than 1 s. Images were collected using NIS Elements and a Photometrics Evolve EMCCD camera  using 10–30 ms exposures and EM gain setting of 700.3D image stacks of Fig. S1A) designed in our laboratory and fabricated by Fotofab  using our original computer-aided designs . Two micromanipulators  were used, one to hold the paddle, the other to hold the microinjection needle . Needles were pulled from borosilicate glass , and tips broken to an angle with forceps. The outside diameter of the tip measured 30–70 microns. Tips were loaded with a mix of 0.5 mg/ml rhodamine B isothiocyanate-dextran (70 kD) and 0.4%, 0.1 µm carboxylate-modified microspheres  mixed with U-46619  or 0.01 units/ml thrombin (Sigma-Aldrich). Needles were placed within the duct of Cuvier  of b107/+;Tg(kdrl:GFP)la116chrna1 transgenics and small volumes (up to 25 ηL) of the bead/dextran mix were delivered. The rhodamine-labeled dextran was used to confirm that contents were flowing properly out of the pipette tip and into the lumen of the vessel. The concentration of beads in the injecting needle was kept to <0.5% as increasing the concentration beyond this led to vascular occlusion. At this concentration and volume there was no effect on vascular flow rate. Using a Nikon sweptfield confocal scan head, piezo driven Z-stage and a 10X high NA (0.5) objective, two color Z-stacks were collected  with a Photometrics Evolve camera (20 Z-planes/second). A quad pass filter  was used for these studies in order to maximize speed. The illumination was scanned in sequential frame mode and there was no evidence of bleed through between the channels. Using these parameters, a complete 2-color stack was collected in 5 seconds. Z-stacks were collected every 2 minutes post-injection. The zebrafish were imaged for up to 2 hours with no motion, and good image registration.By convention, vascular injections into either the cardinal vein or heart of embryonic zebrafish have been performed by tricane anesthesia and embedment in agarose. However, it was essential to be able to image at high resolution using multiple imaging modalities. Thus the embryos need to be mounted directly on the glass substrate at the base of a petri dish  for use with high numeric aperture, short working distance lenses. As we wish to look at effects of vascular agonists, it was deemed important not to render the embryos hypoxic by embedment in agarose. We therefore developed methods for injection using immobilization paddles . Due to the extremely rapid frame rate and short exposure times, the images of individual cells remained exact, without smearing as shown in the representative time sequence . Red blood cell flow velocity was quantified by first segmenting individual cells by size, shape and intensity , followed by an autoregressive tracking algorithm which assumed a continued vector for travel (P<0.05), for 3 dpf and 5 dpf embryos, respectively.Scientific CMOS cameras have similar quantum efficiencies as conventional cooled CCD cameras in the spectral range being used here. As a result of the reduced read noise of the CMOS detector technology, it possible to read from these cameras much more rapidly than with other commercially available CCD devices. We were able to image a 1 mm section of the dorsal Fig. 1A 2 (TXA2) analogue, U-46619. TXA2 is the main arachidonic acid metabolite generated by the sequential action of cyclooxygenase (COX) and thromboxane synthase (TXS); and has been shown to elicit diverse physiological effects in the vasculature system including smooth-muscle contraction and proliferation, endothelial dysfunction, and platelet activation and aggregation P<0.05, ,,In order to determine the quantitative capabilities of the imaging system to measure increases in blood flow velocity, we tested the response of 3 and 5 dpf zebrafish embryos to the stable thromboxane AThe degree of constriction or relaxation of a vessel (i.e. vessel diameter) is a key determinant of vascular resistance and as such, regulates regional blood flow. According to Poiseuille’s equation, flow is related to radius to the fourth power and thus very small decreases in vessel radius can dramatically reduce blood flow. Accurate real-time measurement of dynamic changes in vascular resistance, therefore, requires the ability to generate high speed, 3D reconstructions of the vascular network over time with sufficient resolution to detect small changes in diameter. Measurements derived from single plane images of the vessel wall are subject to errors caused by even very minute focal drifts in the Z-axis. The point scanning confocal and multiphoton microscopes typically used for optical sectioning are less suitable than multi-pinhole scan heads for live cell/tissue imaging because of the relatively slow scan speed along with the associated phototoxicity of these devices when compared with multi-pinhole systems. Even when used in resonant mode this frame rate is limited to 28 fps at high resolution. Furthermore, the currently available photomultiplier tube (PMT) detectors used in point scanning systems, have much lower quantum efficiencies (30%) compared with modern back-thinned electron multiplying CCD cameras (∼90%). We investigated whether the effects of U-46619 on blood flow velocity could be mediated by changes in vascular resistance by examining its effects on the diameter of the dorsal aorta in 5 dpf zebrafish embryos using high speed, slit-scanning confocal imaging. We were able to image the entire depth of 5 dpf larvae Fig. 5E.2 receptor agonists inducing increases in vascular permeability in the aorta, lung, heart, kidney, spleen, and testis In order to assess endothelial barrier function in the zebrafish larvae, we used fluorescence based time-lapse imaging techniques to track extravasion of fluorescently labeled tracer particles microinjected into the vascular space. We first tested our ability to measure disruption of barrier function using the coagulation serine protease, thrombin (29 µM). We observed leakage of fluorescent microspheres from the vascular space after exposure to thrombin in all 8 of the 5 dpf larvae studied Fig. 6A.2 agonist (U-46619) which has well-characterized effects on vascular tone in other vertebrate models We used high speed, multidimensional optical microscopy to dynamically assess metrics of vascular function (blood flow and vascular reactivity) in real time in unanaesthetized zebrafish embryos. Until recently, the high speed detectors required to make these measurements (video-rate and CCDs devices) were only fast enough to measure either slower peripheral blood flow, or flow after treatment to slow heartrate . Erythrocyte tracking methods such as digital motion analysis Zebrafish form fully lumenized vessels with intact barrier function in the dorsal aorta, cardinal vein and intersegmental vessels, as early as 54 hours post fertilization Figure S2). Vascular endothelium contains all the molecular machinery required to generate contractile force via the actomyosin motor, and has been shown to play a role in capillary constriction in a number of vascular beds in other vertebrate models ,Beyond the technical advances described above, our observation that 5 dpf embryos regulate aortic blood flow in response to a vasoconstricting agent (U46619), at a point in development when there is only limited expression of vascular smooth muscle The actions of TXA2 are mediated by a G-protein-coupled receptor (TxA2 receptor) triggering activation of phospholipase Cβ via Gq/11, and ultimately inducing increases in intracellular Ca2+ and cell contraction 2+/calmodulin-dependent assembly of actin stress fibers, cell contraction, disruption of cell–cell contacts, and intercellular gap formation par genes have been cloned and identified as homologs of mammalian PARs1–32+ response in Xenopus oocytes in situ in 5 dpf zebrafish larvae.It is well recognized that the endothelium modulates vascular tone via production of vasoactive mediators including endothelin and NO, while also acting as a semi-permeable barrier between the vascular lumen and surrounding tissue. Disruption of the endothelial barrier has been shown to be mediated via Cachrna1 deficient transgenic model we have employed  will not live once nutrients from the yolk are exhausted. However, if measurements are made on anaesthetized fish, then the age of imaging can be extended out further, such that flow velocities in animals with varying degrees of vascular smooth muscle coverage can be compared.Our data suggest that the scientific opportunities presented using an integrated suite of high speed imaging techniques are extensive, and are penetrant in attempting to answer temporally important questions in vascular physiology. A limitation of the approach used here is that it will work best on embryonic trunk vessels, because of the restriction in the depth of penetration of wide field, non-multiphoton microscopy. While the working distance of the 10X objective used in these studies would work on older embryos, the Figure S1Immobilization paddles (A) were designed in our laboratory and fabricated by Fotofab  using our original computer-aided designs (CAD. B). Two micromanipulators  were used (C), one to hold the paddle (right), the other to hold the microinjection needle (left).(TIF)Click here for additional data file.Figure S2Transmission electron microscopy shows that the trunk region of the dorsal aorta of the zebrafish embryo was devoid of smooth muscle cells. This image, taken from the midpoint of the region of the aorta used for time based imaging, demonstrates that no smooth muscle cells are present around the vessel at 5 dpf. Skeletal muscle cells are clearly visible, as is the pronephros (PN). Erythrocytes (RBC) are abundant within the vessel, and endothelial cells are clearly evident. No smooth muscle cells can be detected. .(TIF)Click here for additional data file.Movie S1The movie is divided into two repeating sections, each section shows the blood flow along the aorta over a 1.5 second period (the time lapse sequence has been slowed down 10 fold). The first section simply shows the erythrocytes moving along the vessel. The pulsatile nature of the flow is readily apparent. In the second sequence, the red cells have been segmented out and are overlaid with round spots  which are shown to faithfully track the erythrocyte passage along the vessel. It is these segmented spots that were used for the quantitative analysis of flow.(MOV)Click here for additional data file.


In order to identify genetic causes of VACTERL association , we have collected DNA samples from 20 patients diagnosed with VACTERL or with a VACTERL-like phenotype as well as samples from 19 aborted fetal cases with VACTERL. To investigate the importance of gene dose alterations in the genetic etiology of VACTERL association we have performed a systematic analysis of this cohort using a 180K array comparative genomic hybridization (array-CGH) platform. In addition, to further clarify the significance of PCSK5, HOXD13 and CHD7 genes in the VACTERL phenotype, mutation screening has been performed. We identified pathogenic gene dose imbalances in two fetal cases; a hemizygous deletion of the FANCB gene and a  unbalanced translocation. In addition, one pathogenic mutation in CHD7 was detected, while no apparent disease-causing mutations were found in HOXD13 or PCSK5. Our study shows that although large gene dose alterations do not seem to be a common cause in VACTERL association, array-CGH is still important in clinical diagnostics to identify disease cause in individual cases. VACTERL association (OMIM #192350) is described as the non-random association of multiple congenital anomalies affecting the specific organ systems intended in its letters; V vertebral anomalies, A anal atresia, C cardiac defects, T tracheoesophageal fistula, E esophageal atresia, R renal anomalies and L limb defects. The diagnosis is based on clinical observation, with a minimum of three anomalies in the spectrum required for diagnosis and a reported prevalence of VACTERL association in infants in different studies between 1/10, 000 and 1/40, 000 At present, no common etiology of VACTERL association is known and a heterogeneous background to the phenotype is likely The aim of the study was to determine the role of gene dose alterations in the etiology of VACTERL association through screening analysis using array-CGH in patients and fetal cases. We also wanted to clarify the role of PCSK5, HOXD13 and CHD7 variants in the VACTERL phenotype since PCSK5 and HOXD13 genetic variants have been reported in VACTERL patients Patient participation was approved after obtaining written consent from the parents. Samples from fetal cases were obtained from a biobank to which the parents had agreed on storage of fetal tissue samples for research and clinical purposes. Ethical approval for the study was obtained from the regional ethics committee at Karolinska Institutet, Stockholm.Altogether, 39 patients and fetal cases with either VACTERL or a VACTERL-like phenotype were included in the study . Twelve Genomic DNA was isolated from either peripheral blood or tissue samples according to standard procedures. For fetal cases, DNA was isolated from routinely preserved fresh frozen heart, liver, lung, or spleen tissue using the Gentra Puregene Blood Kit  in combination with Proteinase K  with slight modifications to the manufacturer's protocol.http://projects.tcag.ca/variation/), DatabasE of Chromosomal Imbalance and Phenotype in Humans using Ensembl Resources  and the clinical database at the Department of Clinical Genetics, Karolinska University Hospital, comprising approximately 2000 analyzed patient samples. Detected CNVs overlapping CNVs reported in DGV or frequently observed in the clinical database were considered to be likely benign. Specifically, a visual inspection of probes located in PCSK5, HOXD13, the FANC genes  and CHD7 was carried out. The data discussed in this section have been deposited in NCBI's Gene Expression Omnibus http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE51958).Screening for gene dose alterations was carried out using a 180K custom oligonucleotide microarray with whole genome coverage (Oxford Gene Technology) used in a clinical setting at the Department of Clinical Genetics, Karolinska University Hospital. Genomic DNA isolated from blood or tissue and sex-matched pooled reference DNA isolated from healthy controls  was used for analysis. Sample labelling , hybridization and slide washing  was performed essentially according to the manufacturer's recommendations. Slide scanning was performed using a 3 µm-resolution microarray scanner and the Feature Extraction software v 10.7.3.1 (Agilent Technologies) was used for initial data analysis followed by analysis with the CytoSure Interpret Software v 3.3.2 (Oxford Gene Technology). For detection of copy number variants (CNVs), the standard probe cut off levels used in routine diagnostics at the Department of Clinical Genetics, Karolinska University Hospital (log2-ratios above 0.35 for duplications and below -0.65 for deletions) were applied. For an overall screening analysis, CNVs with a minimum of three consecutive probes with deviating log2-ratios were identified. After visual inspection, CNVs were compared to variants reported in the Database of Genomic Variants  was performed as previously described in individual cases as specified in Confirmation of the FANCB deletion detected with array-CGH was performed at Centogene GmbH, Rostock, Germany using the MLPA-P113-A2 kit from MRC Holland according to the manufacturer's recommendations.http://www.interactivebiosoftware.com) to predict variant pathogenicity. Alignment of protein sequences for PCSK5 was performed using the UniProtKB database (http://www.uniprot.org/) and included sequences from 6 vertebrates.DNA isolated from blood or tissue was used for exon and exon/intron boundary amplification of the genes PCSK5 (exons 1–39), HOXD13 (exons 1–2) and CHD7 (exons 2–38) with PCR using standard procedures. Mutation screening in PCSK5 was performed in all patients while screening of HOXD13 and CHD7 was performed in a subset of patients . All pri20-primers according to the manufacturer's protocol. Subsequent PCR using cDNA from patient V7 as well as a healthy control in parallel reactions using a forward primer located in exon 12 and a reverse primer located in exon 14 (Ensembl accession number ENSG00000171316) was carried out. Fragment sizes were compared using agarose gel electrophoresis. DNA sequencing of the amplified fragment was performed as described above.As DNA from the parents was unavailable in patient V7, total RNA was isolated from peripheral blood of the patient  to investigate a potential effect of a detected variant on CHD7 splicing. cDNA synthesis was performed with the SuperScript® III First Strand Synthesis System kit  using oligo(dT)http://cbg.garvan.unsw.edu.au/pina/) To test for protein-protein interactions between proteins encoded by genes in detected pathogenic CNVs or CNVs of unclear significance and VACTERL candidate genes, searches were performed in the PINA: Protein Interaction Network Analysis Platform database  confirmed deletion of the complete FANCB gene in the fetus, a finding associated with the diagnosis of X-linked VACTERL  that was found in 1/95 healthy blood donors  and two predicted to be benign , that were not detected in 95 healthy blood donors , Table 3http://www.uniprot.org/).A paternally inherited heterozygous 9 bp-deletion in a polyalanine region of HOXD13 was identified in patient V4 , resultide novo mutation (c.3202-1G>C) predicted by the Alamut program to affect splicing, was identified in patient V15 (CHD7 database (www.chd7.org). Of the corresponding publications CHD7 splicing. The splice prediction function in the Alamut v2.0 software did not predict the mutation to strongly affect splicing.A heterozygous ient V15 , Table 4Searches in the PINA database to firstly identify possible interactions between proteins encoded by genes in the 9p-deletion and 18q-duplication, and secondly between proteins encoded by genes in the detected gene dose alterations  and VACTERL candidate genes, resulted in a number of putative interactions presented in FANCB was identified together with a dup(16)(p12.3p13.11) that has been associated with malformations FANCB gene deletions in males with Fanconi anemia presenting as VACTERL have been reported twice, in both cases the aberrations were detected by array-CGH screening FANCB deletion alone with certainty can explain the fetal malformations. In summary our results support published data that clearly pathogenic gene dose imbalances are rarely found in VACTERL patients We have performed array-CGH on DNA from 39 patients and fetal cases with VACTERL diagnosis or a VACTERL-like phenotype. In two out of the 19 fetal cases pathogenic gene dose alterations were identified. In one fetal case, a terminal 18q-duplication and 9p-deletion was shown to be caused by a  unbalanced translocation inherited from the father who was a balanced carrier. A clinical overlap between the VACTERL and trisomy 18 phenotypes can be observed Mutation screening of PCSK5 and HOXD13 was initiated after reported mutations in VACTERL cases The apparent low number of findings in genetic studies of VACTERL may be the result of “true” VACTERL cases  being due to other than genetic causes such as epigenetic factors, disruptive processes during pregnancy or maternal disease. Recent twin studies support a low proportion of genetic abnormalities found in VACTERL patients The results of our study show, that array-CGH may be considered as a diagnostic tool in patients with VACTERL association. To search for additional causative mutations we plan systematic high throughput-sequencing in VACTERL patients.Table S1Summary of clinical findings in patients and fetal cases with VACTERL association or a VACTERL-like phenotype(DOC)Click here for additional data file.Table S2Summary of genetic analyses performed in patients and fetal cases(DOC)Click here for additional data file.Table S3Protein-protein interactions between proteins encoded by genes in detected CNVs and VACTERL candidate genes*(DOC)Click here for additional data file.Table S4Copy number variants of unclear clinical significance detected by array-CGH(XLS)Click here for additional data file.


The tetracycline antibiotics are widely used in biomedical research as mediators of inducible gene expression systems. Despite many known effects of tetracyclines on mammalian cells–including inhibition of the mitochondrial ribosome–there have been few reports on potential off-target effects at concentrations commonly used in inducible systems. Here, we report that in human cell lines, commonly used concentrations of doxycycline change gene expression patterns and concomitantly shift metabolism towards a more glycolytic phenotype, evidenced by increased lactate secretion and reduced oxygen consumption. We also show that these concentrations are sufficient to slow proliferation. These findings suggest that researchers using doxycycline in inducible expression systems should design appropriate controls to account for potential confounding effects of the drug on cellular metabolism. The tetracycline family is a class of broad-spectrum antibiotics that have been used clinically since the mid-twentieth century. Since then, they have found application beyond their anti-microbial activity in both the clinic and biomedical research The canonical prokaryotic target of the tetracyclines is the bacterial ribosome, the inhibition of which blocks bacterial protein synthesis In this study, we expanded upon these findings to determine potential confounding effects of the tetracyclines–particularly doxycycline (Dox), the predominantly used compound–at concentrations commonly employed in inducible gene expression systems: 100 ng/mL - 5 µg/mL. We found that these concentrations of drug can significantly alter the metabolic profile of the cell, as well as reduce the proliferative rate, though the effect size depends upon the particular cell line used. These data strongly suggest that researchers using Dox-inducible systems should carefully optimize experiments to minimize potentially confounding effects of the drug, and design additional controls as needed.To look in an unbiased way at the effects of Dox on cells in culture, we performed gene expression analysis on MCF12A cells–an untransformed breast epithelial line–treated with the drug at 1 µg/mL or with a vehicle control. Metabolic pathway enrichment analysis ) revealed several pathways, including oxidative phosphorylation and glycolysis, to be significantly enriched in the Dox-treated cells . Many ofBecause treatment with Dox alters expression of genes involved in glycolysis and oxidative phosphorylation, we tested whether Dox treatment causes corresponding functional metabolic changes in MCF12A cells. As shown in To determine whether the effect of Dox on cellular metabolism is dose-dependent, we measured lactate production rates  and oxygTo assess whether the Dox-mediated metabolic changes are cell type-specific or broadly generalizable to human cell lines, we repeated the metabolic assays on 9 different human cell lines–both transformed and untransformed–and observed enhanced glycolysis in a majority of them  . There iin vitroPrevious studies have shown that Dox can impair cell proliferation 1 arrest, propidium iodide (PI) staining showed modest enrichment of cells in this phase of the cell cycle in MCF12A cells , lung cancer cell lines H157 and H1437 from Dr. Steven Dubinett (UCLA), and LNCaP prostate cancer and HeLa cervical cancer cell lines from Dr. Steven Bensinger (UCLA), and all other cell lines from Dr. Frank McCormick (UCSF); all cell lines have been well characterized RNA extraction was performed using RNeasy (Qiagen) according to manufacturer’s guidelines. Samples were analyzed using the Affymetrix Genechip U133 Plus 2 array by the UCLA Clinical Microarray Core Laboratory. Resulting data are available through NCBI’s Gene Expression Omnibus (GEO Series accession number GSE45029). To identify metabolic pathways associated with Dox treatment, we used the GSEA algorithm and metabolic pathway annotation defined by the Kyoto Encyclopedia of Genes and Genomes (KEGG) (release #58). We collapsed gene expression probes based on enzyme activity (Enzyme Commission [EC] numbers) rather than on gene identity to avoid unequal representation of equivalent enzymatic function within pathways, thus emphasizing potential flux through the network. The metric used for gene ranking was the signal to noise ratio (SNR) between the Dox treatment and control. The probesets were collapsed by average SNR for GSEA, and by maximum absolute SNR for each enzyme in the heat maps. Probeset annotation was based on UniGene build #230 and UniGene identifiers were mapped to each EC using the gene names provided by KEGG.Media was collected from culture plates and analyzed for glucose and lactate concentrations using the Bioanalyzer 4 . Cells were treated with Dox or vehicle control for 96 hours. Fresh media was added to plates between 20 and 24 hours before sample collection. Values were normalized to both cell number and time interval. Some statistical variability was observed among experiments, particularly with respect to glucose, because the effect size measured at these concentrations of Dox is small compared to the error of the assay. Lactate is a more consistently sensitive readout of glycolytic activity.Basal oxygen consumption rates were measured using the XF24 Extracellular Flux Analyzer (Seahorse Bioscience). Cells were treated with Dox or vehicle control for 96 hours, unless otherwise indicated. 18 hours prior to assay, cells were seeded at 50,000–80,000 cells per well (optimized for each cell line) in 24-well proprietary assay plates with between 3 and 8 replicates per treatment group. Oxygen consumption values were normalized to total protein using BCA Protein Assay Kit (Pierce). Because plating density around the oxygen sensors affects readings, there is considerable well-to-well variability. Because cell morphology can alter the distribution of cells in the well, different cell lines are measured with varying sensitivity.Cells were seeded in 6 well plates at 50,000 cells per well and treated with Dox . Cells were counted 24, 72, and 96 hours after plating using a particle counter (Beckman Coulter).6 cells/mL and incubated for 20 minutes at room temperature in the dark. Analysis was performed using the BD FACSDiva software on an LSR II flow cytometer (BD Biosciences). PI fluorescence was recorded in the PE channel. For cell cycle analysis, flow cytometry data was imported and analyzed using the ModFit LT software.Cell cycle analysis was performed using propidium iodide (PI) staining. In brief, cells were trypsinized and resuspended in hypotonic DNA staining buffer  at a concentration of 10Cell viability was measured using the FITC Annexin V Apoptosis Detection Kit II (BD Pharmingen) according to the manufacturer’s protocol. Cells were incubated with Annexin V and PI for 15 min and then analyzed by flow cytometry as previously described. Annexin V fluorescence was recorded on the FITC channel and PI on the PE channel. Data was analyzed using FlowJo. Cells in early apoptosis were indicated by Annexin V positive, PI negative staining (lower right quadrant) while cells in late apoptosis were marked by Annexin V/PI double positive staining (upper right quadrant).Figure S1Metabolic gene sets are enriched upon treatment with Dox. Enrichment plots for the top ranked KEGG-defined pathways in MCF12A cells treated with Dox at 1 µg/mL, compared to vehicle control.(TIF)Click here for additional data file.Figure S2Dox treatment alters expression of genes involved in major central carbon metabolism pathways. Heat maps highlight changes in expression of genes in A) the Glycolysis-Gluconeogenesis & Pentose Phosphate Pathway  and in B) KEGG-defined Oxidative Phosphorylation after Dox treatment at 1 µg/mL in MCF12A cells, compared to vehicle control. Entries are ranked by SNR and are collapsed by enzyme function; the constituent gene with the maximum absolute SNR is shown in the heat map. Regulated enzymes in glycolysis are shown in blue and those in gluconeogenesis are shown in orange.(PDF)Click here for additional data file.


High genetic diversity at both inter- and intra-host level are hallmarks of RNA viruses due to the error-prone nature of their genome replication. Several groups have evaluated the extent of viral variability using different RNA virus deep sequencing methods. Although much of this effort has been dedicated to pathogens that cause chronic infections in humans, few studies investigated arthropod-borne, acute viral infections.We deep sequenced the complete genome of ten DENV2 isolates from representative classical and severe cases sampled in a large outbreak in Brazil using two different approaches. Analysis of the consensus genomes confirmed the larger extent of the 2010 epidemic in comparison to a previous epidemic caused by the same viruses in another city two years before (genetic distance = 0.002 and 0.0008 respectively). Analysis of viral populations within the host revealed a high level of conservation. After excluding homopolymer regions of 454/Roche generated sequences, we found 10 to 44 variable sites per genome population at a frequency of >1%, resulting in very low intra-host genetic diversity. While up to 60% of all variable sites at intra-host level were non-synonymous changes, only 10% of inter-host variability resulted from non-synonymous mutations, indicative of purifying selection at the population level.Despite the error-prone nature of RNA-dependent RNA-polymerase, dengue viruses maintain low levels of intra-host variability. Dengue is the most frequent arthropod-borne human viral infection in tropical and sub-tropical regions, with one hundred million people at risk annually. In the last two decades, Brazil has been responsible for more than 60% of the total reported dengue fever cases in the Americas, experiencing severe yearly outbreaks of serotypes 1 to 3 Dengue has limited intra-genotype diversity, which is modulated by two main processes: (i) the host’s immune response, which exerts a selective pressure on the virus, and (ii) the bottlenecks at transmission  et al.quasispecies diversity with unprecedented resolution. Work by our group and others has employed 454 pyrosequencing, a method for deep sequencing, for whole-genome shotgun sequencing of RNA viruses causing chronic infections, such as hepatitis C virus (HCV), human immunodeficiency virus (HIV), and simian immunodeficiency virus (SIV) Our current understanding of dengue intra-host diversity has been mostly limited to the analysis of single genes of dengue serotypes 1 to 3 using conventional cloning and Sanger sequencing methods The current project was conducted after Hospital das Clínicas -University of São Paulo’s Institutional Review Board (CAPPesq) approval, under protocol #0652/09. Written informed consent was obtained from all participants. Written informed consent was also obtained for children through their parents.Serum samples were obtained from patients with clinically suspected dengue fever treated at the Ana Costa Hospital in Santos, coastal region in the State of Sao Paulo, Brazil, during the 2010 DENV2 epidemic To determine the viral load, RNA was extracted from 140 µl of plasma using the Qiagen Viral RNA mini kit  according to the manufacturer’s instructions. RT-PCRs were performed in duplicate using SuperScript® III Platinum® SYBR® Green One-Step RT-qPCR with ROX kit  and pan-dengue primers covering all 4 serotypes Aedes albopictus cell line (ATCC number CRL-1660). After the onset of a cytopathic effect (CPE) associated with DENV infection, supernatants were collected and subjected to RT-PCR in order to confirm the serotype. To discriminate between primary and secondary dengue virus infections, the antigen-binding avidity of specific IgG was measured using a modified Dengue ELISA IgG kit (Focus Technologies) Several samples were also isolated in cell culture using the established C6/36 500 µl of plasma from acutely ill patients were centrifuged at 3,000 rpm for 5 min to remove cellular debris followed by centrifugation at 14,000 rpm for 60 min to concentrate the virus. To evaluate if the viral population diversity found in plasma is maintained after isolation in insect cell culture, we isolated viruses from one acutely infected patient and sequenced the viruses (i) direct from plasma and (ii) after one-week culture in C6/36 cell line. Viral RNA was extracted from 200 µl of plasma or cell culture supernatant containing the concentrated virus using the QIAamp Viral RNA Mini Kit  according to the manufacturer’s instructions. Primers were designed to amplify the complete open reading frame with three to six overlapping PCR amplicons of approximately 2–4 kb . Initially, reads were trimmed to remove short and low-quality reads. Using a phylogenetically related DENV2 genome as reference (GenBank ID GU131864), reads were assembled and the resulting consensus sequences were then used as each subject’s sequence in a reference guided alignment to assess intra-host variability. The single nucleotide polymorphism (SNP) analysis was performed using CLC's SNP analysis tool, applying the following parameters: window length = 7, maximum gap and mismatch count = 2, minimum central quality base = 30, minimum average quality for window bases = 25, minimum coverage = ×100, and minimum variant frequency = 1% (defined as the percentage of nucleotides that differ from the reference). Parameters used in this analysis were chosen to include variants more likely to be biologically significant, based on previous studies that used Roche/454 data In addition to the samples sequenced in 454 Roche, one different plasma sample was sequenced on the Illumina MiSeq using direct sequencing as previously described E. coli DH5alpha quimio-competent cells. Thirty-one clones were sequenced on the ABI3100 using Big-Dye7 Terminator , with M13 forward and M13 reverse primers. Chromatograms were analyzed in CodonCode Aligner v.3.0 (available at http://www.codoncode.com/) with a Phred quality score of 20 as cut-off for trimming of low-quality sequences and manually aligned and inspected using SeAl .To compare the variability obtained by deep sequencing methods to traditional cloning and Sanger sequencing, the capsid gene and part of the prM gene of the sample ACS538 were subjected to traditional Sanger sequencing. Viral RNA was extracted as described above and single strand cDNA was generated with random hexamers using the High Capacity cDNA Reverse Transcription Kit  followed by amplification with the TaqPlatinum Hi Fidelity kit  using primers described in http://tree.bio.ed.ac.uk/). Selection pressures were evaluated by comparing the rate of non-synonymous changes per non-synonymous site (dN) to the rate of synonymous changes per synonymous sites (dS) inferred for entire polyprotein as well as for specific sites (by codon) of all the consensus genomes by single-likelihood ancestor counting (SLAC), using the open-source HyPhy package Consensus complete genome sequences obtained by sequencing on the GS Junior and the MiSeq were aligned to globally sampled DENV2 genomes . Selection pressure analysis performed across the whole coding region and also by codon revealed no signs of positive selection (w = 0.03) as well as no codon under selection. The mean genetic diversity of the population (π) across the entire polyprotein was 0.002 for nucleotide and 0.0008 for amino acid sequence, indicative of protein conservation. Changes in 84 nucleotide sites were distributed throughout the coding region, and only nine were non-synonymous with three of them exclusively found in ACS542  . SurprisTo evaluate Dengue viruses in an epidemiological context, a Bayesian phylogenetic tree was constructed using 124 complete genomes comprising all five DENV-2 human genotypes and the eleven genomes sequenced from the 2010 DENV2 Santos outbreak   and clade 2  . By mappOn average, 44,000 reads per sample (±20.800) were generated with an average coverage of ∼1226 reads per nucleotide in Roche/454 . BetweenHomopolymer regions can induce erroneous base calls on the Roche/454 platform, therefore all SNPs and DIPs that mapped to those regions were disregarded in subsequent analyses. Between 32 and 85 DIPs for each sample sequenced were recovered by 454/Roche and only two DIPs by Illumina. More than 90% of 454/Roche DIPs mapped to homopolymer regions and about 80% of them were identified as deletions in homopolymers of A, followed by a much lower proportion of Ts, Cs and Gs, respectively . The preThe level of intrahost variability obtained through Illumina direct sequencing was also evaluated for the sample ACS380 and was comparable to the observed through 454 pyrosequencing  and S4.In parallel, the entire capsid and part of the prM gene of ACS538 (400 base pairs) was amplified using similar conditions and 31 clones were sequenced. The resulting 12378 nucleotides sequenced revealed twelve changes randomly distributed among the clones. One change was observed twice, but the remaining eleven were singletons. None of the changes were detected by deep sequencing and the unique variable site in the capsid recovered by deep sequencing was not observed in Sanger sequences probably due to the very low frequency (1.6%).Overall, the two deep sequencing methods revealed between 10 and 44 variable sites per genome, resulting in approximately 0.002% of variability in all sequenced sites  were in the first and second codon positions, resulting in non-synonymous changes. High proportions of non- synonymous mutations at the intra-host level have previously been reported for DENV1 −5 respectively) . Among the seven non-synonymous variants, four were into Domain II (EDII). With exception of I129F/V for DGV37 where variant phenylalanine was at a frequency of 6%, the remaining non-synonymous variations occurred in a frequency below 2% and none of them mapped into known immunogenic regions or were related to infection. Three non-sense changes were found , but they appeared in a frequency below 2% in all cases (ctively) . AlthougDespite such a low number of intra-host variability, particular sites showed some variability in distinct viral populations . Among fTo date, within-host diversity of RNA viruses has been studied most comprehensively in HIV-1 and HCV bona fide and artificial variants since the type and rate of the error introduced by each platform is different. For example, insertion and deletion (DIPs) errors occur more frequently on the 454/Roche platform, especially in homopolymer regions, while the Illumina platform is more prone to mismatch errors One of the challenges in studying viral diversity using massive parallel sequencing is the discrimination between Although the results described in this report are not based on large number of patients to carry out statistical analysis, some considerations can be discussed.−4s/s/y bona fide mutations. Parameswaran et al., also used deep sequencing to evaluate intra-host DENV2 variability in Nicaragua, but although they also noticed low intra-host variability, they observed 10 to 100× more variable sites across the genome compared to our results, with no evidences of mixed infection Despite the generally accepted high substitution rate of dengue viruses, estimated at 6.5×10−5, which is consistent with previous results reporting DENV1 intra-host variability as 7.2×10−5et al. used cloning and Sanger sequencing to evaluate variability, they also applied rigorous methods to validate variants eventually leading to results comparable to our study.By considering the number of reads with variability at any given site per total coverage at this site, the mean diversity of intra-host viral population was 4.8×10With few exceptions, most studies estimating DENV variability have used cloning and Sanger sequencing Two SNPs found by Sanger (both from A to G) were also present in 454/Roche reads. However, they mapped in homopolymer regions of 3 and 4 Adenines and were in frequencies of 0.37 and 0.4% respectively (data not shown). Thus, according to the parameters adopted here to consider a variant to be true , these variants were dismissed. We therefore suspect that much of the changes reported by studies that used Sanger sequencing without posterior validation of the variants represent artifacts generated during cDNA synthesis and PCR amplification Arboviruses usually harbor less nucleotide and amino acid variation than observed in other RNA viruses due to alternate replication in mammalian and mosquito cells in vitro culture is largely unknown. To address this question, we compared deep sequenced viruses directly from human plasma to the same viruses after one-week of culture in C6/36 mosquito cells. As expected, the consensus sequences were identical, but the sites showing variability at the intra-host level were different. These findings indicate that different (low) selection pressures are exerted by C6/36 culture It is not unusual to use in-vitro cell culture to amplify viruses obtained from infected individuals prior to genetic and molecular studies There is little  evidence that intra-host genetic variability is linked to disease severity i) common variable sites are a consequence of de novo mutations in less constrained sites (i.e. mutation hotspots). Mutation hotspots often reflect the mechanisms of generating mutations at a particular site  ii), minor variants are not immediately eliminated by genetic drift and could be maintained during transmission to a new host. Although variable shared sites were found as minority population within the host, it has been demonstrated that minor variants can be passed between individuals regardless of their fitness, and sometimes for extended time-periods Although only few sites exhibited variability in our study, some were consistently identified among distinct viral populations. Detection of the same variants in viruses circulating during the same epidemic suggests two possible and non-exclusive explanations:  against more than 33 thousand cases reported in Santos coastal cities in a short five month period (http://www.cve.saude.sp.gov.br/htm/zoo/den09_import_autoc.htm). Although viruses sequenced in this study were all sampled in the city of Santos, it is a regional hub that is heavily trafficked by residents of neighboring cities.Interestingly, the separation of Santos viruses into two sub-clades and the high diversity found among the sequences in comparison to viruses sampled in previous epidemics  sampled by Roche/454 GS Jr. The main graph shows the absolute number of DIPs found per nucleotide obtained for the 10 viruses sequenced in Roche/454. The small graph at the top right specifies the proportion of DIPs in relation to homopolymer size.(TIF)Click here for additional data file.Table S1Primers used to amplify the complete genome and also the capsid gene.(DOC)Click here for additional data file.Table S2GenBank accession numbers of Dengue viruses serotype 2 used to build the phylogenetic tree.(DOC)Click here for additional data file.Table S3Intra-host variability (SNPs). Reference positions are according to the ACS380 genome (JX286526), and the details of the amino acid changes are in (DOC)Click here for additional data file.Table S4Variable non-synonymous sites at intra-host level.(DOC)Click here for additional data file.