Spring-assisted cranioplasty is performed to correct the long and narrow head shape of children with sagittal synostosis. Such corrective surgery involves osteotomies and the placement of spring-like distractors, which gradually expand to widen the skull until removal about 4 months later. Due to its dynamic nature, associations between surgical parameters and post-operative 3D head shape features are difficult to comprehend. The current study aimed at applying population-based statistical shape modelling to gain insight into how the choice of surgical parameters such as craniotomy size and spring positioning affects post-surgical head shape.Twenty consecutive patients with sagittal synostosis who underwent spring-assisted cranioplasty at Great Ormond Street Hospital for Children  were prospectively recruited. Using a nonparametric statistical modelling technique based on mathematical currents, a 3D head shape template was computed from surface head scans of sagittal patients after spring removal. Partial least squares (PLS) regression was employed to quantify and visualise trends of localised head shape changes associated with the surgical parameters recorded during spring insertion: anterior–posterior and lateral craniotomy dimensions, anterior spring position and distance between anterior and posterior springs.Bivariate correlations between surgical parameters and corresponding PLS shape vectors demonstrated that anterior–posterior (Pearson’s Population-based analysis of 3D post-operative medical images via computational statistical modelling tools allowed for detection of novel associations between surgical parameters and head shape features achieved following spring-assisted cranioplasty. The techniques described here could be extended to other cranio-maxillofacial procedures in order to assess post-operative outcomes and ultimately facilitate surgical decision making. Craniosynostosis is a congenital condition characterised by premature fusion of one or more cranial sutures during infancy. This can result in aesthetic and/or functional problems due to skull growth restrictions, often requiring early surgical intervention to reshape the skull –3. In thIn recent years, computer-assisted analysis of 3D medical image data has been employed to improve the head shape description –12 and tOne of these procedures is spring-assisted cranioplasty (SAC), used to correct the head shape in infants with sagittal synostosis –38, wherIn this study, a nonparametric SSM technique –44 was ePopulation-based analysis of 3D post-operative medical image data using computational SSM tools was expected to detect and untangle novel associations between each of the surgical parameters and the achieved head shape outcomes, which may ultimately impact on surgical decision making.Twenty consecutive patients with non-syndromic, single suture, sagittal synostosis  who underwent SAC at Great Ormond Street Hospital for Children  were prospectively recruited for this study between May 2015 and 2016. Patient age at time of spring insertion was 5.2 ± 1.2 months and springs were removed when the patients were 9.5 ± 1.4 month old. Written parental consent was obtained during pre-operative clinic for all patients for acquisition of 3D head scans and their use in research.Clinical details about SAC Figs. , 3 can bAP) and the lateral (LAT) lengths; the distance from the coronal suture to the anterior spring (CorToAnt) as well as the distance between the anterior and posterior springs (AntToPost).Figure pre-insertion, Fig. post-removal, Fig. N-point registration algorithm in 3-matic  using the same landmarks as for the cutting plane. The registered scans were then used for statistical shape modelling.Since sagittal patients do not routinely undergo computed tomography scanning at our centre , 48, 3D Linear measurements were automatically computed on the STL files using the “meshcube” function in the Morpho-package of R . This function calculates the corners of the bounding box comprising the STL mesh, which can then be translated to head width, length and height measurements.AP, CorToAnt and AntToPost were normalised as percentages of pre-insertion head length, while LAT was represented as a percentage of pre-insertion head width, as indicated in Eqs. Pre-operative head width and length were used to normalise the recorded surgical parameters according to head size. Statistical shape analysis was performed to assess how surgical parameters at spring insertion Fig.  related www.deformetrica.org) [i [mathematical currents [M, which parameterises all 3D head shape feature information based on the common basis shape M allows extraction, quantification and visualisation of dominant 3D shape features most associated with a chosen response parameter via partial least squares regression (PLS) [Based on the twenty previously registered post-removal 3D head scan computational surface meshes, a post-removal template shape ica.org) –44 was u shape i . Within currents , which acurrents , 51. Forailed in –44. Gause et al. . Each paon (PLS) , 51.PLS shape modes [most correlated with the surgical parameters of interest [AP, LAT, CorToAnt, AntToPost) were extracted. Further, each patient head shape was projected onto the respective PLS shape mode in order to obtain a PLS shape vector   in combiPLS shape vectors most related to differences in normalised AP, LAT, CorToAnt and AntToPost (after removing size effects) and the corresponding surgical parameter were evaluated via bivariate correlation analyses. Normality of the data was assessed using Shapiro–Wilk test. For parametric data, Pearson’s r correlation coefficient was used, while Spearman’s p values <0.05. All statistical analyses were performed using R.Mean values and standard deviations  by LAT). The first spring was located at a distance of 31 ± 4% from the coronal suture, while the average distance between the anterior and posterior springs was Population values of normalised surgical parameters recorded during spring insertion are reported in Table Initial PLS analysis, regressing 3D head shape with post-removal head volume AP, LAT, CorToAnt and AntToPost) and their corresponding PLS shape vectors after accounting for volume differences. In addition, computed 3D models for the extreme cases of small (−3 SD) and big  with a prominence on top of the head, whereas big AP values were related to bigger bi-parietal widening  was associated with bigger bi-parietal widening of the posterior part of the head  was associated with localised prominences on top of the head, while positioning both springs further apart led to more globalised widening of the head with a more rounded profile in the back of the head  affects global and local post-surgical head shape outcome. Combining non-invasive 3D head shape scanning with nonparametric statistical shape modelling and PLS regression, it was found that craniotomy dimensions and positions of springs on average had significant effects on head shapes achieved at the time of spring removal. While previous studies have used SSM to predict the shape of missing anatomical bony parts , 34 or cSpecifically, we computed a 3D template head shape based on a cohort of SAC patients and employed PLS to quantify and visualise trends of localised head shape changes associated with the four surgical parameters the surgeon needs to choose when performing SAC Fig. b.AP resulted in prominences on top of the head  determined the initial spring opening. Since the forces that the compressed springs exert on the skull bone are proportional to their opening , smaller LAT values resulted in bigger forces and thus more effective widening of the head  with the objective of reducing the patient’s prominent forehead . However, Fig. AntToPost) resulted in localised head shape changes, apparent in the lateral view in Fig. As far as spring position is concerned, surgeons often place the anterior spring close to the coronal suture  when the anterior–posterior craniotomy length was big, the width of parasagittal osteotomies was narrow, the anterior spring was positioned far from the coronal suture and the separation between both springs was big. Overall, population-based 3D statistical shape modelling allowed for quantification and visualisation of trends in achieved head shape outcomes depending on each of the selected surgical parameters.LAT were found to be more effective in our cohort. However, the minimum width between parasagittal osteotomies is restricted by the fact that extreme care must be taken while performing the cuts not to damage the vein that runs below the fused sagittal suture, called the sagittal sinus [It must be noted that although trends discovered here can already facilitate surgical decisions, surgeons might face physical restrictions when performing SAC. For example, in order to maximise spring opening from insertion to removal and obtain maximum head widening, small values of al sinus . FurtherTherefore, “small” and “big” values of the surgical parameters described in this study should be understood within the limits of values reported in Table PLS shape vectors and corresponding surgical parameters were strong and the computed 3D models showed logical trends, in line with changes observed in individual patients and with expert clinical opinion.The main limitation of the current study is the relatively small sample size. Future studies should increase the number of patients in order to create more robust predictive models which could also consider factors such as patient age or severity of the pathology when analysing the effect of surgical choices in more detail. However, we believe that our cohort of twenty patients with the same diagnosis of non-syndromic single suture sagittal synostosis who have been operated by the same surgical team following the same protocols is suited well to investigate associations between surgical decisions and outcomes. Despite the small sample size, correlations between With this in mind, we believe that the proposed image-based computational methodology can be applied to other disciplines and surgical procedures for relating surgical parameters and post-operative results—the ultimate aim being facilitating surgical decision making to improve surgical outcome.In this study, 3D handheld scanning in combination with computational statistical shape modelling was employed to relate surgical parameters with long-term global and local 3D head shape features in sagittal patients undergoing spring-assisted cranioplasty. Using partial least squares regression, it was found that craniotomy dimensions and position of springs have a significant effect on local 3D head shape features about 4–5 months after initial surgery. The methodology described here could also be implemented for understanding long-term shape implications of cranio-maxillofacial surgery, which are of paramount importance when performing surgery in growing children. In conclusion, this study demonstrated that an image-based computational methodology involving statistical shape modelling and partial least squares regression provides a powerful platform to untangle the average effect of individual surgical choices in order to guide surgeons in optimising their procedural approach.


Ischemic muscles isolated from mice exposed to O2 after birth exhibit increased oxidative stress levels and reduced expression of superoxide dismutase 1 (SOD1) and vascular endothelial growth factor (VEGF). Pro-angiogenic cells (PACs) have been shown to have an important role for postnatal neovascularisation. We found that neonatal exposure to O2 is associated with reduced number of PACs in adults. Moreover, the angiogenic activities of both PACs and mature mouse aortic endothelial cells (MAECs) are significantly impaired in mice exposed to hyperoxia after birth. Our results indicate that neonatal exposure to high oxygen levels leads to impaired ischemia-induced neovascularization during adulthood. The mechanism involves deleterious effects on oxidative stress levels and angiogenic signals in ischemic muscles, together with dysfunctional activities of PACs and mature endothelial cells.Adverse perinatal conditions can lead to developmental programming of cardiovascular diseases. Prematurely born infants are often exposed to high oxygen levels, which in animal models has been associated with endothelial dysfunction, hypertension, and cardiac remodeling during adulthood. Here we found that adult mice that have been transiently exposed to O For instance, a Swedish cohort study documented a 7% higher risk of dying from cardiovascular disease in young adulthood for each week of increased prematurity3. Preterm birth has also been associated with higher blood pressure, increased incidence of hypertension, and signs of vascular dysfunction in adolescents and young adults2. Infants born preterm are often exposed to high concentrations of O2, especially when compared to the relative hypoxic condition of the intrauterine life4. This can lead to increased oxidative stress5, a factor that is thought to be involved in several diseases of prematurity including retinopathy and bronchopulmonary dysplasia6. It has been suggested that exposure to high oxygen levels in infants born prematurely could also contribute to alter cardiovascular function during adulthood. In animal models, perinatal hyperoxia (a model of prematurity) has been associated with the adult development of endothelial dysfunction, hypertension, and cardiac remodeling8. However, the specific mechanisms that are involved in that pathophysiology are largely unknown. Moreover, how prematurity and transient neonatal exposure to high oxygen levels might modulate the physiological response to cardiovascular stresses in adults remains to be determined.Increasing evidence suggests that adverse perinatal events can induce developmental programming of future diseases during adulthood, particularly in the cardiovascular system9. Postnatal neovascularization is a complex phenomenon that necessitates the activation, proliferation and migration of mature endothelial cells (angiogenesis)10. Vascular endothelial growth factor (VEGF), an endothelial cell specific mitogen, has been shown to be a critical limiting factor for the induction of angiogenesis11. In addition, nitric oxide (NO) is recognized as an essential factor for endothelial function, VEGF-induced angiogenesis13, and ischemia-induced neovascularization14. Recently, it has been proposed that postnatal neovascularization does not only rely on the sprouting of pre-existing vessels, but also involves the contribution of bone marrow-derived pro-angiogenic cells (PACs)16. Evidence suggests that these cells are mobilized from the bone marrow into the peripheral blood in response to tissue ischemia. PACs migrate and reach sites of ischemia where they can promote neovascularization after differentiation into mature endothelial cells, or more often through paracrine secretion of growth factors and cytokines17.One of the most important adaptive mechanisms in the cardiovascular system is the response to ischemia and hypoxic stress. The capacity of the organism to counteract the negative effects of ischemia following vascular occlusion depends in large part on its ability to develop new vessels (neovascularization)9. Cardiovascular risk factors such as aging, cigarette smoke exposure, diabetes and hypercholesterolemia have been associated with impaired ischemia-induced neovascularization and reduced number of PACs, both in animal models and in humans17. Preterm birth is increasingly recognized as a risk factor associated with the development of cardiovascular diseases later in life2. However, whether prematurity and/or perinatal adverse conditions can modify the physiological response to ischemia in adults is currently unknown. Here we used an animal model of prematurity and tested the hypothesis that neonatal exposure to hyperoxia might impair ischemia-induced neovascularization during adulthood. We also investigated potential mechanisms involved in that pathophysiology, including the effects of perinatal hyperoxia on the functional activities of mature endothelial cells and PACs.Unfortunately, conditions leading to the development of atherosclerosis and vascular occlusions in patients are also often associated with impaired neovascularization in response to ischemia2 or room air (controls) from day 2 to 14 after birth. When the mice reached adulthood (8–10 weeks old), hindlimb ischemia was surgically induced by femoral artery removal. Compared to controls, mice exposed to O2 after birth showed a significant impairment of blood flow recuperation after hindlimb ischemia  in ischemic hindlimb muscles at day 21 after surgery . Moreover, the number of arterioles . Interestingly, the expression of the angiogenic factor VEGF was found to be significantly reduced in the ischemic muscles of mice that had been exposed to O2 after birth . VEGF levels were similar between groups in non-ischemic muscles  isolated from adult mice. As shown in Fig. 2 after birth exhibited impaired tubule formation  compared to MAECs isolated from control mice. Moreover, NO expression (DAF staining) was significantly reduced in MAECs originating from O2–exposed mice  from day 2 to 14 after birth18. Since organ maturation continues after birth in rodents, the immediate neonatal period in these animals is thought to reflect a developmental time equivalent in humans to the last trimester of gestation. Hyperoxia in this model replicates the ex utero marked rise in PO2  occurring at delivery, a phenomenon that is accentuated in premature infants treated with high levels of oxygen. Even short-term (2–5 hours) exposure to hyperoxia has been associated with organ dysfunction after ischemia in some models19. Neonatal exposure to hyperoxia in rodents is a recognized model to study classical  O2-related complications of prematurity21. Moreover this model has also been used to document developmental programming of future diseases in the cardiovascular system, including hypertension7 and cardiac dysfunction8. Our study extends these previous findings by showing that neonatal exposure to hyperoxia also significantly impairs the future physiological response to ischemia (i.e. neovascularization) in adults. This could have important clinical implications since prematurity has been associated with a higher risk of dying from cardiovascular disease in young adulthood3. Therefore improving neovascularization and blood flow recovery in the context of tissue ischemia could constitute an important therapeutic target in this population.To our knowledge, the present study is the first documentation of the negative effect of neonatal hyperoxia on ischemia-induced neovascularization during adulthood. We used an established mouse model of prematurity, where pups were exposed to hyperoxic conditions  in ischemic muscles. Clinically, the reduction in neovascularization was also associated with increased hindlimb ischemic damages. The mechanisms involved in the inhibition of ischemia-induced neovascularization are potentially diverse. Interestingly, neonatal hyperoxia has previously been associated with increased blood pressure levels in adult rats whereas both angiogenesis24 and vasculogenesis (EPC function)25 have been shown to be impaired in animal models of hypertension. In humans, hypertension is more prevalent among adults born preterm2 and preterm-born individuals exhibit an enhanced antiangiogenic state in adult life that is specifically related to elevations in blood pressure26. However, here we found that blood pressure levels were not significantly increased in mice that had been exposed to hyperoxia after birth. Compared to previous findings, this might reflects differences in the animal model used (mice vs. rats) and/or be related to the fact that animals were studied at a relatively young age (8–10 weeks old) in the current study. In any case, our data suggest that the deleterious effect of neonatal hyperoxia on ischemia-induced neovascularization in adults is not due to hypertension.We used a well-characterized mouse model of hindlimb ischemia27 and are often exposed upon birth to high concentrations of O2 and pro-oxidant conditions6. This oxidative injury is thought to be an important factor involved in short-term complications of prematurity such as bronchopulmonary dysplasia and retinopathy of prematurity28. It has also been associated with reduced pulmonary angiogenesis in neonates29. Interestingly, our results indicate that adult mice that have been exposed to hyperoxia after birth also exhibit increased oxidative stress in ischemic muscles compared to control mice  can impair the activity of VEGF33, a critical limiting factor involved in the initiation and the maintenance of angiogenesis11. Here we found that increased oxidative stress levels in ischemic muscles of mice exposed to hyperoxia after birth was associated with a significant reduction of VEGF expression  could contribute to impair VEGF activity and ischemia-induced neovascularization in adult mice exposed to neonatal hyperoxia. However the mechanisms involved in the developmental programming of defective antioxidant defense in adults, especially in the context of tissue ischemia, remain to be defined.An important factor that could be involved in the modulation of neovascularization is oxidative stress. Infants born prematurely have reduced antioxidant defensesice Fig. . Angiogeion Fig. . It is p17. The effect of prematurity on the number of PACs is unclear. Previous studies have found increased, reduced or equal number of PACs in preterm infants compared to term controls37. However, preterm PACs seem more vulnerable to exogenous factors such as oxidative stress37. To our knowledge, no study has examined the effects of prematurity and/or neonatal hyperoxia on PAC number and function beyond the neonatal period. Here we found that previous neonatal exposure to hyperoxia is associated in adult animals not only with defective angiogenic activities of mature endothelial cells, but also with a significant reduction in the number and the functional activities of PACs. Although the mechanisms involved in that physiopathology are currently unknown, the deleterious effects of neonatal hyperoxia on PACs could be at least in part related to increased ROS levels. Indeed, previous reports have shown that excessive oxidative stress leads to impaired PAC angiogenic activities38. In future studies, it will be interesting to determine whether defective antioxidant defense mechanisms are found in PACs isolated from adults born preterm, which could contribute to impair PAC function and neovascularization in these individuals.The results of the present study suggest that PACs could also be involved in the inhibition of neovascularization in adults that have been exposed to hyperoxia after birth. PACs are recruited into ischemic tissues where they can facilitate neovascularization directly through incorporation into new vessels, or indirectly by secreting angiogenic factors in a paracrine fashionIn conclusion, our study demonstrates for the first time that transient neonatal exposure to high oxygen levels leads to impaired ischemia-induced neovascularization during adulthood. We propose that this occurs through deleterious effects on oxidative stress levels and angiogenic signals in ischemic muscles, together with dysfunctional activities of mature endothelial cells and PACs. Our study sheds light on a novel mechanism that could contribute to increase the incidence and morbidity of cardiovascular diseases in individuals born preterm. The development of strategies to therapeutically improve neovascularization in this population could have important clinical implications.2 by a mixture of medical grade 100% O2 and room air with an oxycycler ProOx model 110, Biospherix, Lacona, NY) as previously described39. Nursing dams were rotated between the hyperoxia and normoxia groups every 24 h to avoid maternal oxygen toxicity. Fourteen days after birth, exposure to hyperoxia was ended and all mice were then maintained in normoxic room air until the end of the study. For the measurement of angiogenic growth factors in the serum, enzyme-linked immunosorbent assays (ELISA) were performed using the mouse angiogenesis ELISA strip assays from Signosis  according to the manufacturer’s instructions.The animal protocol was approved and all experiments were conducted in accordance with the Comité Institutionnel de Protection des Animaux (CIPA) of the Centre Hospitalier de l’Université de Montréal (CHUM). Time-dated pregnant female C57Bl/6 mice were obtained from Charles River , maintained in a 12 h light-dark cycle and fed ad libitum. At day 2 after birth, pups were divided in two groups: the control group was maintained in normoxic conditions whereas the second group was exposed to hyperoxic conditions  system 40. Low and/or no perfusion was displayed in dark blue, whereas the highest perfusion interval was displayed in red. To account for variables such as ambient light and temperature, the results are expressed as the ratio of perfusion in the left (ischemic) vs. right (non-ischemic) hindlimb. Ischemic damage of foot and toes was evaluated using a scale from 0 (no necrosis) to 4 (amputation). Blood pressure was measured using a tail-cuff pressure instrument 41.When mice reached 8–10 weeks of age, unilateral hindlimb ischemia was surgically induced after anesthesia with 2% isoflurane as previously described41.Whole ischemic hindlimb muscles were immediately fixed in tissue-fix overnight. Identification of endothelial cells was performed by immunostaining for mouse platelet endothelial cells adhesion molecule-1 (PECAM-1 or CD31) with a rat monoclonal antibody . Capillaries, identified by positive staining of CD31 and appropriate morphology, were counted by a single observer blinded to the treatment regimen under a 200x magnification to determine the capillary density. Serial sections were cut at three different levels, and representative fields were analyzed by counting the number of capillaries in each field. Arterioles were identified using a Modified Verhoeff Van Gieson Elastic Stain Kit . Serial sections were cut at three different levels, and positive vessels identified by the presence of a continuous internal elastic laminae were analyzed for the entire section under a 100x magnification41. After bones had been carefully removed, ischemic muscles were put in a 25% sucrose solution for 10 minutes and were frozen in eppendorf tubes at −80 °C for 24 h, molded in OCT and kept at −20 °C. 3 mm frozen sections were made at three different levels in the ischemic muscles. Sections were labeled with 10 mM DHE  for 30 minutes. Intensities of fluorescence were measured and analyzed using computer-based software (ImageJ) with the same threshold for all sections under a 100x magnification. The specificity of the test was confirmed by pre-incubating the section with superoxide dismutase polyethyleneglycol (PEG-SOD) 500 U/ml for 1 h (data not shown).To evaluate superoxide production in ischemic muscles, dihydroethidium (DHE) fluorescence labeling was performedHindlimb muscles were rinsed in PBS, frozen in liquid nitrogen and stored at −80 °C. Protein extracts were obtained after homogenization of muscles in ice-cold RIPA lysis buffer. The membranes were probed with the following antibodies: 1:1000 VEGF , 1:1000 SOD-1  and 1:5000 alpha-tubulin . Protein expression was quantified by high-resolution optical densitometry (image J software). Results are expressed as density values normalized to the loading control.41.The percentages of PACs contained in the total viable cell population derived from the spleen was measured by flow cytometry  using the fluorescence-coupled cell markers CD34-FITC, VEGFR-2 (Flk1)-PE and CD117 (c-kit)-APC . Cell phenotypes were determined by the analysis of 300 000 events41.Seven days after hindlimb ischemia, mouse bone marrow mononuclear cells were isolated from the femora, tibiae and humeri bones by flushing the bone marrow cavities using culture medium (medium 200 (Life Technologies) supplemented with 18% fetal bovine serum  and low serum growth supplement ). After red blood cell lysis and washing, bone marrow mononuclear cells were plated on 0.005% fibronectin (Sigma). After 4 days in culture, non-adherent cells were removed by thorough washing with PBS. Adherent cells were stained with 1,10-dictadecyl-3,3,30,30 tetramethyllindocarbocyanine perchlorate-acetylated low-density lipoprotein . Bone marrow PACs were characterized as adherent cells that were positive for DiI-acLDL uptake42.A monolayer of confluent human umbilical vein endothelial cells  was prepared in 24 well plates. HUVECs were pretreated for 16 h with tumor necrosis factor-α , fixed and stained with DAPI . PACs were labeled with DiI-AcLDL and 15 000 PACs were added to each well (2 wells/mouse) and incubated for 3 h at 37 °C. Non-attached cells were gently removed by PBS washing and adherent PACs were fixed with 2% paraformaldehyde and counted in three random fields per well32. The thoracic aorta was opened longitudinally and cut into 2 mm-long explants. The aortic segments were placed face-down on a Matrigel-coated 6-well plates  and incubated in DMEM supplemented with 10% FBS, 10% Newborn Calf serum, 1% penicillin-streptomycin (Life Technologies), 90 ug/mL heparin (Sigma), 50 ug/ml endothelial cell growth supplements (VWR), and 100 U/mL fungizone (Life Technologies) at 37 °C in a 95% air/5% CO2 incubator. The vessel segments were removed once cell outgrowth was observed (around day 7). 1–2 weeks later, the cells were detached with dispase (BD Biosciences) and plated onto 1% gelatin–coated 25 cm2 flasks. Cells were used at early passage (P2).Endothelial cells were isolated from the thoracic aorta using an explant technique42.Endothelial cells were plated (20 000) in 96-well plates that had been pre-coated with 50 μl of growth factor reduced Matrigel Matrix (BD Biosciences) and cultured at 37 °C for 6 h with 50 ng/ml of VEGF. Capillary-like tubes were then photographed under an inverted microscope and all side branches were counted by a single investigator in a blinded manner42.Cell migration was assessed using a modified Boyden chamber assay. Polyvinylpyrrolidine-free polycarbonate filter Transwell inserts  were coated with 0.1% gelatin. Inserts were placed in a 24-well plate containing medium with 50 ng/ml of VEGF. Cells were allowed to migrate from the upper to the lower chamber for 6 h at 37 °C. The number of cells that had migrated was counted in three different representative high power (200x) fields per insert (2 inserts/condition)41.Intracellular generation of NO was visualized with diaminofluorescein-2-diacetate . Cells were incubated with DAF-2DA for 30 minutes. Fluorescence intensities were quantified using imageJ, with the same threshold for each experimentAll results are presented as mean ± SEM. Statistical significance was evaluated by unpaired t-test. A value of P < 0.05 was interpreted to denote statistical significance.All data generated or analyzed during this study are included in this published article.Supplemental Figure


SWS1) in all owls we studied, but two other color vision genes, the red-sensitive LWS and the blue-sensitive SWS2, were found to be under strong positive selection, which may be linked to the spectral tunings of these genes toward maximizing photon absorption in crepuscular conditions. We also detected the only other positively selected genes associated with motion detection in falcons and positively selected genes associated with bright-light vision and eye protection in other diurnal raptors (Accipitriformes). Our results suggest the adaptive evolution of vision genes reflect differentiated activity time and distinct hunting behaviors.Owls (Strigiformes) represent a fascinating group of birds that are the ecological night-time counterparts to diurnal raptors (Accipitriformes). The nocturnality of owls, unusual within birds, has favored an exceptional visual system that is highly tuned for hunting at night, yet the molecular basis for this adaptation is lacking. Here, using a comparative evolutionary analysis of 120 vision genes obtained by retinal transcriptome sequencing, we found strong positive selection for low-light vision genes in owls, which contributes to their remarkable nocturnal vision. Not surprisingly, we detected gene loss of the violet/ultraviolet-sensitive opsin ( After the K-T mass extinction, birds experienced an explosive radiation, diversifying and adapting to many different terrestrial and aquatic niches from the tropics to the poles12+ concentration by a Na+/Ca2+-K+ exchanger (NCKX), and thereby restores the cGMP concentration through activating guanylyl cyclase activating proteins (GCAPs) and hence guanylyl cyclase (GC), leading to the re-opening of CNG channels. The restoration of cGMP concentration incorporated with an efficient inactivation of each of the activated components involved in the phototransduction cascade is considered to be important for photoresponse recoveryVision in vertebrates begins with the absorption of light in the retinal photoreceptor cells (rods and cones) and activates a series of biochemical reactions known as the phototransduction cascade, which converts light into an electrical signal. Rods are specialized for vision in dim light and cones mediate vision in bright light. The phototransduction cascade process in rods and cones are similar except for their use of different isoformsde novo assembly using Trinity softwareRH1)  and its sister clade, Australaves   using rewareRH1) . In addiwareRH1)  from thewareRH1) , Table 1PDE6B, CNGB1, CNGA1, SAG and SLC24A1) involved in the phototransduction pathway in rods . SLC24A1 showed the strongest positive selection signal  based on the branch model, and the ω value was also determined to be significantly larger than one (P = 0.047) based on the likelihood ratio test with ω fixed to one in the null model. SLC24A1 encodes Na+/Ca2+-K+ exchanger, extruding free calcium in the outer segment of rods and allowing the restoration of the cGMP concentration. The positive selection observed in five dim-light vision genes involved in the activation and recovery of photoresponse may help to enhance the sensitivity to light and therefore possibly contribute to high visual acuity and high temporal resolution of vision in dim light conditions. In addition to this molecular adaptation, for visual sensitivity, the evolution of rod-dominated retina and forward-facing eyes in owls are also considered to be specialized for visual sensitivity612Among 119 vision genes examined in owls, we found 9 genes to be under positive selection, including 5 dim-light vision genes  in owls. Intriguingly, strong signals of positive selection were detected in the red-sensitive LWS (long-wavelength-sensitive) gene and the blue-sensitive SWS2 (short-wavelength-sensitive) gene along the ancestral branch of owls based on the branch-site model, and a number of positively selected sites were identified  and S223A (corresponding to S292A in bovine rhodopsin) were found, respectively , in which another amino acid change T272A (corresponding to T269A in bovine rhodopsin) reducing λmax by 15 nm in LWS was found , the only extant owl species that has had the λmax of cone visual pigments spectrophotometrically measuredLWS  and a long-wavelength shift λmax 463 nm in SWS2  have previously been reportedLWS and the long-wavelength shift of SWS2 in Tawny Owl seem to approach the dominant wavelengths in twilight. One previous study shows that the twilight spectral composition in forests or woodlands presents two dominant wavelengths, in which one ranges from 400 nm to around 570 nm with a λmax peak around 460 nm, and another one beginning around 630 nm and peaking around 670 nm, with a deficiency in middle wavelengths (570–630 nm)LWS and SWS2 in owls may likely help owls to maximize photon absorption by tuning their photoreceptor spectral sensitivities to the dominant wavelengths of light from 400 nm to around 570 nm in twilight to increase total sensitivity at relatively low light levels. This is consistent with the well-known crepuscular activities of many extant owlsLWS and SWS2 may imply an unappreciated significance of color vision during their crepuscular activities. We could not exclude the possibility of the adaptation of LWS and SWS2 in owls for night color vision, as reported in hawkmoths and geckosThe short-wavelength shift of SWS1 (violet-sensitive or ultraviolet (UV)-sensitive) transcripts were not found in any of the six owl species studied or the two species of Accipitriformes (the cinereous vulture (Aegypius monachus) and black-winged kite (Elanus caeruleus)), suggesting an inactive function of SWS1 in these species. The putative loss of SWS1 in owls is consistent with the results of previous studies indicating that UV vision is not found in the Tengmalm’s owl (Aegolius funereus) and UV-receptors are not found in the Tawny Owl 18SWS1 in owls may suggest their reduced need for violet or UV vision in dim-light conditions. Mammals that live in dim environments tend to lose their SWS1RH2 transcripts were not found in the grass owl (Tyto longimembris), consistent with the pseudogenization of RH2 in the barn owl RH2 belong to the family Tytonidae, suggesting even further reduction in daylight or color vision. Similarly, a more reduced visual pathway in Tytonidae has been observed when compared with another family (Strigidae) of owlsGUCA1C from the five owl species in Strigidae. GUCA1C is a cone-specific protein and is involved in photoresponse recovery through activating guanylyl cyclase. The loss of GUCA1C in Strigidae might be due to its pseudogenization as has been found in the mouse (Mus musculus)ABCA4 and PCDH15) that seem to play an important role in maintaining normal photoreceptor function in owls. ABCA4 is expressed in both rods and cones and is thought to prevent accumulation of the toxic substances N-retinylidene-PE generated during the phototransduction process, allowing the released all-trans-retinal to enter the visual cyclePCDH15 putatively plays a role in the development and maintenance of photoreceptor cells29We also found two PSGs , similar to owls, suggesting that falcons underwent a parallel adaptive evolution to dim-light conditions. While falcons are normally active in daytime, their hunting under relatively low-light conditions (e.g. crepuscularity) has been frequently reported35363712LWS and S223A in SWS2) found in owls that resulted in the short-wavelength shift of LWS and the long-wavelength shift of SWS2  were found to be under positive selection among 120 vision genes examined in Accipitriformes. One of these genes, CNGB3, is involved in the phototransduction pathway in cones, while the other five genes are all involved in maintaining normal vision function. Specifically, using the branch-site test model, a strong positive selection of CNGB3  along the ancestral branch of Accipitriformes was found. CNGB3 encodes the β subunit of CNG channels  was from Tytonidae and the other five species  covered all three known subfamilies  of StrigidaeFalco subbuteo and Falco tinnunculus) of Falconiformes and five species from five genera of Accipitriformes including Elanus caeruleus, Aegypius monachus, Accipiter virgatus, Circus melanoleucos and Butastur indicus were used. We also included one species (Upupa epops) from Upupiformes and one species (Picus canus) from Piciformes, both belonging to the superorder Coraciimorphae, the most closely related relative to owlsTyto alba), peregrine , saker falcon , white-tailed eagle , bald eagle , speckled mousebird (Colius striatus), cuckoo roller (Leptosomus discolor), bar-tailed trogon , rhinoceros hornbill (Buceros rhinoceros), carmine bee-eater (Merops nubicus) and downy woodpecker (Picoides pubescens).15 bird species were used for transcriptome sequencing in this study. The six owl species used in this study included both extant families (Tytonidae and Strigidae) of Strigiformes. One species . All experimental procedures carried out in this study were approved by the National Animal Research Authority of Northeast Normal University, China  and the Forestry Bureau of Jilin Province of China . The experimental procedures were performed in accordance with an animal ethics approval granted by the Northeast Normal University.Otus bakkamoena), in which its cDNA library was sequenced by Illumina HiSeq2000 .Total RNA extractions were performed using TRIzol Reagent (Life Technologies), following the manufacturer’s protocol and instruction. 50–100 mg of tissue from each individual was used. The quality and quantity of RNA was assessed using Thermo Scientific NanoDrop 2000  and Agilent 2100 Bioanalyzer . The retina cDNA sample was originally pooled with cochlear cDNA  with an equal quality from the same individual for cDNA library construction. cDNA library from each individual was separately prepared using NEBNext Ultra RNA Library Prep Kit for Illumina , according to manufacturer’s protocol. Specifically, Magnetic oligo (dT) beads (NEBNext Poly(A) mRNA Magnetic Isolation Module, NEB, Co) were used for enrichment of the poly (A) mRNA. The enriched mRNA was fragmented into small pieces, which were used for the synthesis of the first strand of cDNA by using superScript II reverse transcriptase and random hexamers, and subsequently the second strand of cDNA was synthesized by using buffer, dNTPs, RNaseH, and DNA polymerase I. The cDNA was purified using AMPure XP beads, and was subjected to end repair, adapter ligation and agarose gel electrophoresis filtration. Then, the suitable fragment size 250–350 bp was selected as templates for PCR amplification, which was used for cDNA library preparation. Paired-ending sequencing of cDNA library of all 15 species was performed using Illumina HiSeq2500  except for one species (de novo assembly. Reads from each library were assembled separately. De novo assembly was performed using Trinity softwareCircus melanoleucos (SRR3203217), Asio otus(SRR3203220), Tyto longimembris (SRR3203222), Upupa epops(SRR3203224), Bubo bubo(SRR3203225), Elanus caeruleus (SRR3203227), Otus scops(SRR3203230), Falco tinnunculus(SRR3203231), Butastur indicus (SRR3203233), Accipiter virgatus(SRR3203234), Aegypius monachus(SRR3203236), Falco subbuteo(SRR3203238), Picus canus(SRR3203240), Athene noctua(SRR3203242), Otus bakkamoena(SRR3203243).Raw reads were first preprocessed by removing adaptor sequences, and low-quality reads, this included reads with more than 10% unknown nucleotides and reads with the percentage of the bases with quality scores Q30 is lower than 15%, were removed. In this way, high-quality clean reads were obtained and used for Gallus gallus annotated vision genes were retrieved from Aimgo (http://amigo.geneontology.org/amigo) using “vision” as a search keyword. Using this approach, 124 vision genes were found. Subsequently, the protein sequences of the 124 genes of Gallus gallus were used as the query sequences to blast against each of our 15 species unigene pools using Tblastn 2.2.29 with an e-value cut-off of 1e−5. The unigene sequences obtained were then annotated by blasting against the NCBI nr/nt database using the online Blastn, and only the unigene sequences with the same gene annotation as that of the query sequences of Gallus gallus were used for subsequent analyses. In addition to the vision gene list from Aimgo, an additional 18 vision genes involved in the phototransduction cascade based on previous literatureFalco peregrinus, Falco cherrug and Gallus gallus) of our 15 species, and subsequently these sequences were used as the query sequences to blast against our 15 species unigene pools by using Blastn 2.2.29. Using the two approaches, we identified 142 vision genes in total. In addition to the 142 vision genes from our 15 species, we also downloaded the sequences of another 11 species mentioned above from NCBI using the published accession numbers.Vision gene sequences were aligned using ClustalW algorithm that is implemented in the DNAman software , and was manually inspected one by one for quality. Species with short sequences or low sequence identities  were removed. In addition, the genes shared by no more than two of our four focal bird taxa were excluded for subsequent analyses. After such pruning, a total of 120 vision genes with high-quality alignment results were retained and their coding sequences were used for subsequent positive selection analyses.Adaptive evolution of vision genes along the ancestral branches and clades of Strigiformes, Coraciimorphae, Falconiformes and Accipitriformes were analyzed using a maximum-likelihood method implemented in the Codeml program in PAML 4.8a package149500 < 1) includes codons that are conserved throughout the tree, and site class 1 (ω1 = 1) includes evolutionarily neutral codons. Site classes 2a and 2b include codons that are evolutionarily conserved (0 < ω0 < 1) or neutral (ω1 = 1) on the background branches, respectively, but are assumed to be under positive selection (ω2 > 1) on the foreground branches. When the LRT suggests positive selection, the Bayes empirical Bayes method was used to estimate the posterior probabilities that each codon belongs to the site class of positive selection on the foreground branches.In addition to the branch model, we also used the branch-site model to detect positively selected sites along the foreground branches that were pre-specified. For this, Test 2, which compares the modified model A with the null model, with ω = 1 fixed, was used. In the modified model A, four classes of sites are assumed and only the foreground branches are assumed to have experienced positive selection. Among the four classes of sites, site class 0  To guarantee that only the orthologous gene sequences were used, the annotations of PSGs sequences from all species studied were confirmed by blasting their cDNA sequences and amino acid sequences against NCBI nr/nt database using the online Blastn and Blastp with the default parameter settings. II) For the PSGs identified based on the clade model C, we further used a revised null model (M2a_rel) for LRT since M2a_rel is considered to better account for among-site variation in selective constraint and to reduce false positives compared with the standard null model M1aIII) We realigned the gene sequences of PSGs using webPRANK (http://www.ebi.ac.uk/goldman-srv/webprank/)IV) Recombination is considered to lead to a high rate of false positive in positive selection inferencehttp://www.datamonkey.org/) and reanalyzed their positive selection based on the gene partitions of those PSGs in which the recombination events were found. V) To avoid being trapped at a local optimum, we respectively changed initial parameter values of kappa and omega to see whether the results of the positive selection inference were stable. After examining of PSGs by the five steps, any PSGs that did not pass any one of the five tests were excluded for subsequent analyses, and in this way about half of original PSGs were filtered out and 22 PSGs retained.After positively selected genes (PSGs) were identified based on the branch model, branch-site model and clade model C mentioned above, these PSGs were further analyzed for robustness as several factors have been known to affect the result of positive selection analyses and lead to false positive results. LWS, SWS2, RH2 and RH1) were reconstructed at all ancestral nodes to analyze the spectral changes along different branches. Ancestral sequences reconstructions were carried out using the empirical Bayes approach implemented in PAML 4.8Ancestral amino acid sequences of four visual opsins . Three primer pairs were used to amplify the complete coding sequence of RH1 gene,  was used following manufacturer’s instruction. The different exons of RH1 gene were respectively amplified in a total volume of 25 μl using l μl of template DNA, 1.25 μl of each primer (10 μM), 12.5 μl Q5TM super fidelity 2× Master Mix and 9 μl ddH2O. PCR reactions consisted of an initial denaturation at 98 °C for 30 s, followed by 35 cycles of 98 °C for 10 s, 54–63.3 °C for 30 s .


Genome sequence analysis was performed on multiple culture time point samples from six monoclonal isolates, and single nucleotide polymorphism (SNP) variants emerging over time were detected. Out of a total of five positively selected SNPs, four represented nonsense mutations resulting in stop codons, three of these in a single ApiAP2 transcription factor gene, and one in SRPK1. To survey further for nonsense mutants associated with culture, genome sequences of eleven long-term laboratory-adapted parasite strains were examined, revealing four independently acquired nonsense mutations in two other ApiAP2 genes, and five in Epac. No mutants of these genes exist in a large database of parasite sequences from uncultured clinical samples. This implicates putative master regulator genes in which multiple independent stop codon mutations have convergently led to culture adaptation, affecting most laboratory lines of P. falciparum. Understanding the adaptive processes should guide development of experimental models, which could include targeted gene disruption to adapt fastidious malaria parasite species to culture.Cultured human pathogens may differ significantly from source populations. To investigate the genetic basis of laboratory adaptation in malaria parasites, clinical Plasmodium falciparum isolates were sampled from patients and cultured  All species adapt to their environment through the process of natural selection. In particular, many organisms grown in laboratory culture have adapted to an extent that they differ significantly from their original source populations. Given that many microbes occurring in nature are unable to be grown under culture conditions1in vitroPlasmodium falciparum more than forty years agoP. knowlesi that have recently been adapted6P. falciparum lines have been used for most studies to date, and it is unclear how well they represent parasites in natural infections, as many clinical isolates do not show robust continuous growth in vitroin vitroApiAP2 transcription factor gene were detected in particular cultures that had lost ability to differentiate into sexual stages, enhancing asexual parasite replicationFor over a century it has been possible to culture blood stages of different human malaria parasite species isolated from clinical infections for a few replication cycles in human erythrocytes Here, genome sequence analyses identify malaria parasite genes in which mutations are associated with culture adaptation. Sequencing of multiple time point samples was conducted on new clinical isolates that adapted successfully to culture, and novel single nucleotide polymorphism (SNP) alleles were shown to have emerged, most of which lead to premature stop codons. Subsequently, a scan for nonsense mutation alleles in genome sequences of long-term adapted laboratory strains was conducted, and compared with sequences of a large global sample of uncultured clinical isolates. Together, these results have identified several putative master regulator genes in which multiple independent nonsense mutations are specifically associated with culture adaptation.P. falciparum parasites from 46 different Gambian malaria patients were introduced into standard parasite culture conditions, out of which 11 (24%) of the isolates were successfully grown for at least 48 days. Of these, for analysis we selected six isolates that each contained single genotypes, as determined by PCR amplification and typing of the highly polymorphic msp1 and msp2 gene loci . As an indication of sample quality, out of 944,270 of the MalariaGEN globally ascertained SNPs, at least 99.65% were successfully genotyped in each time point sample . At Day 0, each isolate had a distinct sequence that was unrelated to any other, and equally unrelated to the 3D7 reference genome sequence  and increased in frequency so that the mutants were in a majority by the end of the culture period. This was a nonsense mutant leading to a stop codon S931X which would truncate the ApiAP2 protein by more than half its length (ApiAP2 transcription factor gene (PF3D7_1342900) that was noted in the line above, with a separate premature stop codon mutant K622X  and a non-synonymous substitution E396K in DOC2 , likely to be within the same genome as they emerged in parallel and represented a majority by the end of the culture period (ApiAP2 gene (PF3D7_1342900) emerged and increased in frequency until day 56  showed emergence of novel SNP alleles during culture, resulting from five independent mutations . The chas length . In the nt K622X . This mue period . In the l day 56 .ApiAP2 transcription factor gene . A Pfam search on the full gene sequence identified three predicted AP2 domains , all of which are downstream of the stop codons and will not be translated in the mutant parasites . Two of the ApiAP2 mutants stopped increasing in frequency later during culture , illustrating a need to examine parasites that have been culture adapted over longer periods to identify mutants that become fixed.Overall, four out of the five novel SNPs emerging during culture adaptation of these clinical isolates represented nonsense mutations. Of these, 3 occurred in different locations of the same arasites . AlthougP. falciparum lines derived from seven original clinical isolates were studied  in which nonsense mutant alleles were very rare overall, and non-randomly associated with subtelomeric regions   .ApiAP2 gene (PF3D7_1222400), three different nonsense mutation alleles were detected respectively in the long-term cultured lines T9-94, CS2 and the pair of related clones W2 and Dd2 (ApiAP2 gene (PF3D7_1222600) in clones of HB3 , a different mutation arising in each of three different cultured isolates. One isolate also had a nonsense mutation allele in a protein kinase gene (SRPK1). Following this, analysis of globally available data revealed three other genes in which nonsense mutation alleles are only seen in adapted, cloned, strains and not in clinical isolates. Two of these are other ApiAP2 genes, and one is the Epac gene encoding a guanine nucleotide exchange factor. Considering all results together, so it is remarkable that four particular genes (three members of the ApiAP2 family and Epac) show multiple independent loss-of-function mutations in different cultured lines, and none in sequences of uncultured parasites.This study has identified key genomic changes associated with E. coli adapting to high temperaturesLoss-of-function mutations have been shown to commonly occur during culture adaptation of bacteriaApiAP2 transcription factor genes, it is likely that disruption would interfere with the expression of many other genesApiAP2 genes  in two laboratory strains were previously shown to prevent gametocytogenesis10ApiAP2 gene (PF3D7_1222400) with three independent nonsense mutations in laboratory strains identified here, in its intact state is predicted to encode a transcription factor with a very high number of gene targetsApiAP2 in P. falciparum without a homologue detected in rodent malaria parasitesApiAP2 gene (PF3D7_1342900) with three different nonsense mutations identified in the new clinical isolates encodes a transcription factor preferentially interacting with G-boxes [(A/G)NGGGG(C/A)], which is a putative regulator of heat shock genes during the asexual blood stage cycle18The four genes identified here with convergent nonsense mutants selected in culture probably have major roles in parasite growth. For the three repeatedly mutated SRPK1 and with a nonsynonymous change in the DOC2 gene outcompeted wild-type parasites and also began to replace the ApiAP2 (PF3D7_1342900) mutant parasites. The gene product SRPK1 catalyzes phosphorylation of the parasite PfSR1 (Ser/Arg-rich) protein, involved in mRNA splicing in the nucleusSRPK1 gene transcript is expressed in P. falciparum asexual stages and at an even higher level in gametocytesP. falciparum by targeted disruption have not succeeded, suggesting that it was essential as appears the case for other related genesSRPK mutant here indicates either that disruption is adaptive in certain conditions of initial culture establishment, or that it is tolerated in the context of a particular nonsynonymous alteration in DOC2, a gene that has been shown to affect microneme exocytosis which is important for erythrocyte invasionIn one of the cultured clinical isolates, parasites with a novel premature stop codon in 2+, which mediate signalling pathways regulating microneme secretion in the merozoite. An increase in cAMP apparently activates protein kinase A and Epac, the latter then activating Rap1 which increases cytosolic Ca2+ level through the phospholipase C pathwayEpac gene have occurred in a large proportion of long-term adapted laboratory strains, and it is possible that its disrupted function is partly complemented by the role of protein kinase A in the culture environment. Epac may also have additional functions as a cyclic nucleotide binding proteinEpac and the ApiAP2 genes identified here . To address this experimentally, assays need to be developed to reliably phenotype gametocytogenesis responses to relevant induction treatments in culture, as processes previously used have been crude and experimentally variableFaster growth of asexual blood stage malaria parasites could be due to changes in various developmental parameters, including decreased cycle time, increased numbers of merozoites produced per cycle, or higher merozoite invasion rates into erythrocytesApiAP2 gene with a different stop codon mutant emerging in each of three Gambian clinical isolates was intact in all long-term culture adapted lines, although some of these had premature stop codon alleles in either of two other ApiAP2 genes. This suggests that not all alleles selected in the short term may go to fixation, and that other mutants might take over in a culture line over time, a pattern consistent with the trajectory of frequency changes seen in the clinical isolates here. A future priority is longer-term analysis of sequence evolution in culture, as this approach has given unique insights into adaptive processes in bacteria38Genomic analyses have revealed loss-of-function mutations in pathways that affect the replication of yeast under particular culture conditions323235P. falciparum cultures might have the potential to identify additional changesP. falciparum genome, only five  are identified to have nonsense SNP mutants causing premature stop codon alleles that have emerged in culture. With the exception of SRPK1, these genes all showed independent nonsense mutations in multiple strains. This suggests that the current study might have identified most of the genes that confer a very substantial growth advantage with a loss-of-function mutation. Although the long-term cultured laboratory strains were originally of diverse origins, almost all of them are now mutants having loss-of-function in Epac or ApiAP2 genes. Understanding the impact of this profound convergent evolution in culture is vital, as the derived strains may have some limits as models of malaria parasites. It will be important to understand the impact of these adaptations, as this may guide the development of better experimental models for malaria, including culture adaptation of other malaria parasite species. Despite efforts undertaken in different laboratories over many years, long-term cultures of P. vivax have not yet been established, and culture of either P. malariae or P. ovale has been rarely attempted as it is regarded as unlikely to succeed. The results presented here encourage functional studies of parasites early during ex vivo cultivation, and raise the possibility that targeted deletion or editing of Epac or ApiAP2 genes might provide a fast-track to culture adaptation of otherwise fastidious parasites.The present study focuses on single nucleotide variants that are robustly called, whereas development of methods to accurately detect novel indels and structural variants in Illumina sequences of The sample collection, laboratory culture and parasite genome sequencing for this study was reviewed and approved by the joint Ethics Committee of the Gambian Government and the MRC Gambia Unit. Written informed consent was obtained from all subjects or their attending guardian at the clinic before enrolment, with additional verbal assent of children being confirmed when guardians provided written consent. The methods were carried out in accordance with the relevant guidelines and regulations.P. falciparum parasitaemia of >5000 per microliter as estimated by thick blood film examination, with a thin blood film examination to confirm the parasite species as P. falciparum alone. A heparinized venous blood sample of up to 5 ml was collected from each patient and centrifuged to remove the plasma. Separation of the erythrocytes from leukocytes was performed using Nycoprep density gradient centrifugation, following which erythrocytes were washed and resuspended at 50% hematocrit in RPMI 1640 growth medium, supplemented with 25 mmol/L HEPES, 2 mmol/L l-glutamine, 25 mmol/L D-glucose, 25 mg gentamicin/L, 10 mg hypoxanthine/L and 10% albumax. Infected erythrocytes were either put directly into in vitro culture or cryopreserved for culture at a later date.Patients with malaria were sampled from four health centers within the Greater Banjul area in The Gambia , between September 2009 and November 2010. Each patient had axillary temperature of >37.5 °C or a history of fever in the previous 48 h, and 2, 3% CO2 and 96% N2. Samples were checked every day by thin blood film examination, and fresh medium and uninfected blood group O erythrocytes were added when necessary to maintain hematocrit at 2% and parasitemia below 10% of erythrocytes infected. Isolates were grown in culture for up to 92 days, with samples for parasite genome sequence analysis being taken at multiple time points during the culture period, depending on the parasitemia and volume of culture material available at various times. DNA was extracted from 200 μl packed erythrocytes using the QIAamp DNA Blood Mini Kit (Qiagen). To identify isolates that were apparently single genotype infections, the pre-culture (day 0) DNA sample from each isolate was screened by genotyping highly polymorphic repeat regions of msp1 and msp2 genesClinical isolates were cultured in complete RPMI 1640, using standard methodswww.malariagen.net/data/pf3k-pilot-data-release-4) following methods based on those previously publishedP. falciparum genomes, including the 35 cultured timepoints sampled here, being mapped to the 3D7 reference genome sequence version 3.1  with the BWA algorithm. Samtools mpileup called a total of 4,305,966 potential SNPs . Low quality SNPs were filtered out if any of the following condition was not met, for each position: base quality score threshold superior to 27, inside coding regions, represented by more than 10 reads in a single sample or more than 1% of all reads, total coverage is within the 5th and 99.5th percentiles of the coverage distribution, uniqueness score greater than 26 . A final global dataset of 944,270 high-quality SNPs was used , covering coding regions of the core genome, with highly polymorphic gene families var, rif and stevor being excluded from analysis.Library preparation, sequencing and quality control was completed following protocols as described previouslyP. falciparum samples was examined, using the Pf3K project release 3.1 . This contained sequences of 2483 P. falciparum clinical samples of diverse geographical origin, after removing 29 that were annotated as “culture adapted”, with the same pipeline for SNP calling used as above . Different types of SNP types in coding regions  were determined with the R package VariantAnnotationhttp://r-project.org).Novel SNPs arising during culture adaptation were identified at nucleotide positions where there was no minor allele detected at day 0, but where an alternative nucleotide appeared in an isolate during culture and subsequently increased in frequency to represent more than 20% of the mapped sequence reads. The quality of the mapped sequence reads for each of these cases were inspected visually using the LookSeq softwareHow to cite this article: Claessens, A. et al. Culture adaptation of malaria parasites selects for convergent loss-of-function mutants. Sci. Rep.7, 41303; doi: 10.1038/srep41303 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


DMD) NB transcript in normal skeletal muscle. To achieve depth of coverage required for the analysis of this lowly expressed gene in muscle, we designed a targeted RNA-Seq procedure that combines amplification of the full-length 11.3 kb DMD cDNA sequence and 454 sequencing technology. A high and uniform coverage of the cDNA sequence was obtained that allowed to draw up a reliable inventory of the physiological alternative splicing events in the muscular DMD transcript. In contrast to previous assumptions, we evidenced that most of the 79 DMD exons are constitutively spliced in skeletal muscle. Only a limited number of 12 alternative splicing events were identified, all present at a very low level. These include previously known exon skipping events but also newly described pseudoexon inclusions and alternative 3′ splice sites, of which one is the first functional NAGNAG splice site reported in the DMD gene. This study provides the first RNA-Seq-based reference of DMD splicing pattern in skeletal muscle and reports on an experimental procedure well suited to detect condition-specific differences in this low abundance transcript that may prove useful for diagnostic, research or RNA-based therapeutic applications.We have analysed the splicing pattern of the human Duchenne Muscular Dystrophy ( Alternative splicing (AS) is a key mechanism for generating tissue or developmental stage-specific proteomic diversity in eukaryotes1DMD gene encodes dystrophin, a cytoskeletal protein of 427 kDa accounting for only approximately 0.002% of the total striated muscle protein content but playing an essential role in muscle fiber integrity and function. Loss-of-function mutations cause Duchenne muscular dystrophy (DMD), the most common and severe form of progressive muscular dystrophy in childrenDMD transcript can be categorized among the low abundance transcripts relative to other muscle transcripts. In adult skeletal muscle tissues, its concentration is estimated to be 5–10 molecules per nucleus as compared to 25,000–50,000 copies per nucleus for the highly abundant muscle transcript encoding the myosin heavy chainDMD gene is remarkable by its length  and genomic structure. About 99% of the gene is made of introns, some of them being very long introns exceeding 200 kb, while the translated coding sequence, which is fragmented into 79 exons, is only 11.3 kb. Seven independent tissue-specific promoters encode three full-length isoforms (including the Dp427m muscle one) and four N-terminally truncated proteinsThe DMD transcript.There is no clear picture of the global splicing profile of the muscle transcript. Available data are not readily comparable because techniques of different sensitivity were used in previous studies that generally focused only on specific regions of the gene78912DMD-targeted RNA-Seq protocol and the 454 technology to allow accurate and comprehensive inventory of physiological ASEs of the DMD transcript in human skeletal muscle.Massively parallel RNA sequencing (RNA-Seq) has become a powerful technology to explore the complexity of mammalian transcriptomesDMD transcript was globally insufficient (50X–200X) despite the huge amount of reads produced, to allow reliable detection of ASEs present at low level. This observation prompted us to set up a DMD-targeted RNA-Seq approach (DMD coding sequence (11.3 kb) with the Roche 454 sequencing technology. Typically 454 reads are >300 bp long and are likely to span more than two exon junctions that helps to identify non-canonical splicing events. To assess the performance of our procedure, a first comparison has been made between the data obtained from the analysis of one skeletal muscle sample using our targeted approach  and the data from the IBM project . Visualization of the aligned reads to the DMD Dp427m isoform showed differences in DMD mRNA sequencing depth and coverage  reads per base that would theoretically allow ASEs as low as 1% to be reliably detected (>10 reads).Before designing our experimental workflow, a preliminary analysis of RNA sequencing data of human skeletal muscle tissue available in the Illumina’s Body Map (IBM) transcriptome projectapproach  that comcoverage . With thDMD canonical splice junctions as well as potential new ones from junction-spanning reads. Compared to TopHat2, a popular mapper suitable for processing relatively short reads, STAR performed better both in the total mRNA-Seq and our DMD-targeted mRNA-Seq datasets (DMD transcriptThe Spliced Transcripts Alignment to a Reference software (STAR), specially designed for aligning long readsdatasets  for the DMD transcript. We applied our established DMD-targeted RNA-Seq protocol, and the splice junction reads obtained both for the 78 canonical junctions and novel splice junctions in the 4 samples are detailed in the Four independent skeletal muscle tissue samples from normal young men were used to establish the splicing pattern of the muscular In addition to exon skipping events, the RNA-Seq analysis has revealed the usage of alternative splice sites located either at proximity of the natural ones or deep in the introns . The relDMD transcripts. Regarding the detected 3′ alternative ss, all are exonic and their calculated scores were lower than the scores of the adjacent natural splices in all cases except for the exon 76 + 59 alternative 3′ss, which exhibits scores (HSF: 89.39 and MES: 9.59) significantly higher than the natural 3′ss of exon 76 (HSF: 69.15 and MES: 3.81) (DMD exon 54 that deletes one amino acid (p.Glu2625). This novel junction was reliably detected in all four analysed skeletal muscle samples (11–28 junction reads).All three pseudoexons displayed high 5′ and 3′ splice site scores, thus supporting their use to some extent in the S: 3.81) . When usi.e. a minimum of 5 reads in 2 out of the 4 samples analysed)  . InteresDMD gene was a priori a good candidate for the occurrence of multiple alternative splicing events. A literature survey supports this common assumption with many reports describing alternative splicing events in the DMD gene since its discovery in 1986 , and gene regions explored (most studies focused on specific regions), that makes qualitative and quantitative comparisons between these different datasets difficult. A comprehensive and unbiased inventory of the DMD splicing events in skeletal muscle was lacking.Over half of the multi-exonic human genes are believed to have splice variants in 1986 . HoweverDMD gene. The deep RNA sequencing strategy was reasonably expected to expand the repertoire of ASEs in the DMD gene. Yet surprisingly, the vast majority of the 79 DMD exons were found to be constitutively spliced in skeletal muscle under physiological conditions. Hence, our results would indicate that the frequency of ASEs previously reported in the DMD muscular transcript may have been overestimated, possibly due to methodological considerations.The recently developed deep-sequencing technologies allows a far more precise quantification of transcript levels and their isoforms than other methodsDMD gene, skipping of only a single or a limited number of exons were characterized  that share some characteristics with the recently described microexons in neurons, a set of highly conserved short exons, which are strongly regulated by RNA-binding proteins (RBPs) and functionally modulate tissue-dependent protein-protein networks33Exon-skipping was the most common form of the ASEs detected . Notwithstanding the large number of exons in the cterized . SkippinDMD transcripts. The inclusion of this out-of-frame extra-exon may represent a post-transcriptional control in cells that normally do not express the dystrophin protein (like lymphocytes), while a low inclusion rate is observed in skeletal muscle transcripts (3%). Due to the size of the DMD introns, multistep splicing events (recursive or nested splicing) are frequently used to splice out a single intronThe RNA-Seq analysis has revealed the usage of alternative splice sites located either in the close vicinity of the natural sites or deep in the introns , which aDMD after exon skipping. All are exonic alternative 3′ splice sites that induce partial exon deletion when used, and the major one involves a NAGNAG tandem site in DMD exon 54. NAGNAG motif occurs in 30% of human genes and appears to be functional in at least 5% of human genes37DMD gene. Splicing at this site deletes the polar amino acid glutamine at position 2625 of dystrophin repeat 21 which forms part of the region involved in specific protein/lipid interactions that favours homogeneous dystrophin distribution along the membraneDMD gene.As previously reported in other human genesDMD splicing pattern in normal muscle. The strategy used allows the analysis of the whole 11.3 kb-cDNA sequence all at once that may prove useful for mutational analysis or monitoring the impact of splicing interventions on transcript structure in patients with Duchenne muscular dystrophy. Undesirable alternative splicing events may impact the outcome of exon skipping or trans-splicing approaches. Unlike other isoforms2627DMD gene is tightly regulated in skeletal muscle to preserve the production of a full-length transcript and a 427 kDa dystrophin protein, even if some protein domains are considered to be functionally dispensableDMD gene represents a paradigm for extreme splicing conditions that illustrates perfectly how accurate the process of splice site selection shoud be to include the 79 exons into the mature transcript concomitantly with the repression of near perfect pseudoexons or cryptic 3′ss activation. A large piece of work remains to be done to elucidate the splicing regulation in this huge gene, which will contribute to reveal new insights into basic splicing mechanisms.In conclusion, this study provides the first RNA-Seq-based reference of Two striated muscle tissue samples (n°20316 and n°27563) from healthy male individuals  were obtained from the Myobank-AFM . Two additional control skeletal muscle total RNA samples from 20 year-old males were purchased from Clontech .® II (Thermo Scientific) and oligodT primers for RNA-Seq experiments.Total RNA was extracted from biopsies using the SV total RNA extraction kit (Promega) and RNA integrity was checked using the Agilent RNA 6000 Nano kit with Agilent 2100 bioanalyzer (Agilent Technologies). 700 ng of total RNA were used to synthesize template cDNA by reverse transcription with the SuperscriptDMD cDNA as a single long fragment (11.3 kb) by using the GoTaq® Long PCR Master Mix (Promega) and primers located in exon 1 of the muscle isoform (Dp427m) (5′-CTTTCCCCCTACAGGACTCAG-3′) and in the 3′UTR (5′-CCAAATCATCTGCCATGTGG-3′). Cycle parameters were programmed as 94 °C for 2 min, followed by the first 10 cycles of 93 °C for 15 s, 58 °C for 30 s, and 68 °C for 11 min and a 20 s auto-extension of elongation time for cycles 11–35. After verification of amplicon size by agarose gel electrophoresis, LR-PCR reactions were purified using Qiaquick PCR purification kit (Qiagen) and quantified using the NanoDrop 2000 spectrophotometer (Thermo Scientific).One fifth of the RT product was used as template to amplify the full-length ® Fluorometer and pooled in equimolar amounts into a single sample prior to emulsion PCR amplification and sequencing in parallel on the Roche GS Junior 454 sequencer giving a mean total number of 69,060 reads (387 bp mean length) per library. The data obtained were then sorted according to their tag sequences.For each library, around 1 μg of purified PCR product were fragmented by nebulization (2.1 bars of nitrogen for 1 min) and subjected to library preparation with the Rapid Library Preparation Kit (Roche) according to manufacturer’s instructions. The different samples were labeled via ligation of Multiplex Identifiers (MID) oligonucleotide adaptors to allow multiplexing. Three MID-containing libraries were quantified using the Qubithttp://galaxyproject.org)DMD mRNA coverage, as well as a list of identified splice junctions and their counts. Only new junctions covered by a minimum of 5 reads in at least two out of the four biological replicates were considered for further analysis. The Percent-Spliced-In (PSI) was calculated for each splicing event using intron-centric metricshttps://github.com/pervouchine/ipsa). For the comparison of the DMD-targeted versus publicly available total mRNA sequencing data from the Illumina Human Body Map project 2.0 , raw reads were mapped to the Human X chromosome sequence with Bowtie2 (v2.default parameters)http://www.umd.be/HSF3/), which uses the weight matrix modelhttp://genes.mit.edu/burgelab/maxent/Xmaxentscan_scoreseq_acc.html) based on the maximum entropy principleRaw reads were edited and filtered prior to analysis. A dedicated analysis pipeline was developed using the Galaxy framework  and primers hybridizing to upstream and downstream exons (sequences available upon request). For conventional RT-PCR, the 30 cycles-amplified products were separated on 1.8% agarose gels and spliced products were quantified with the Quantity One (v.4.6.9) software (Bio-Rad). For semi-quantitative PCR, fluorescein-labeled forward primers were used and 26 cycles were done. Capillary electrophoresis analysis was performed using 1 μl of PCR product added to 18 μl of formamide and 0.5 μl of ROX 400 HD fluorescent size standards (Applied Biosystems). Amplified products were separated on an ABI 3130 XL DNA analyzer and the peaks areas were measured by the GeneMapper v4.0 software. Ratios of splicing isoforms were determined as the peak area of one specific isoform divided by the total peak areas for the two or three detected isoforms. Data represent the mean +/−SD of at least three independent assays.How to cite this article: Bougé, A.-L. et al. Targeted RNA-Seq profiling of splicing pattern in the DMD gene: exons are mostly constitutively spliced in human skeletal muscle. Sci. Rep.7, 39094; doi: 10.1038/srep39094 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Intercommunication of Dopamine Receptors (DRs) with their associate protein partners is crucial to maintain regular brain function in human. Majority of the brain disorders arise due to malfunctioning of such communication process. Hence, contributions of genetic factors, as well as phenotypic indications for various neurological and psychiatric disorders are often attributed as sharing in nature. In our earlier research article entitled “Human Dopamine Receptors Interaction Network (DRIN): a systems biology perspective on topology, stability and functionality of the network”   Specifications TableValue of the data•The data hints an overlapping disease spectrum of neurological disorders in DRIN.•The data pinpoint towards common contributing genetic factors of neurological disorders in DRIN.•The data might encourage researchers to identify additional drug targets in DRIN for future drug discovery endeavors.1The data highlighted 25848 associations between 431 proteins and 4312 diseases, disorders and clinical or abnormal human phenotypes in Dopamine Receptors Interactions Network (DRIN) 22.1Genes were mapped to respective disease data using DisGeNet database P-values were further corrected by the bonferroni correction (P≤0.05) to determine a list of significant disease association in our data.N=Total number of genes in DisGeNet; n=Total number of genes in DRIN; m=Each disease associated genes in DisGeNet; x=Each disease associated genes in DRINTo identify significant enrichment of diseases in our data two-sided hypergeometric test 2.2Na=number of elements in set A; Nb=number of elements in set B; Nc=number of elements in intersecting setOverlapping of genes between two diseases was measured using Jaccard or Tanimoto coefficient metrics Based on the similarity matrix, the disease sets were further classified into different clusters using Neighbor Joining (NJ) algorithm which is a bottom-up or agglomerative clustering method to identify similar group of elements in the data 2.3The pathways information and transcription factor (TF) regulatory data were collected from the Molecular Signatures Database (MSigDB) 2.4http://www.drugbank.ca/) database A comprehensive search was made in DrugBank v4.0 (


Arabidopsis thaliana) can be used to study the canalization during vascular cambium regeneration and new vasculature formation. We monitored localized auxin responses, directional auxin-transport channels formation, and establishment of new vascular cambium polarity during regenerative processes after stem wounding. The increased auxin response above and around the wound preceded the formation of PIN1 auxin transporter-marked channels from the primarily homogenous tissue and the transient, gradual changes in PIN1 localization preceded the polarity of newly formed vascular tissue. Thus, Arabidopsis is a useful model for studies of coordinated tissue polarization and vasculature formation after wounding allowing for genetic and mechanistic dissection of the canalization hypothesis.Synchronized tissue polarization during regeneration or de novo vascular tissue formation is a plant-specific example of intercellular communication and coordinated development. According to the canalization hypothesis, the plant hormone auxin serves as polarizing signal that mediates directional channel formation underlying the spatio-temporal vasculature patterning. A necessary part of canalization is a positive feedback between auxin signaling and polarity of the intercellular auxin flow. The cellular and molecular mechanisms of this process are still poorly understood, not the least, because of a lack of a suitable model system. We show that the main genetic model plant, Arabidopsis ( Development and patterning of vascular tissue require signaling and directional flow of the plant hormone auxin125MONOPTEROS (MP) and the auxin transport protein PIN1The auxin flow direction has been shown to depend on the asymmetric position of the PIN-FORMED (PIN) auxin transporters at the plasma membranes of transporting cells7891011121317123192Vascular tissue disruption in experimental systems leads to regeneration processes. In nonwoody dicotyledonous plants, vasculature is regenerated in the wound neighborhood of primary tissues2356242527282932343637Arabidopsis thaliana), making it impossible to use large collections of genetic material available for this model. Nonetheless, artificial weights applied to the apical parts of immature inflorescence stems of Arabidopsis (stems with primary tissue architecture) increase the basipetal auxin transport, stimulate stem growth, and promote secondary growth in basal parts of these stems41424445464740Thus far, studies on vascular cambium regeneration and accompanying changes in auxin distribution, flow directionality, and cellular polarity of PIN transporters have been hampered by the inability to induce and follow these processes in Arabidopsis  were also analyzed . Such plHistological analysis of both nonincised controls and wounded stems revealed significant differences in vessels organization . A serieIn wounded stems of Arabidopsis, the regenerated vessels were much shorter  than thoAn interesting regeneration manner was observed in deeply wounded stems, few days after wound (DAW), in which the so-called circular vessels developed . In the The histological analysis revealed different ways of the vascular cambium regeneration and the gradual vasculature reconstruction in wounded Arabidopsis stems .AtHB8 gene is known as a vessel formation marker50AtHB8::GUS transgenic line in Wassilevskija (Ws) backgroundAtHB8 gene expression was found in the wounded stem regions  vessels in unwounded controls was also analyzed and compared . The lenIn the circular vessels, the PIN1 localization rearranged in an unprecedented way . At earlPhaseolus vulgaris) or pea (Pisum sativum)235De novo formation and regeneration of vascular tissues are instances of intercellular communication and coordinated tissue repolarization during developmental processes. More than 30 years ago, the canalization hypothesis had been proposed postulating the crucial role of the plant hormone auxin in these processes5The major herbaceous model plant Arabidopsis is a well established model system for many molecular and genetic studies, including vascular development and secondary xylem formation31535442444546472240via promotion auxin channel formation. The canalization hypothesis-predicted behavior is observed during de novo formation and regeneration of vasculature, especially of new regenerated vessels after incision, i.e. wounding or grafting2356355Auxin is regarded as a primary signal that induces vascularization PIN1 was expressed in cells around the wound whereafter auxin channels emerged and was localized less polarly at the plasma membranes. PIN1 expression became increasingly stronger and more focus, when more defined auxin channels began to emerge. In a few days, the subcellular PIN1 position was gradually stabilized and restricted only to cell sides along the presumable direction of the auxin flow. Probably, this polarized PIN1 position marked the prospective perforation plates in fully regenerated vessels.Originally, the feedback between auxin and its flow had been proposed to be realized by an auxin effect on the polar, subcellular localization of the PIN auxin transport proteins that, in turn, determine the auxin flow directionality2132PIN expression and PIN polarity217abp1 mutants has been shown to be due to mutations in the neighboring gene, necessitating additional analyses of the loss-of-function abp1 phenotypesThe impact of auxin on the polar PIN1 positioning might be part of a mechanism for auxin-regulated canalization58In the course of our research on regeneration and canalization, we observed the interesting phenomenon of the so-called circular vessels that form isolated islands of differentiated vessel-like cells, indicative of small vortexes of circular auxin flow. Development of the circular vessels has been described previously3637386365DR5, the auxin transporter PIN1, and the vessel identity marker AtHB8.Here we show that Arabidopsis comes out as a suitable model system for the analysis of vascular cambium regeneration that typically occurs in trees and provides the possibility to study this spectacular example of coordinated tissue differentiation and repolarization . This prArabidopsis thaliana (L.) Heynh. ecotype Columbia 0 (Col-0) and transgenic lines DR5::GUSDR5::GFPpPIN1::PIN1:GFPAtHB8::GUS in Wassilevskija (Ws) backgroundhttp://www.arabidopsis.info/BasicForm). Individual plants were grown in pots with a mixture of soil and vermiculite  in growth chambers and under stable conditions as describedWild-type I STEP was done to obtain a closed ring of active vascular cambium and secondary tissue architecture in immature inflorescence stems  were analyzed as well and used as additional controls for stems with weight-induced secondary growth. In mature stems, secondary growth developed in the basal parts. These observations and statistical analyses were done on a total of 46 mature plants .For detailed histological examination, 3-mm basal segments of the wounded stems were processed by fixation, embedding, and sectioning as describedThe histochemical analysis was done as described previously52l-lysine (Menzel-Glaser), and processed for immunolocalization. Briefly, slides were washed with phosphate buffered saline (PBS) (pH. 7.4), 2 × 15 min at RT, incubated first with 2% (w/v) blocking solution  for 30 min at RT and then with a primary antibody  in the humid chamber at 4 °C overnight. Then, the slides were washed with PBS, 5 × 10 min at RT, incubated with the fluorescence-labeled secondary antibody  for 4 h at RT, again washed in PBS, 5 × for 10 min at RT. Finally, they were mounted with antifade FluoroMount Aqueous Mounting medium (Sigma-Aldrich) and analyzed with an epi-fluorescent or confocal laser-scanning microscope.Immunolocalization was done as described previouslyin vivo. The DR5::GFP and pPIN1::PIN1:GFP transgenic lines were used. Sections from non-fixed samples of incised stems were handly cut with a sharp razor blade, mounted in 25% (w/v) glycerol solution, and analyzed with a confocal laser-scanning microscope.Changes in expression of genes regulating auxin response and tissue polarity were analyzed t-test . Transition from primary to secondary tissue architecture in weight-applied immature stems was analyzed on a total of 432 plants. Differences in vessel lengths were compared: 100 individual cells from both vessel types  were selected and measured along the tangential dimensions. Additionally, regenerated vessels of 383 wounded stems were analyzed. The number of vessel cells in circular vessels was evaluated. In total, 156 plants with circular vessels were found in wounded stems. Statistical analyses were done with Office Excel 2003 (Microsoft).Statistical significances were evaluated with the Student’s DR5::GFP and pPIN1::PIN1:GFP) was excited with a multiband argon laser at wavelength of 561 nm and 488 nm, respectively. The acquired images were processed with ZEN 2008 and ZEN 2012 Light Edition softwares.Samples of wounded stems were analyzed with a stereomicroscope (NIKON MSZ1500) equipped with the charge-coupled device camera (DS-Fi1). Semi-thin sections were examined with a transmitted light microscope OLYMPUS BX41 equipped with fluorescence filters. PIN1 localization visualized by fluorescence-labeled anti-rabbit Cy3, and auxin response in wounded areas was analyzed using a confocal laser-scanning microscope ZEISS LSM5 Exciter or Zeiss Observer.Z1. Fluorescence of Cy3 and GFP .


Early diagnosis of cerebral venous and sinus thrombosis (CVT) is currently a major clinical challenge. We proposed a selective MR black-blood thrombus imaging technique(MRBTI).MRBTI was performed on 23 patients with proven CVT and 24 patients with negative CVT by conventional imaging techniques. Signal-to-noise ratio (SNR) was calculated for the detected thrombus and contrast-to-noise ratio (CNR) was measured between thrombus and lumen, and also between thrombus and brain tissue. The feasibility of using MRBTI for thrombus volume measurement was also explored., MRBTI correctly identified 113 out of 116 segments with proven CVT with a sensitivity of 97.4%. In 527 out 531 segments, CVT was ruled out correctly with a specificity of 99.3%. Quantification of thrombus volume was successfully conducted in all patients with CVT, and mean volume of thrombus was 10.5 ± 6.9 cc.With effectively suppressed blood signalThe current findings support that MRBTI allows direct selective visualization of thrombus as opposed to indirect detection of venous flow perturbation and can be used as a promising first line diagnostic imaging tool.