Circulating immature platelet fraction (IPF) reflects real-time thrombopoiesis and correlates with platelet recovery from thrombocytopenic presentations. To understand the dynamics of IPF in platelet transfusions, we quantified the %-IPF in single-donor platelet components (SDP) during prolonged storage. %-IPF significantly increased from baseline by day 5 post-donation. Absolute IPF counts (A-IPC) had similar significant increments. However, gamma-irradiation suppressed the increments of %-IPF and A-IPC by >50%. Ultrastructural analysis of SDP units at day 10 showed well preserved morphology of immature platelets. Our findings suggest that IPF might actively expand ex-vivo and may have a longer shelf life than their mature counterparts. Closer study of IPF may be of critical clinical importance for transfusion practices. Circulating immature platelets are newly released from the bone marrow, contain higher concentration of RNA than mature platelets, and are measured by automated hematology analyzers equipped with a reticulocyte detection channel as the percent immature platelet fraction (%-IPF) The clinical significance of %-IPF and A-IPC in platelet transfusion has not been elucidated. A recent report showed that transfusion of hematopoietic-progenitor-cell transplanted children with IPF-rich platelet units significantly reduced the overall number of platelet transfusions required by patients compared to those receiving IPF-poor units SDP specimens were obtained from our local blood suppliers: American Red Cross  and LifeShare Community Blood Services . All units were from healthy volunteers, leukoreduced, collected by apheresis, stored in PVC bags  with ACD-A (10–15%) as anticoagulation TM controls  were measured after the analysis of every 50 samples. XbarM control program was also applied to re-assure the equipment's quality control as recommended by the manufacturer . A-IPC and H-IPF counts (H-IPC) were obtained by multiplying the %-IPF or %-H-IPF times the optical platelet count respectively.All aliquots were tested for optical platelet (fluorescent) count, mean platelet volume (MPV), platelet-large cell ratio (P-LCR), %-IPF and %-High (H)-IPF within 1 hour of sampling using the Sysmex hematology analyzer XE-5000 according to manufacturer's protocols . Sysmex e-Check (XE)Three 1 mL aliquots from day 3 and three from day 10 were fixed by immersion in quarter-strength Karnovsky fixative solution for 2 hours at room temperature. After washing, specimens were post-fixed for 2 hours in an unbuffered 1:1 mixture of 2% osmium tetroxide and 3% potassium ferrocyanide. After rinsing with distilled water, specimens were soaked overnight in an acidified solution of 0.25% uranyl acetate. Specimens were then rinsed in distilled water, dehydrated in ascending concentrations of ethanol, passed through propylene oxide, and embedded in a Poly/Bed 812 embedding media . Thin sections were cut on a RMC MT6000-XL ultramicrotome . These were mounted on Gilder square 300 mesh nickel grids  and sequentially stained with acidified methanolic uranyl acetate and stable lead staining solution. Sections were subsequently coated on a Denton DV-401 carbon coater , and examined in a JEOL 1200EX electron microscope .t test. All p values are two-sided with a type I error rate of 5% and a p<0.05 set for significance.All statistics were performed using Prism 6 . Results are presented as mean ± SD unless otherwise specified. Intergroup data comparisons were performed using the Student 9/L) was 20.0±4.0. As shown in 9/L) were stable and did not significantly decrease until day 7 . On the contrary, %-IPF and A-IPC (×109/L) significantly increased by day 5 compared to day 0  and %-IPF further increased on day 7 while A-IPC seemed to reach a plateau .%-IPF on day 0 was 1.58±0.3% similar to the reference range we established for the Sysmex XE-5000  which correlates with ranges reported for other analyzers To test if the %-IPF/A-IPC increment in storage was due to random variation among units or A-IPC expansion ex-vivo, we followed %-IPF and A-IPC changes of SDP from five donors for 14 days. As shown in 9/L) in the five SDPs also increased by day 10 . Analysis of additional parameters obtained with each run showed significant increments of MPV and P-LCR . On day 5 irradiation did not affect platelet counts; however, %-IPF, A-IPC, %-H-IPF, and H-IPC were significantly reduced by 50% in the irradiated group .To understand IPF morphology in SDP during storage, we performed SDP ultrastructural analysis focusing on the internal structure and granule integrity of immature platelets using transmission electron microscopy. As shown in Together %-IPF and %-H-IPF increments in the same units followed for two-weeks suggest ex-vivo expansion as a plausible explanation. A recent report indicated that the increment of %-IPF in storage represents non-specific artifacts of platelets degranulation Analysis of other parameters using our analyzer showed increments of %-H-IPF and H-IPC which implies that immature platelets are capable of expansion ex-vivo. Alternatively, a less mature platelet fraction than H-IPF might exist in peripheral blood that replenishes H-IPF but cannot be detected by the analyzer used. However, increases in both PLCR and MPV at day 10 along with data obtained through TEM showing morphologically intact immature platelets in day 10 samples lend support to the ex-vivo expansion or the greater stability of this platelet fraction in prolonged storage. It will be of interest to determine if the immature platelets observed on day 10 samples are still functional and capable of giving origin to mature platelets. Along these lines, the optimal growth/preservation conditions required by immature platelets in storage need to be elucidated.Ionizing irradiation is known to damage DNA and RNA and inhibit protein synthesis. However, ionizing irradiation of apheresis platelets has not been shown to have a clinically significant or demonstrably adverse effects on platelet function L degradation that antagonizes the pro-apoptotic effect of Bak How immature platelets give origin to mature ones is not understood but it has been shown that ex-vivo generated murine megakaryocytes infused into mice generate up to 200 platelets/cell in as little as 5 minutes Platelet transfusions prevent bleeding in patients with thrombocytopenia, but the clinical significance of %-IPF requires scrutiny. Animal studies have shown that newly formed murine immature platelets have increased hemostatic activity compared to mature ones as demonstrated by their increased response to thrombin and higher expression of surface P-selectin


In this study, a novel method of graphite chemical exfoliation to create graphene oxide (GO) is reported. Here, new oxidants were examined: a mixture of perchloric and nitric acids and potassium chromate. Furthermore, an effect of oxidation time, temperature of oxidation, and ultrasonication on graphite exfoliation degree was investigated. The obtained GOs were next reduced with glucose, used as a reducing agent. Detailed analysis of the materials indicated that when graphite was oxidized for 24 h at 50 °C, 5-layered graphene was prepared. An effect of sonication process was also examined, and it was found to enhance the exfoliation to bilayer graphene. Furthermore, when time and temperature were increased to 48 h and 100 °C, respectively, graphite was exfoliated to single-layer graphene. Therefore, it is believed that the proposed route can be applied for the preparation of graphene or few-layered graphene with defined number of layers upon the process parameters optimization and in a bulk scale. The materials were characterized with atomic force microscopy, Fourier-transform infrared spectroscopy, Raman spectroscopy, and X-ray diffraction. Perchloric acid, nitric acid, hydrochloric acid, and ethanol were obtained from Chempur. K2CrO4 (6 g) was added. The mixture was then heated to 50 °C and reaction was operated for 24 h. The obtained mixture was next filtrated through polycarbonate (PC) membrane (Whatman 0.2 μm) and washed three times with ethanol (200 mL) and 10 % hydrochloric acid (200 mL) to remove residual metal ions, and finally with distilled water until pH of the solution was 7. Finally, the material was dried in air at 100 °C for 24 h.Here, three types of graphene oxides (GOs) were prepared. In the first procedure of GO1 synthesis, 1 g of graphite was dispersed in a mixture of perchloric and nitric acids , and next, K2CrO4, an ultrasonication process was performed for 6 h at room temperature. After oxidation process, the purification, filtration, and drying steps were realized as in the first procedure, and finally GO2 was obtained.In the second method, prior heating a mixture of graphite, perchloric acid, nitric acid, and KIn the third route, the time and the temperature of oxidation process were increased to 48 h and 100 °C, respectively. The sonication, purification, filtration, and drying steps were carried out as in the second procedure to obtain GO3. After the purification process of the each graphene oxide, the content of impurities was determined with thermogravimetric analysis. It was estimated that GO1, GO2, and GO3 contain 0.11, 0.19, and 0.08 wt% of contaminants, respectively (data are not presented here).Reduced graphene oxide (RGO) was synthesized with glucose using as a reducing agent  . The measurements were done in air under ambient conditions. Raman measurements were performed on an In-Via Raman microscope (Renishaw) with excitation laser wavelengths of 785 nm. Raman spectra were obtained from individual flakes deposited on SiO−1. The peaks between 1,190 and 1,380 cm−1 are related with C–OH stretching vibration  of GO1, GO2, and GO3 is 3.7, 3.6, and 3.7 Å, respectively. It is enhanced compared to that of graphite (3.4 Å)  at the Brillouin zone center  determines relative defect content in the carbon lattice  preparation, glucose was used as a reducing agent. The obtained materials  were analyzed with Raman spectroscopy to characterize their structure, in terms of number of graphene layers and lattice defects. Figure 2/Si with approximately 1 nm rms roughness, would be 0.8–1.2 nm. The enhanced thickness of monolayer graphene may be attributed to the interaction between the sample and the tip (Gupta et al. To confirm the morphology of RGO1, RGO2, and RGO3, atomic force microscopy was applied. Figure In summary, novel method of chemical exfoliation of graphite was presented. Here, novel oxidants were examined: a mixture of perchloric and nitric acids and potassium chromate, and an effect of oxidation time, temperature of oxidation, and ultrasonication in exfoliation degree were investigated. The presented methodology leads to creation of graphene with controlled number of layers: single-, bi-, and 5-layered graphene in a bulk scale.


In this work micro temperature and humidity sensors are fabricated to measure the junction temperature and humidity of light emitting diodes (LED). The junction temperature is frequently measured using thermal resistance measurement technology. The weakness of this method is that the timing of data capture is not regulated by any standard. This investigation develops a device that can stably and continually measure temperature and humidity. The device is light-weight and can monitor junction temperature and humidity in real time. Using micro-electro-mechanical systems (MEMS), this study minimizes the size of the micro temperature and humidity sensors, which are constructed on a stainless steel foil substrate (40 μm-thick SS-304). The micro temperature and humidity sensors can be fixed between the LED chip and frame. The sensitivities of the micro temperature and humidity sensors are 0.06 ± 0.005 (Ω/°C) and 0.033 pF/%RH, respectively. They are favored because of their high reliability, low power consumption, and little-to-no-maintenance requirements. They have been adopted for a long time in various devices and computers as visual indicators of signal integrity and operational status. An LED is a solid-state semiconductor device that directly converts electrical energy into light. It is a strong candidate for the next generation of general illumination applications. Following further improvements, LEDs have recently been identified as illumination devices. High-power, high-brightness light emitting diodes are being used in an increasing number of lighting applications because of their excellent color saturation and long lifetimes. Preventing them from overheating is challenging task for thermal designers. The thermal management of packages depends on external cooling mechanisms, heat dissipation, and thermal interfaces. Under a fixed cooling condition, the rate of increase of the junction temperature of an LED increases with thermal resistance, reducing the luminescent efficiency declines. Therefore, the effective thermal design of LED packages with low thermal resistance is essential to improving the performance of LEDs  estimateAs well as temperature, humidity factor significantly influences LED packaging. Moisture causes delaminating in electronic packages. LED packages are molded using polymeric materials. Such moisture diffuses into the package, which therefore swells. The package swells after absorbing moisture. Furthermore, the mismatching coefficients of moisture expansion (CMEs) induce hydro-mechanical stress in the package . Tan 6]6] invest2.2.1.In this study, the micro temperature sensor was a resistance temperature detector (RTD). Among the various advantages of an array of thin film RTD sensors include a small volume, high accuracy, short response time, and mass production capacity. As Pt and Au are the conventionally adopted sensing materials in temperature sensors, the former is expensive while the latter has a higher conductivity and flexibility. Therefore, in this work, Au is used in the device . As the tR represents the resistance at t °C; iR is the resistance at i °C, and αT is the sensitivity (1/°C).When the temperature of the RTD varies linearly, the relationship between the measured resistance and the change in temperature can be expressed as:2.2.C is capacitance (F); ε0 is the dielectric constant of a vacuum; ε is the dielectric constant of the environment; A is cross-sectional area of the electrode (m2), and d is distance between the two electrodes (m).The three main classes of humidity sensors are ceramic, electrolyte-based and polymer-based. Polymer-based sensors are either of the capacitance type or of the resistance type. Although the measurement range of the polymer-based sensor is not as large as that of the ceramic-based sensor, it is simply fabricated, low-cost, and highly accurate because of the high degree of polymer polymerization. It is useful for developing rapid IC processes. The polymer must have high resistance and a low dielectric constant. As the amount of vapor that is absorbed by the polymer increases, the dielectric constant increases and the increase in the capacitance can be derived using Hα is the sensitivity of the humidity sensor (C/%RH) [ (C/%RH) .3.The frame of the LED is a very important conductor of heat in an LED chip, so an insulating medium is installed between the frame of the LED and the LED chip to increase thermal resistance. The authors’ earlier study involved the development of a flexible micro temperature sensor that could be installed between an LED chip and the frame. The design in the present work is based on that earlier work . In this4.2 and the sensing area of the micro humidity sensor was 18,900 μm2. Flexible micro temperature and humidity sensors were fabricated using MEMS. Such micro sensors have the advantages of (1) small size, (2) mass-producability, (3) multi-functionality, and (4) flexible but precise measurement positions.In The junction temperature of the LED can be determined from the voltage based on the thermal resistance measurement theory. A CREE® EZ1000 chip and input currents ranging from 0 to 350 mA were used. Data obtained by thermal resistance measurement differed from those obtained using a micro temperature sensor. The data experimented is shown in In the humidity portion, the capacitance that was measured by the humidity sensor was 21.98 pF both before and after packaging. This result suggests that the LED in this study was well-packaged.5.In this study, flexible micro temperature and humidity sensors that could be set between an LED chip and an LED frame was developed, and a 40 μm-thick stainless steel foil was used to conduct heat between the LED chip and the frame. Flexible micro temperature and humidity sensors were fabricated using MEMS. These sensors have the advantages of: (1) small size, (2) high sensitivity, (3) mass-producability, (4) multi-functionality, and (5) flexible but precise measurement positions. A future study will adopt flexible micro temperature and humidity sensors to evaluate the operating parameters of LEDs and thus could improve their design and performance.


Phaeocystis, respectively low and high DMSP producers. On an annual basis, the particulate DMSP (DMSPp) production simulated in 1989 is mainly related to Phaeocystis colonies (78%) rather than diatoms (13%) and nanoflagellates (9%). Accordingly, sensitivity analysis shows that the model responds more to changes in the sulfur:carbon (S:C) quota and lyase yield of Phaeocystis. DMS originates equally from phytoplankton and bacterial DMSP-lyase activity and only 3% of the DMS is emitted to the atmosphere. Model analysis demonstrates the sensitivity of DMS emission towards the atmosphere to the description and parameterization of biological processes emphasizing the need of adequately representing in models both phytoplankton and bacterial processes affecting DMS(P) dynamics. This is particularly important in eutrophied coastal environments such as the SNS dominated by high non-diatom blooms and where empirical models developed from data-sets biased towards open ocean conditions do not satisfactorily predict the timing and amplitude of the DMS seasonal cycle. In order to predict future feedbacks of DMS emissions on climate, it is needed to account for hotspots of DMS emissions from coastal environments that, if eutrophied, are dominated not only by diatoms.We developed a module describing the dimethylsulfoniopropionate (DMSP) and dimethylsulfide (DMS) dynamics, including biological transformations by phytoplankton and bacteria, and physico-chemical processes (including DMS air-sea exchange). This module was integrated in the MIRO ecological model and applied in a 0D frame in the Southern North Sea (SNS). The DMS(P) module is built on parameterizations derived from available knowledge on DMS(P) sources, transformations and sinks, and provides an explicit representation of bacterial activity in contrast to most of existing models that only include phytoplankton process (and abiotic transformations). The model is tested in a highly productive coastal ecosystem  dominated by diatoms and the Haptophyceae  Dimethylsulfide (DMS) is a volatile sulfur (S) compound that plays an important role in the global S cycle and may control climate by influencing cloud albedo through the emission of atmospheric aerosols a), nutrients, irradiance or mixed layer depth) are uncertain and generally regional in scope In marine ecosystems, phytoplankton are the primary producers of dimethylsulfoniopropionate (DMSP), the precursor of the DMS e.g. . HoweverSeveral mechanistic models of different biological complexity  that is one of the most intense DMSP producers Phaeocystis globosa that develops between the spring and summer diatom blooms  bacterial transformation, and DMS emissions as field observations also report important DMS concentration Accordingly, we integrated a module describing the DMS(P) cycle into the existing ecological MIRO model oms e.g.  in respoa.In this paper, we first describe the concepts behind the DMS(P) mathematical model and its coupling with the ecological MIRO model (MIRO-DMS). The model is then applied in the SNS to describe the seasonal evolution of DMS(P) and the associated DMS emission to the atmosphere, and provide an annual budget of DMS(P) fluxes. Sensitivity tests on parameters are conducted to identify key microbial controls of DMS(P) production and how these change the emission of DMS to the atmosphere. Finally, we test the applicability of several published empirical relationships that predict DMS from other variables such as Chl Phaeocystis colonies The MIRO-DMS model results from the coupling between a module describing the DMS(P) dynamics and the existing ecological MIRO model developed to represent the dynamics of the ecosystem of the North Sea dominated by Phaeocystis colonies), two zooplankton FT (meso- and microzooplankton) and one bacteria FT involved in the degradation of dissolved and particulate organic matter (each with two classes of biodegradability) and the regeneration of inorganic nutrients 4) in the water column and the sediment. Equations and parameters were formulated based on current knowledge of the kinetics and the factors controlling the main auto- and heterotrophic processes involved in the functioning of the coastal marine ecosystem .The ecological MIRO model, describing C, N, phosphorus (P) and silica (Si) cycles, assembles four modules describing the dynamics of three phytoplankton Functional Types  cycle  and its Phaeocystis colonies (OP) expressed in mgC m−3) as in the MIRO model and a specific DMSP:C quota (SC) is attributed to the three phytoplankton types. The DMSPp (in mmolS m−3) state equation is:The DMSPp is a constitutive compatible solute produced by phytoplankton cell nμ represents the growth of different phytoplankton types (in mgC m−3 h−1), nlysis is the phytoplankton lysis (in mgC m−3 h−1)  derived from the literature (nsed correspond to the loss of DMSPp due to diatoms and Phaeocystis colonies sedimentation (in mgC m−3 h−1)  and diatoms (DA) (in mgC m−3 h−1)  are not subject to grazing where flux1+2,  and SCn terature . sedn co) flux4, . In the ) flux3, . Phaeocy−3) is:nlysis  and uptakeDMSPd is the bacterial uptake of DMSPd (in mmolS m−3 h−1)  for their C and S needs: BC is the bacterial biomass, bmx is the bacterial growth, SBC are monomeric C substrates available for bacteria and sbck is the half-saturation constant for the bacterial consumption of SBC (in mgC m−3).The description of Sneed, flux5, BCy is the bacterial growth efficiency and BCSC is the bacterial S:C ratio  state equation is:DMS is produced from enzymatic cleavage of DMSP by phytoplankton 1, nlysis is the phytoplankton cellular lysis (in mgC m−3 h−1), bacterial_lyase is the enzymatic cleavage of DMSPd in DMS by bacteria   flux9, .43-Although bacterial degradation of DMS is important uptakeDMS) will cover the bacteria S needs if uptakeDMSPd (Eq. 3) is not sufficient. In the model, uptakeDMS is described from the consumption of carbon by bacteria and the DMS content of bacterial C substrates, according to:Based on that, we assume that bacterial uptake of DMS , K0 is the photooxidation rate in the surface Z is DMS at depth z. As a first approximation, the ultraviolet A (UVA) penetration in the water column is considered equal to that of photosynthetic active radiation (PAR), as PAR attenuation in the studied coastal area is mainly governed by detrital particulate and colored dissolved organic matter. This assumption corresponds to a maximum water penetration of UVA and tends to overestimate the DMS loss by photooxidation. surface  and kD iDMS) is determined based on the surface DMS concentration and the gas transfer velocity (k) of DMS at in-situ temperature (kDMS):The DMS air-sea flux  of 600 and ScDMS is the Sc of DMS computed according to Saltzman et al. where kT is sea surface temperature (°C).where k600 (cm h−1) was computed from a parameterization (u10 in m s−1) that we derived from the binned data reported by Yang et al. rization  as a funu10 data were extracted from the National Centers for Environmental Prediction (NCEP) Reanalysis Daily Averages Surface Flux (http://www.cdc.noaa.gov/) for one station in the North Sea (3.75°E 52.38°N).http://saga.pmel.noaa.gov/dms/) and correspond to data available in the SNS between 51.0°N–52.5°N and 1.5°E–4.5°E For this application, the MIRO-DMS model was implemented in the SNS using a multi-box 0D frame delineated on the basis of the hydrological regime and river inputs  [60]. InValidation of the MIRO ecological model is given by Lancelot et al. Phaeocystis colonies, and summer diatoms  concentrations are analyzed in parallel to the evolution of the planktonic compartments (phytoplankton and bacteria) . The phy diatoms . Spring Chla m−3  in April 1 month . In asso 1 month .Phaeocystis bloom  concentrations show low values except during the spring is bloom . Simulat1 month, , i.e. ines bloom  and reac mgC m−3  correspo−3; The time lag of about 10 days between the simulated DMSPp and DMSPd  representing a DMS flux similar to bacterial DMSP-lyase activity. The importance of phytoplankton DMSP-lyase was previously reported in the area by Stefels and Dijkhuizen Phaeocystis bloom MIRO-DMS estimates the total annual phytoplankton production of DMSPp at 50 mmolS mPhaeocystis, which in itself releases almost 50% of DMSPp throughout the year. The sedimentation of DMSPp amounts to 4.3 mmolS m−2 y−1. Bacterial uptake accounts for the majority the removal of both DMSPd and DMS inducing a rapid decrease of their concentrations in the water column. The consumption of DMSPd is sufficient to sustain the total bacteria S need (10.8 mmolS m−2 y−1), and provides up to 16% of bacteria C requirements. In agreement with previous findings −2 y−1 and results in S products other than DMS (mainly SO42− and MeSH). Although only 8% (3.2 mmolS m−2 y−1) of the DMSPd consumed by bacteria is cleaved to DMS, this flux represents 50% of annual DMS input and is similar to phytoplankton DMSP-lyase activity (DMSPd results from phytoplankton cell lysis (68%) and zooplankton grazing (32%). The dominant process is the cell lysis of activity .−2 y−1) of the DMS pool is consumed by bacteria and transformed in SO42− or DMSO. Kiene and Bates DMS represents <1% of the DMSPp production in the water column in agreement with Archer et al. Bacteria also consume directly DMS and about 83% (5.3 mmolS mk600 parameterization). In particular, the impact of phytoplankton S:C quota determining the maximal DMSP production of the ecosystem, the importance of phytoplankton DMSP-lyase that represents the direct transformation pathway of DMSP into DMS and the DMS(P) bacterial uptake and lyase activity were tested.Annual S budget simulated in the BCZ points both phytoplankton and bacteria as key controlling factors of the DMS production. However the relative importance of these processes will results from their description and parameterization in the model. Sensitivity analyses were then carried out to estimate the impact on the atmospheric emission of DMS of the description of several biological processes compared to physical processes , but also within a FTs (spring versus summer diatoms).In our model, phytoplankton S:C quotas were fixed, corresponding to the mean values of measurements for Haptophyceae and diatoms reported by Stefels et al. r factor  without r factor , any modr factor ; 8a and tor . For low phytoplankton S:C ratio  similar to the DMS flux that comes from bacterial enzymatic cleavage  with little impact on DMSPd concentration.In the model, DMSPd is released in the water column by phytoplankton lysis and grazing processes (Eq. 2). The modification of the phytoplankton DMSP-lyase activity affects the F Test 8,  will decDMS. However, as shown in Sensitivity tests were then conducted by varying the bacterial S:C quota between extreme values reported in the literature i.e. 1∶37 and 1∶196 molS:molC . In our DMS increases  and 375 µmolS m−3 and 40 µmolS m−3 for a fraction of 50%. The simulated DMS emissions to the atmosphere also increase with an annual FDMS of about 0.24 and 0.32 mmolS m−2 y−1, respectively, compared to 0.19 mmolS m−2 y−1 in the reference simulation (−2 y−1 compared to 3.2 mmolS m−2 y−1 in the reference simulation), and the increase of FDMS mainly results from the decrease of bacterial DMS uptake and the accumulation of DMS in the water column. This is confirmed by results obtained by modifying only DMSPd (Test 15) or DMS bacterial uptake (Test 16). These results are consistent with the observations that suggest that bacterial DMS uptake may be a quantitatively important sink for DMS from the surface ocean In a third series of tests, we modified the percentage of bacteria able to use DMSPd and/or DMS as S source. The hypothesis of 100% used in the reference simulation was based on the observation that most marine bacteria have the genetic capability to demethylate DMSP ncreases . The maxncreases  simulatemulation . This inDMS.In the model, bacterial cleavage of DMSP in DMS represents 10% of the uptake of DMSPd not assimilated by bacteria. To test the importance of bacterial DMSP-lyase activity, this fraction was set to 25% inducing an increase of almost two fold of both the concentration of DMS and FDMS resulting from the maximal hypothesis of bacterial uptake (RatioBCS = 1). This is particularly important for the direct bacterial uptake of DMS. Similarly, the bacteria state variable lumps both bacteria and Achaea that might also be important for the demethylation/demethiolation processes Due to their importance on both DMSPd and DMS transformation, bacterial processes need to be accurately described and/or parameterized in ecosystem models. Note that in the present version of the model we only considered one bacterial community, and we did not individually represent the DMS- or DMSP-consumers although this simplification also induces possible uncertainties and underestimation of FDMS is also function of the k600 that depends on the intensity of wind speed and how it is translated into turbulence (depending on the parameterization). Additional tests were performed to estimate the sensitivity of the simulated atmospheric emission of DMS to wind speed and k600 parameterization  release experiments in the SNS, and this parameterization has been shown to be also applicable in open ocean conditions k values of Yang et al. DMS by eddy-covariance during 2 experiments in the Pacific Ocean and 3 experiments in the Atlantic Ocean. The k600 values of Nightingale et al. 10>8 m s−1  wind speed is low , the annual average of k600 computed from the Yang et al. −1) is only ∼9% lower than the one computed using the Nightingale et al. −1).The net annual Fry close . The two>8 m s−1 , and suc the SNS  when [DM the SNS . Since wDMS, several empirical relationships have been derived from DMS field data and variables such Chl a, NO3−, T, primary production, solar radiation, or mixed layer depth that can be derived at higher spatial and temporal resolution from climatologies, remote sensing or models. We tested if some of these empirical relationships that are assumed universal and generic were applicable to the SNS that is representative of a temperate eutrophied coastal system. Several empirical parameterizations that allow to compute DMS concentration in marine waters  and compared to DMS concentration obtained with the MIRO-DMS, and with the available DMS observations in area  used in these relationship was computed based diatoms and non-diatoms (nanoflagellates and Phaeocystis colonies) Chl a simulated by the MIRO-DMS model. Results obtained with both relationships largely overestimated DMS concentrations in the area during Phaeocystis bloom , well below the maximum values in the SNS, up to 25 µg L−1  but also within a FT (spring versus summer diatoms) to describe DMSP and DMS concentrations in marine ecosystems. Due to their elevated S:C quota and their major contribution (50%) to the annual primary production, Phaeocystis colonies are responsible of 78% of the annual production of DMSP in the BCZ. This work is an additional modelling effort to explicitly include bacterial processes in transforming DMS(P), and shows their contribution in processing DMSP and as a sink of DMS that is much higher than DMS removal by photooxidation and FDMS.The application in the BCZ of the newly developed biogeochemical model MIRO-DMS shows that modelled Faa values were related to diatoms unlike eutrophied coastal environments such as the SNS where high biomass is not associated to diatoms. Therefore, future projections of FDMS and the investigation of the potential feedback on climate require to use modeling tools that accurately represent DMS(P) dynamics in coastal environments that are hotspots of DMS emissions, in particular, in eutrophied coastal environments dominated by high biomass non-diatom blooms. Further, bacterial processing of DMS(P) needs to be correctly represented in models. The potential feedbacks of DMS emissions on climate will depend on the impact of climate change on the phytoplankton composition and biomass, as postulated by the CLAW hypothesis versus bacterial consumption/transformation).Current empirical relationships to predict DMS from Chl


TTC28 using deep coverage WGS data from 92 CRC tumor-normal sample pairs. In two cases the event had targeted NOVA1 gene (p=0.025). In addition, a germline retrotransposition event from TTC28 to GABRA4 was found to be a common polymorphism in the Finnish population. Thus while some events may be tumorigenic, others are likely to be neutral. Our data contradict a recent study where a similar signal in TTC28 was interpreted as a common inactivating translocation. While much work remains to be performed to understand the biological significance of retrotranspositions in cancer, accurate identification of these events is a prerequisite for success.L1 element retrotranspositions have been found to alter expression of genes neighboring the insertion sites, potentially involving them in tumorigenesis and tumor progression. In colorectal cancer (CRC), L1 insertions have been found to target genes with a role in tumorigenesis. Structural changes such as L1 insertions are identifiable by whole genome sequencing (WGS). In this study, we observed frequent somatic L1 retrotranspositions originating from  The role of transposable elements (TEs) in tumorigenesis and tumor progression is, despite active research -4, stillODZ3, ROBO2, PTPRM, PCM1, and CDH11 that have a role in tumorigenesis [L1 retrotranspositions have been reported in several tumor types including colorectal cancer , 13. Theigenesis .TTC28 intron 1 - that was recently reported to be the site for the most frequent structural change in CRC  is a state-of-the-art method for analyzing structural variations in the genome  and is gf cases; ). The chTTC28  WGS in the first intron of TTC28 were observed in WGS data of 52 out of 92 (57%) CRC cases. Deep sequencing coverage (>40x) facilitated identification of multiple retrotransposition events in some of the cases. A total of 17 cases with two separate retrotransposition events were identified. In addition, we observed four cases that harbored three events, and two cases with four events. Insertion sites are illustrated in Figure 2=54.2, df=1, p=1.81e-13), compatible with previous literature on retrotranspositions [neuro-oncological ventral antigen 1 (NOVA1) (p=0.025).A total of 83 somatic L1 insertions originating from a specific L1 element residing in the first intron of SGIP1, NOVA1 and ARHGEF4. In each case, we identified two PCR products of varying length: one matching the expected wild type allele size and a larger one representing the allele containing also the L1 insertion. In two cases (SGIP1 and NOVA1), we were able to see sequence corresponding to the truncated 5' end of the L1 element. In SGIP1 case, we observed 449 bp of L1 sequence originating from 5484 bp downstream from the start of ORF1. Similarly in NOVA1, 246 bp of inserted L1 sequence originating from 5472 bp downstream from ORF1 start was identified. For the ARHGEF4 case, we observed 235 bp of sequence from 6162 bp downstream from ORF1. Finally, in SGIP1 and ARHGEF4 cases, we also observed a poly(A) tail at the 3' end.We successfully validated three somatic L1 insertions by Sanger sequencing. Insertion sites of the validated somatic events were in introns of TTC28 were observed in WGS data; at GABRA4 and rp11-136O12  were found to share significantly longer haplotypes around the insertion locus than other cases, indicating a shared founder haplotype . As expected, evidence for shared haplotypes at TTC28 (chr 22) were not observed in cases sharing the TTC28-GABRA4-retrotransposition . The 3/92 (3.3%) WGS cases with the germline L1 insertion at rp11-136O12 (chr 8) were found not to have a significantly longer haplotype , suggesting more ancient or independent origins.The four WGS cases sharing the germline retrotransposition at TTC28 L1 element  observed by TCGA [TTC28 was depicted as a frequent target for inactivating translocations [TTC28 are in fact L1 retrotranspositions, based on the evidence presented here.The frequent breakpoint signals in  by TCGA  and thosocations . The intNOVA1. Given the large number of potential insertion sites, even two hits to a single gene was somewhat unexpected (p=0.025) and might reflect selective value. NOVA1 has been identified as a splicing factor playing a role in neuronal splicing program, but it is also expressed in fibroblasts [SRSF6 have been associated with colorectal cancer [NOVA1 in CRC remains to be studied. We also found that TTC28-GABRA4 retrotransposition is a common polymorphism in the Finnish population. Thus this particular event is not likely to be oncogenic.Retrotranspositions may have oncogenic effects , or theyroblasts . Other sl cancer . The posTTC28 aberrations in CRC, as well as provides a valuable lesson in interpretation of WGS data. The TTC28 events that we observed are frequent, and some may be involved in tumorigenesis while others are likely to be neutral. Much work remains to be done to unravel the biological consequences of retrotranspositions in cancer. Accurate identification of these events is a prerequisite for success.To summarize, our study sheds light on the nature of In total, 92 familial CRC cases from 89 families  fulfilling the following criteria were included in this study: (i) at least one CRC case in a first degree relative, (ii) negative for any known high penetrance CRC mutation, and (iii) availability of sufficient amount of DNA, and (iv) microsatellite stable tumors. Seventy-nine of these 92 cases, and the additional 90 cases, used in the validation are part of a previously described population-based collection of Finnish CRC cases , 25. TheThe study has been reviewed and approved by the Ethics Committee of the Hospital district of Helsinki and Uusimaa (HUS). Signed informed consent or authorization from the National Supervisory Authority for Welfare and Health has been obtained for all the study participants.www.bioinformatics.babraham.ac.uk/projects/fastqc). Paired-end sequencing data was aligned using BWA 0.6.2 [Whole-genome sequencing was performed on the Illumina HiSeq 2000 platform with paired-end reads of length 100 bp. Each normal and tumor DNA sample was sequenced to at least 40x median coverage. Sequencing data quality was evaluated with FastQC . Here it was observed that the presumed translocation calls corresponded to insertions of sequence originating from the TTC28 locus elsewhere in the genome, instead of a translocation. Whole-genome sequences of all tumor and normal samples at the breakpoint position were visually inspected in RikuRator. An L1 insertion to a specific locus was called when at least three paired-end reads supported the insertion. This inspection also revealed the germline insertions that were initially removed by the above somatic filtering.The most frequently involved gene in DELLY translocation calls was TTC28 were examined by first calculating the sequencing coverage in tumors around the 3' end of the L1 element  was validated by Sanger sequencing in four samples. Primer pairs comprising each insertion junction were designed using Primer3Plus ([http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi/); primer sequences and PCR conditions are available upon request. Fragments were amplified with AmpliTaqGoldVR  and the PCR products were purified using the ExoSAP-IT PCR purification kit . Sequencing reactions were performed using the Big Dye Terminator v.3.1 kit (Applied Biosystems) and electrophoresis was run on 3730xl DNA Analyzer (Applied Biosystems) at FIMM Genome and Technology Centre, Finland. The sequence graphs were manually analyzed using FinchTV v.1.4 .One germline L1 insertion site candidate  were successfully validated by Sanger sequencing. Primers flanking the insertion breakpoints were designed, as previously described. The PCR was performed using Expand Long Template PCR system . The PCR products were run in standard low-melting agarose gel. DNA band corresponding to the allele with the L1 insertion, was extracted from the agarose gel using QIAquick Gel extraction Kit . The sequencing reaction and the analysis of the sequences were performed as previously described.Additional three somatic L1 insertion candidates  or intergenic region (59%). In case of an event targeting a gene, the target gene was uniformly selected from 51573 genes, including protein-coding genes, short and long non-coding RNAs and pseudogenes (Ensembl 71.37). A total of 1000000 permutations were performed. An empirical p-value was derived as the fraction of permutations where any gene was hit two or more times.Significance of


Takayasu arteritis (TA) is a chronic vasculitis of unknown origin, affecting mainly the aorta and its main branches. As a result of the inflammation, stenosis, occlusion or dilatation of the involved vessels may occur and cause a wide range of symptoms. It has been described in association with various auto-immune disorders (mainly inflammatory digestive tract diseases). However, only few cases of TA associated with sarcoidosis have been reported, raising the question of an association by chance.R).We report a case of a 34 year-old woman, with one year history of sarcoidosis, who presented with asymmetric high hypertension revealing inflammatory humeral, axillary and subclavian arteritis related to TA, successfully treated by steroid and immunosuppressive therapy(MethotrexateTA and sarcoidosis may be related, rising the hypothesis that TA or Takayasu arteritis-like granulomatous vasculitis may be, in fact, a complication of sarcoidosis. Takayasu arteritis (TA) is a chronic inflammatory disease of large arteries which progressively develop stenosis, occlusion or aneurismal degeneration. The clinical presentation is characterised by an acute phase with constitutional symptoms, followed, months or years later, by a chronic phase in which symptoms relate to fibrosis or occlusion of vessels. Angiography is the gold standard for diagnosis and for topographical classification and it correlates with symptoms and prognosis. Proinflammatory cytokines and, among these, tumor necrosis factor-alpha  are increased and play a pathogenetic role in the development of disease. Laboratory findings are non-specific. Treatment of active disease is primarily based on corticosteroids but other immunosuppressive drugs are frequently needed. Anti-platelets agents, statins and antihypertensive drugs are frequently considered .Sarcoidosis, too, is a systemic inflammatory disease which can affect virtually any organ system. The lungs are most commonly affected, but often there is also affection of the musculoskeletal system, the cutaneous and central nervous systems, and the heart. Histology shows granulomas consisting of a central zone with macrophages, epithelioid cells, and multinucleated giant cells in addition to activated CD4 lymphocytes, and a peripheral zone with macrophages, fibroblasts, and CD4 and CD8 lymphocytes. Inclusion bodies are commonly found. Vasculitis may be found occasionally in patients with sarcoidosis. It has not been confirmed, however, whether vasculitis is a true manifestation of the disease or an occasional, incidentally concurring abnormality.Concurrence of sarcoidosis and TA were reported in few cases.A 34 year old woman was admitted to the hospital with dyspnea, submandubular gland tumefaction and parotid swelling.On physical examination, we found the tumefaction of the parotid and the submandibular gland. The respiratory frequency was 24 cycles per minute. The rest of the clinical examination was without abnormalities.Laboratory results were as follows: Haemoglobin 10 g/dl, white blood cell count (WBC) 11 200/mm3, platelets 450 000/mm3, erythrocyte sedimentation rate (ESR) 76 mm/h, calcium 12 mg/dl . Muscle enzymes, ANA, rheumatoid factor, VDRL, and complement components C3 and C4 were negative. The angiotensin converting enzyme (ACE) level was 135 UI/L.Chest radiography showed "Bilateral interstitial involvement". Thoracoabdominal CT scan showed "Multiple mediastinal and retroperitoneal lymphadenopathy, with moderate hepatomegaly". The respiratory functional test showed the presence of a restrictive syndrome.The lung fibroscopy was normal. The biopsy confirmed the presence of a non caseating granuloma in the bronchial mucosa. The bronchoalveolar lavage showed a predominantly lymphocytic cell with the elevated Cd4/CD8 ratio.Ophthalmological examination confirmed the presence of dry eye syndrome. There was no uveitis. The salivary gland biopsy confirmed the presence of a granuloma without caseous necrosis.Sarcoidosis was diagnosed, and the patient received prednisolone 60 mg/day. After 1 month of treatment, all complaints have been resolved.Seven months later, when she received 10 mg/day of prednisolone, she consulted for respiratory distress. Her left brachial and radial pulses were absent, and her blood pressure was 180/120 mm Hg as measured from the left arm. Bruits were audible over the course of the left carotid artery and the left and the right subclavian artery. The patient had elevated ERS (100 mm/H).An Ultra Sound Colour Doppler (USCD) was performed showing a circumferential parietal swelling interesting the left subclavian artery reducing his superficies of 76%, demodulating the Doppler spectrum and edema of the vessl wallFigure . These dWe diagnosed the patient having TA associated with sarcoidosis. She received prednisolone 40 mg/day with Methotrexate 10 mg/week and amlodipine 5 mg/say.As a response to treatment, blood pressure was normal 2 weeks after. The USCD control confirmed the therapeutic benefice showing a reduction of the parietal anomalies.About a dozen cases of the association TA and sarcoidosis were reported in the literature. -6; some TA can be associated with Crohn's disease , RCH, syThe association between sarcoidosis and TA or other forms of aortitis may be rare. However, TA or similar forms of aortitis are too rare to indicate that the reported concurrence with sarcoidosis is merely governed by chance. In fact, all these patients had several common features: (a) usually sarcoidosis preceded TA; (b) the time between the diagnosis of sarcoidosis and that of TA was several years (eight or more) in most of the patients; (c) the aorta and/or its major branches was affected; (d) 50% of the patients had uveitis, which is much less common in frank sarcoidosis; and (e) all patients responded to glucocorticoid treatment.Association of TA and sarcoidosis raise a number of questions of a possible link between these 2 diseases. Vascular injury (or "granulomatous vasculitis), and especially the location in the aorta thoracic and/or its branches, could be a complication in the course of sarcoidosis -3,10. HoThe association between sarcoidosis and TA are rare but not accidental; complete vascular clinical examination should be performed in those patients in order to detect asymptomatic underlying inflammatory arteritis.The relatively low prevalence of sarcoidosis, and even more so the rarity of aortitis, suggests that this number is relatively high and too high to be a coincidence; can we say that sarcoidosis and TA appears to be 2 related diseases or that Takayasu arteritis or Takayasu arteritis-like granulomatous vasculitis may be, in fact, a complication of sarcoidosis?Informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.AH, RK, OH, OB and SM: Diagnosis the disease and monitoring the patient RS and MG: Have carried out the radiological explorations.All authors read and approved the final manuscript.


Euglena gracilis is riddled with introns that interrupt almost every protein-encoding gene. The atypical group II introns and twintrons (introns-within-introns) found in the E. gracilis plastid have been hypothesized to have been acquired late in the evolution of euglenids, implying that massive numbers of introns may be lacking in other taxa. This late emergence was recently corroborated by the plastid genome sequences of the two basal euglenids, Eutreptiella gymnastica and Eutreptia viridis, which were found to contain fewer introns.Photosynthetic euglenids acquired their plastid by secondary endosymbiosis of a prasinophyte-like green alga. But unlike its prasinophyte counterparts, the plastid genome of the euglenid Monomorphina aenigmatica, a freshwater species occupying an intermediate phylogenetic position between early and late branching euglenids. The M. aenigmatica UTEX 1284 plastid genome  contains 53 intron insertion sites, of which 41 were found to be shared with other euglenids including 12 of the 15 twintron insertion sites reported in E. gracilis.To gain further insights into the proliferation of introns in euglenid plastids, we have characterized the complete plastid genome sequence of The pattern of insertion sites suggests an ongoing but uneven process of intron gain in the lineage, with perhaps a minimum of two bursts of rapid intron proliferation. We also identified several sites that represent intermediates in the process of twintron evolution, where the external intron is in place, but not the internal one, offering a glimpse into how these convoluted molecular contraptions originate. Pyramimonas parkeaeEuglena gracilis, which has been used as a model organism for numerous studies in the past decades Euglenids are a morphologically diverse group of unicellular freshwater and marine eukaryotes displaying various feeding behaviours, including phagotrophy, osmotrophy, and phototrophy (reviewed in E. gracilis plastid genome (cpDNA) is the presence of over 150 introns  that interrupt almost all of its protein-coding genes , which are introns inserted within other introns. These intricate nested introns must be removed sequentially to result in accurate splicing.One of the most remarkable features of the E. gracilis introns have been found in other Euglena species Euglena (formerly Astasia) longaP. parkeae did not contain any highly-reduced group II introns Eutreptiella gymnasticaEutreptia viridisThe unorthodox Monomorphina aenigmatica (strain UTEX 1284), a freshwater species that branches between Euglena and the earlier-diverging photosynthetic euglenids M. aenigmatica plastid genome  lends further support to the intron-late hypothesis, but also suggests that at least two distinct and independent intron acquisition bursts occurred in the euglenid plastid genomes. We also identify a number of insertion sites with an intermediate stage in the emergence of twintrons, showing how such molecular structures might arise.To better understand intron evolution in photosynthetic euglenids, we determined the complete plastid genome sequence of Monomorphina aenigmatica strain UTEX 1284 maps as a circular molecule of 74,746 bp with an overall 70.6% A+T content  and the gene coding for the small 5S rRNA, which are present in E. gracilis. The 5S rRNA-encoding gene is present in Eta. viridis but absent from Etl. gymnastica and, interestingly, from the prasinophytes P. parkeae and Pycnococcus provasoliirpoA gene reported in some Euglena species psbZ and trnS(uga) does not appear to be present in M. aenigmatica. This location of the genome does not contain an ORF, and while the sequence shares barely-detectable similarity with the alpha subunit of bacterial DNA-directed RNA polymerases, it shares none with the reported euglenid RpoA sequences. The M. aenigmatica cpDNA contains only a single rRNA operon, which contrasts with the three copies found in tandem in the E. gracilis cpDNA. The 1∶1 relative coverage ratio between this operon and the other genomic regions in M. aenigmatica cpDNA assembly excludes the possibility that the single copy observed is the result of an assembly artefact that would have collapsed adjacent operons. Like its E. gracilis counterpart, however, the M. aenigmatica plastid genome features a number of tandem repeats restrained to a single locus, which are likely variable within the population. Interestingly, these variable numbers of tandem repeats (VNTR) are located in the same position, between trnL(caa) and rrs, as those reported in E. gracilis. The two genomes display a high level of synteny, with the 85 genes that they share distributed between four blocks of colinear genes.The chloroplast genome of  content , Table 1M. aenigmatica genome is roughly half the size of that of E. gracilis  with E. longa that is absent from E. gracilis, and which likely has been lost in the latter genome after the split with E. longa. Only one insertion site (psbC.Maen.1) was found to be shared between the four photosynthetic euglenids investigated to date (the psbC gene was lost in E. longa). The remaining 12 insertion sites in M. aenigmatica f′e′d′ A* c′b′a′ (4)-3′ consensuses described in M. aenigmatica genome shares only 6 twintrons with E. gracilis , probably reflecting intermediate stages of twintron evolution, although the possibility that the additional internal introns found in E. gracilis were lost in an ancestor of M. aenigmatica cannot be completely excluded. The six remaining insertion sites where E. gracilis encodes a twintron  were found to encode only a single intron in M. aenigmatica, and in all five cases the intron was orthologous to the E. gracilis external intron.Interestingly, 12 of the 15 sites reported to contain twintrons in gracilis . The psbM. aenigmatica chloroplast genome contains 3 putative reverse transcriptases/maturases. The first two are located within the intron insertion sites psbC.Maen.1  and psbC.Maen.2 , whereas the third (orf240) is free-standing, potentially unique to M. aenigmatica, and highly degenerated . The mat1/mat2 intron-encoded maturases are not unique to M. aenigmatica and are also found in the other euglenid introns that are inserted at cognate sites , the genome of the early branching euglenid Etl. gymnasticapsbC intron found in all four photosynthetic euglenids, as proposed by Doetsch et al.M. aenigmatica insertion sites are shared with E. gracilis , but 21 out of the 23 insertion sites found in Eta. viridis are currently unique to that species, and only one intron is common to all four species  featured only one intron, which eventually became the external intron in a twintron when a second intron was inserted within it. However, no such intermediate stage has ever been described, until now.  viridis , Table 2Thermosynechococcus elongatusDrosophilaE. gracilis plastid genome contain so many of them? The first explanation is perhaps a simple matter of density and mobility: in genomes with high intron mobility, more and more introns are expected to be found. This, in turn increases the likelihood of having an intron insert itself inside another intron. That being said, the psbC twintron cannot be explained in this way since it is present in all four euglenid genomes and likely arose before any intron proliferation. A second potential explanation is that group II intron mobility is often not random. Instead, they propagate at cognate and ectopic sites by processes known as ‘homing/retrohoming’ and ‘transposition/retrotransposition’, respectively. These processes are usually quite stringent, and while the endonucleases or reverse transcriptases involved allow for some polymorphism to occur, the target sites are usually highly conserved. If the target site is contained within a duplicated or repeated sequence however, then a given intron can, in theory, quickly move to occupy most of these loci. It is also possible that the transposition/retrotransposition processes in euglenid genomes are somehow relaxed, thus helping with the intron invasion. A third potential explanation is that in the euglenid plastid genetic system the selection for accurate splicing has been relaxed for some reason, allowing the proliferation of what might otherwise be slightly deleterious introns. It would be interesting to investigate whether the transcription is generally messy in these plastid genomes, and if the mRNAs for intron-interrupted genes form a population of viable and defective molecules. Since previous analyses found a number of splicing variants in E. gracilisGroup II introns are mobile genetic elements known to interrupt target sequences, coding or non-coding. Introns that are inserted in intergenic regions are often transient owing to the relaxed selective pressure resulting from their not interrupting a coding sequence. In contrast, however, introns that are inserted within genes must be properly spliced for the gene to retain its function and any mutation affecting the splicing efficiency of the intron could have a deleterious effect. In twintrons these conditions are compounded, adding an extra layer of complexity to an already complex mechanism. It is therefore not surprising that twintrons are quite rare - other than euglenid plastids, a few isolated cases have been described  using the protocol provided by the manufacturer. Polysaccharides were removed from the extracted DNA as follows. Total DNA (50 µl) was incubated in the presence of 500 µl CTAB buffer  at 60°C, cleaned with 500 µl of chloroform, precipitated with 500 µl of isopropanol, washed with 500 µl of EtOH 80% (vol/vol), then resuspended in water. Total RNA was extracted from liquid NO2-ground and pelleted cells with Trizol (Invitrogen) using the protocol provided by the manufacturer.M. aenigmatica illumina TruSeq library  was prepared from total DNA and sequenced on one lane of illumina HiSeq by the Beaty Biodiversity Research Centre sequencing facilities . Adapters and indexes were removed from the resulting reads and the reads filtered using the CASAVA 1.8.2 pipeline (illumina) under the default parameters. The first 320,000,000 reads  of the filtered reads were assembled in parallel with Ray version 2.0.0 rc4 M. aenigmatica plastid genome were identified as follows. The 500+ bp-filtered contigs were converted to a BLAST Euglena gracilis [EMBL:X70810] and Pyramimonas parkeae [GenBank:NC_012099] were both queried independently against the M. aenigmatica database with TBLASTN from the BLAST package using variable cut-offs to detect potential chloroplast contigs. The potential contigs of interest were pulled out from the multifasta file with the faOneRecord/faSomeRecords command line utilities from the University of California, Santa Cruz (UCSC), and searched reciprocally against the entire NCBI non-redundant database to ascertain their origin.The M. aenigmatica chloroplast contigs were further assembled and polished with CONSED 23 The E. gracilis genome as input queries. Protein-coding genes and open reading frames were annotated with Artemis 14.0 using the built-in tools. Then, each predicted M. aenigmatica protein-coding gene sequence was aligned at the amino acid level against orthologs from euglenids and green algae using the L-INS-i algorithm from MAFFT 6.925 M. aenigmatica plastid genome were searched for specifically by BLAST homology searches using the closest known orthologs. The predicted exon-intron junctions were annotated to preserve homology between orthologs whenever possible. The gene map shown in M. aenigmatica UTEX 1284 plastid genome annotation has been deposited in GenBank [GenBank:JX457480].Genes coding for tRNAs were identified with tRNAscan-SE 1.21 rpoC1 gene is not well conserved, we performed RT-PCR experiments using primers specific to M. aenigmatica to ascertain a number of intron-exon boundaries. The RT-PCRs were performed on total RNA with the Superscript III One-Step RT-PCR kit (Invitrogen) and the resulting products cloned with the StrataClone PCR cloning kit (Stratagene), both using the protocols provided by the manufacturers. The clones grown on LB-ampicillin (50 µg/ml) plates with X-galactose (20 mg/ml) at 37°C overnight were PCR-screened for inserts using the T3/T7 primers flanking the insertion site and the EconoTaq PLUS GREEN (Lucigen) and plated again on LB+ampicillin (37°C/8 hours). Selected clones were cultured overnight at 37°C in 2 ml of liquid LB medium+ampicillin (50 µg/ml) and the plasmids purified with the FastPlasmid Mini kit (5 Prime) using the default protocol. The products were then Sanger-sequenced using the T3/T7 primers flanking the insertion site. Note that we could not produce a reliable RT-PCR covering the insertion site located at position 4 in M. aenigmatica’s rpoC1, and believe that a small unannotated exon might be present within it.Because the M. aenigmatica [GenBank:JX457480], E. gracilis [EMBL:X70810], E. longa [EMBL: AJ294725], Eta. viridis [GenBank:JN643723], Etl. gymnastica [EMBL:HE605038]) were determined as follows. Orthologous protein sequences were aligned with their green algal counterparts using the L-INS-i algorithm from MAFFT and the insertion sites mapped manually on the corresponding sequences. Insertions located within the same amino acid but at different sites within the corresponding codon were considered distinct. We also corrected a number of annotations from the Eta. viridis and Etl. gymnastica accessions numbers  aligning each of the external and internal E. gracilis intron sequences against the target sequence. For M. aenigmatica sequences, we also scanned for the 5′-NUNNG and abcdef(3–8)f′e′d′ A* c′b′a′ (4)-3′ consensuses described in Cognate intron insertion sites between euglenid genomes . The rpoC1.Maen.4 insertion could not be verified by RT-PCR and may contain a short additional exon.(XLSX)Click here for additional data file.Data S1Euglenid plastid genomes EMBL annotations files. The Eta. viridis and Etl. gymnastica original annotations were modified to include additional introns and/or revised insertions sites. The modifications are also indicated by miscellaneous features annotation tags in the revised files for quick localization with Artemis.(ZIP)Click here for additional data file.


A 36-year-old man visited our hospital because of blurred vision and redness of the conjunctiva. Slit-lamp examination showed panuveitis. Two days later, he suddenly experienced dizziness, speech disturbance, paralysis of his right extremities, and gait disturbances. Neurological examinations suggested that his symptoms were caused by a left lateral medullary lesion. He also had erythema mainly on his trunk. Magnetic resonance imaging (MRI) of his brain demonstrated a small infarct on the left side of the medulla oblongata. Clinical presentation and MRI findings were consistent with the diagnosis of a Wallenberg's syndrome. He also had bilateral hilar lymphadenopathy. A skin biopsy showed granulomatous nodular dermatitis compatible with sarcoidosis. He was treated with steroid pulse therapy and his neurological and ocular symptoms immediately improved. Only seven similar cases of intracranical sarcoidosis have been reported, but none had been treated with steroid pulse therapy. We recommend that steroid pulse therapy be considered to treat patients with sarcoidosis with signs of lesions in the central nervous system. Sarcoidosis is characterized by noncaseating granulomas, and in 5% of the patients, the lesions are located in the central nervous system . HoweverChronic posterior ocular sarcoidosis is more difficult to manage, and it can lead to sight-threatening complications, for example, cataracts, glaucoma, and cystoid macular oedema. Eyes with posterior uveitis with chronic sarcoidosis have a poor visual prognosis despite immunosuppressive therapy .Corticosteroids, usually combined with immnosuppresive agents, remain the mainstay therapy for ocular sarcoidosis although refractory cases do occur . The refWe described a patient with ocular sarcoidosis and a cerebral infarction, who had an excellent response to steroid-pulse therapy.A 36-year-old man reported blurred vision and redness of the conjunctiva in both eyes. He was referred to our hospital in January 2009, and our examination showed that his best-corrected visual acuity (BCVA) was 1.2 (20/20) in both eyes. Slit-lamp examination showed ciliary injection and keratic precipitates. Fundus examination demonstrated vasculitis in both eyes Figures .Physical examination showed exudative erythema nodosum on his trunk and both thighs. Chest X-rays and CT scans demonstrated bilateral hilar and mediastinal lymph node swelling. Laboratory tests, including antibodies for rheumatoid factor, Sjögren syndrome, double-stranded DNA, and antinuclear antibodies, were negative. In addition, the serum antiotensin-converting enzyme level was high at 27.0 U/L .Two days after his first visit, he reported dizziness, speech disturbance, clumsiness of his right extremities, and gait disturbances. Neurological examinations showed left ptosis and miosis, hoarseness, dysphagia, right mild hemiparesis, left ataxia of his extremities, and truncal ataxia. He also had hypalgesia/hypotemperature on his right side. These findings suggested a left Wallenberg syndrome. Brain magnetic resonance imaging (MRI) showed an infarct on the left side of the medulla oblongata . LeptomeThe patient was diagnosed with neurosarcoidosis with a brainstem infarct. Steroid pulse therapy was prescribed initially with 1000 mg infusion for three days and then slowly tapered. During the pulse therapy, the patient had laser photocoagulation of the nonperfused areas of the retina. The patient had an improvement of his symptoms after the steroid-pulse therapy.At the last examination on February 2011, the patient was stable with no complications and no recurrence of the ocular and CNS sarcoidosis. And his BCVA was 20/20.Our findings showed that our patient had CNS sarcoidosis of the medulla oblongata, and steroid-pulse therapy was very effective in resolving the symptoms. The mechanism causing the brain infarction in neurosarcoidosis is not completely known but is thought to result from vasculitis of the small vessels, emboli, or inflammation of the large vessels , 17. GraAlthough rare, neurosarcoidosis should be considered in cases of an acute stroke with restricted diffusion of unknown etiology in a young person, even if other stigmata of sarcoidosis are not present. Despite the common finding of vasculitis and microscopic infarction at autopsy, a stroke is usually due to vascular accidents in the cerebral hemisphere and is rarely the presenting sign of neurosarcoidosis  10, 11,,11, 15. Ocular sarcoidosis is not rare in Japan, and it has been found that sarcoidosis responds well to steroid therapy. But 10% of ocular sarcoidosis required periocular corticosteroids to treat the uveitis, and 13% of ocular sarcoidosis required systemic treatment for both the ocular and systemic disease . In addiThe management of resistant uveitis associated with sarcoidosis is a serious therapeutic problem. The toxicity from steroids such as osteoporosis, femur head necrosis, infection, steroidgenic diabetes, corticosteroid-induced glaucoma, and steroid-induced psychosis is difficult to control. This is why clinicians resist the use of high-dose steroids for long periods. Thus, it is important to know that steroid-pulse therapy is effective in treating the ocular inflammation due to sarcoidosis. Because steroid-pulse therapy has fewer side-effect, we recommend steroid pulse therapy for ocular and CNS sarcoidosis.


L1 cell adhesion molecule (CD171) is expressed in many malignant tumors and its expression correlates with unfavourable outcome. It thus represents a target for tumor diagnosis and therapy. An earlier study conducted by our group identified L1 expression levels in primary gastrointestinal stromal tumors (GIST) as a prognostic marker. The aim of the current study was to compare L1 serum levels of GIST patients with those of healthy controls and to determine whether levels of soluble L1 in sera could serve as a prognostic marker.Using a sensitive enzyme-linked immunosorbent assay (ELISA), soluble L1 was measured in sera of 93 GIST patients und 151 healthy controls. Soluble L1 levels were then correlated with clinicopathological data.p < 0.001; Mann-Whitney U test) in sera of GIST patients compared to healthy individuals. Median soluble L1 levels were particularly elevated in patients with recurrence and relapse .Median levels of soluble L1 were significantly higher (These results suggest that high soluble L1 levels predict poor prognosis and may thus be a promising tumor marker that can contribute to individualise therapy. GISTs-kit CD11 gene andof GISTs . First lolerated .There is to date no prognostic serum marker for GISTs. One possible candidate is the neuronal cell adhesion molecule L1 CD171), which is expressed in primary tumors in 74% of cases . L1 is a1, which L1 overexpression has been detected in various tumor cell types of neural, mesothelial, and epithelial origin. Expression profile analysis in multiple human tumors has identified L1 as a molecular marker for differential diagnosis and targeted therapy ,22. Its The aim of this study was to evaluate whether soluble L1 could serve as diagnostic marker and prognostic factor in GIST patients by correlating L1 serum levels to long-term outcome.The study was approved by the Ethics Committee of the Chamber of Physicians in Hamburg, Germany. Written informed consent was obtained from all patients for using tissue and serum samples. For this study, 93 patients with GIST were chosen retrospectively. It was possible to evaluate a rather large number of patients suffering from this rare tumor because of the cooperation of a self-help organization for GIST patients called "Lebenshaus e.v.", Bad Nauheim, Germany, which helped to collect serial serum samples from patients across Germany, Austria and Switzerland. The blood samples were taken from patients at time of diagnosis and every 3 months thereafter. We selected patients on the basis of availability of specimens and did not stratify them due to rare occurrence and different treatment strategies. All data including sex, age, tumor size, mitotic count, tumor location, metastasis, relapse, and imatinib therapy were obtained from the clinical and pathological records. The clinicopathological data from externally treated patients participating in the self-help project were sent regularly by their attending physicians. As healthy controls, 151 blood bank donors were included in the study. Limitations in this study were that we had some missing items in the clinical and pathological data of our patients due to logistic difficulties in collecting data of patients from different hospitals. However, a sufficient number of GIST patients were included in the study.2SO4 and analysed at 450 nm using an ELISA reader. Human L1-Fc protein served as an internal standard for the assay.For the detection of soluble L1, 96-well flexible microtiter plates (Costar 9019) were coated with 50 μl per well of 10 μg/ml of capturing antibody  overnight at 4°C. Wells were blocked with 3% w/v bovine serum albumin  in PBS/T  for 45 min at room temperature and then incubated for 1 h with human sera diluted 1:5 in PBS at room temperature. After five washes with PBS/T, bound protein was detected with biotin-conjugated monoclonal antibody UJ127 - which does not cross-react with the close homolog of L1 (chL1) - followed by streptavidin-conjugated peroxidase using TMB  as substrate. The color reaction was stopped by the addition of 10 mM HTo ensure that the immunoassay was suitable for measuring clinical serum samples, reproducibility, linearity and crossreactivity were examined. The assay showed no crossreactivity with the closed homolog of L1 CHL1, excellent linearity with serial dilutions and a low coefficient of variation (CV) in intra- and inter-assay variability.4P2O7, 1 mM NaF, 1 mM EDTA, 2 mM Na3VO4, 1 mM PMSF, 1% NP-40 plus complete EDTA-free protease inhibitor mixture, pH 7.5) for 30 min at 4°C and then subjected to SDS-Page and Western blot analysis. Equal amount of protein of each sample was mixed with 2x Laemmli Buffer  and boiled at 95°C for 5 min. Protein extracts were separated by 4 - 12% SDS-PAGE  and transferred onto nitrocellulose membrane . For immunoblotting, membranes were blocked with StartingBlock (TBS) Blocking Buffer  and incubated overnight at 4°C with primary 1:1000 biotinylated antibody L1-UJ127 . UJ127 binds to a membrane proximal Fn repeat. This allows detection of both the ADAM-cleaved 180 kDa shed fragment and the 80 kDa membrane-bound fragment presumably generated by plasmin cleavage. After washes with TBST (TBS with 0.1% Tween 20), membranes were incubated with HRP-conjugated streptavidin  (1:500) for 1 h at room temperature. After extensive washes, streptavidin-reactive bands were visualized using enhanced chemiluminescent substrate  on x-ray films .For analysis of L1cam protein, PBS washed GIST882 cells were lysed in RIPA buffer   was used for statistical analysis. Statistical significance was evaluated by the Mann-Whitney U and Kruskal-Wallis test. Cross-table statistics were performed using Fisher's test. Recurrence-free survival was plotted using the Kaplan-Meier method and analysed by the log-rank test. Significance statements refer to p-values of two-tailed tests less than 0.05.SPSSA total of 93 patients suffering from GIST and 151 apparently healthy controls were included in our study. The median age of GIST patients was 61 years (range 28-81 years) and 47 years (range 30-70 years) for the healthy controls. Fifty-one (55%) of the GIST patients were male and 42 (45%) female, Ninety-eight (65%) of the healthy controls were male and 53 (35%) female. Patients` characteristics and clinicopathological data are listed in Table L1 could be detected in sera of GIST patients, representatively shown in Figure Soluble L1 (s-L1) concentrations in the sera of GIST patients were significantly higher than in healthy controls: median (25th and 75th percentiles) soluble L1 values were 1.4 (0.8 and 2.7 respectively) ng/ml in the GIST patients and 0.5 (0 and 1 respectively) ng/ml in the controls  . For additional statistical analysis, patients with high (>2 ng/ml) and low (<2 ng/ml) soluble L1 concentrations were compared by survival curves plotted by the Kaplan-Meier method for recurrence-free survival . Also mitotic count and tumor size could not be significantly correlated with recurrence .Most primary cancers develop loss of expression of adhesion molecules. This allows for a critical step in metastasis to occur: detachment of the invading cell from its neighbors ,28. SomeIn a previous study we could show that L1 expression was immunohistochemically detectable in 74% of GISTs, but not in differential diagnostic important tumors such as smooth muscle tumor, peripheral nerve sheath tumor or desmoid-type fibromatosis. Although c-kit expression is the etablished standard in the identification of GIST, additional prognostic markers are beneficial. To date, most studies regarding L1 expression have been performed at the mRNA level or by immunohistochemistry. To evaluate the role of L1 as a peripheral marker, we here examined L1 levels in serum of GIST patients and healthy controls.Using a specifically developed robust and highly sensitive immunoassay we were able to demonstrate that soluble L1 concentrations were significantly elevated in the blood of GIST patients compared to a healthy control group and other gastric tumors. Furthermore, we observed a significant association between increased soluble L1 levels and recurrence, which predicts worse outcome in GIST. Patients with high soluble L1 concentrations had a poorer recurrence-free survival than patients with lower levels. These observations are in agreement with our previous immunohistochemical study, where a trend towards worse outcome of patients with L1-positive tumors was reported ,34. ElevSoluble L1 concentration could thus become a useful marker for tumor progression and metastasis, which predicts prognosis in GIST.Our results also suggest that L1 may be an important therapeutic target in the treatment of GIST. The efficiency of L1 monoclonal antibody treatment in suppressing tumor growth has been described in nude mice, where intraperitoneal injection of an L1 antibody was found to inhibit intraperitoneal tumor proliferation and dissemination of malignant tumor cells . In thisWe provide evidence that analysis of serum concentrations of soluble L1, comprising the extracellular domain of the L1 molecule, represents a novel and useful approach to assess the malignancy and metastatic potential of GISTs. The availability of a reliable immunoassay, such as the one developed for this study, for measuring serum L1 concentrations will facilitate further studies to establish the clinical applicability of this marker in GISTs and other tumors. These studies should notably investigate L1 levels in other serum samples such as obtained pre- and post-surgery and serially during the course of therapy. Beyond clinical applicability, future work must address mechanistic questions about the functional role of L1 in the tumorigenesis of GISTs and other L1 expressing cancers.The authors declare that they have no competing interests.HZ developed the immunoassay and drafted the manuscript, TR participated in the design of the study and performed the statistical analysis, MW carried out the immunoassays, MT and MK participated in the statistical analysis and collected the clinicopathological data, GW performed the Western Blot, MB helped to recalculate the statistics for the revision, MS and JRI participated in its design and coordination and helped to draft the manuscript. JTK conceived of the study. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/11/189/prepub


Ultrastructural analysis demonstrates that −/−trkb embryos exhibit defects in endothelial cell integrity and perivascular edema. As TrkB is selectively expressed by pericytes and SMCs in the developing cardiac vasculature, we generated mice deficient in TrkB in these cells. Mice with TrkB deficiency in perivascular cells exhibit reduced pericyte/SMC coverage of the cardiac microvasculature, abnormal endothelial cell ultrastructure, and increased vascular permeability. To dissect biological actions and the signaling pathways downstream of TrkB in pericytes/SMCs, human umbilical SMCs were treated with BDNF. This induced membranous protrusions and cell migration, events dependent on myosin light chain phosphorylation. Moreover, inhibition of Rho GTPase and the Rho-associated protein kinase (ROCK) prevented membrane protrusion and myosin light chain phosphorylation in response to BDNF. These results suggest an important role for BDNF in regulating migration of TrkB-expressing pericytes/SMCs to promote cardiac blood vessel ensheathment and functional integrity during development.Pericyte and vascular smooth muscle cell (SMC) recruitment to the developing vasculature is an important step in blood vessel maturation. Brain-derived neurotrophic factor (BDNF), expressed by endothelial cells, activates the receptor tyrosine kinase TrkB to stabilize the cardiac microvasculature in the perinatal period. However, the effects of the BDNF/TrkB signaling on pericytes/SMCs and the mechanisms downstream of TrkB that promote vessel maturation are unknown. To confirm the involvement of TrkB in vessel maturation, we evaluated TrkB deficient ( During late embryonic development, the formation of mature and fully functional blood vessels depends on the tightly regulated association of endothelial cells and mural cells such as pericytes and smooth muscle cells (SMCs). Many growth factors, such as vascular endothelial growth factor (VEGF), regulate endothelial cell migration and survival whereas platelet derived growth factor (PDGF) is involved in the regulation of remodeling and maturation of blood vessels via actions on pericytes and smooth muscle cells pdgf or pdgfrβ result in perinatal lethality, as a consequence of vascular dysfunction caused by mural cell deficiency Pericytes/SMCs are adventitial cells located within the basement membrane of capillaries and post-capillary venules. These contractile cells play an important role in stabilizing nascent endothelial tubes by providing essential survival factors −/−bdnf ) exhibit impaired survival of endothelial cells in intramyocardial arteries and capillaries in the late gestational and early postnatal periods −/−bdnf  mice is restricted to cardiac vessels, reflecting the localized expression of BDNF and its receptor TrkB in the cardiac and skeletal muscle vasculature trkb leads to lethality during late embryonic development Numerous studies have described critical roles for neurotrophins and their receptors in non-neuronal cells, such as endothelial cells, smooth muscle cells, immune cells, and epithelial cells in different organs −/−trkb embryos exhibit a specific deficiency in pericytes/SMCs. Ultrastructural analysis shows that −/−trkb embryos have defects in endothelial cell integrity including discontinuity of the cell membrane, leading to perivascular edema. We also determined that pericyte/SMC-specific deletion of TrkB results in abnormalities of vasculogenesis that significantly contribute to the lethal phenotype observed in −/−trkb mice. Conditional TrkB deletion in SMCs results in marked reduction of pericyte/SMC density in the heart and perinatal lethality. Using cultured SMCs, we further identified that BDNF/TrkB signaling leads to cell migration and contractile force generation by actomyosin phosphorylation, to play a pivotal role in regulating pericyte morphology and migration.Here we demonstrate that TrkB is required for the development of the cardiac microvasculature, and trkb (+/−) mice  on a C57Bl/6 background were intercrossed to generate homozygous animals. For embryo analysis, the gestational age was determined by morphological criteria including limb bud, eye development, length, and weight of the embryo. The genotypes of embryos or neonates were determined using tail-derived DNA and PCR amplification as described by Jackson Laboratories. Trkb floxed (f/ftrkb) animals were generated as described previously trkb gene, which encodes the signal peptide and the first 40 amino acids of the N terminus of TrkB, was floxed. Crossing a f/ftrkb  mouse with a transgenic mouse carrying Cre driven by a smooth muscle actin promoter , generated progeny in which the expression of trkb was selectively eliminated in perivascular cells including pericytes/SMCs. The genotype of each animal was assessed using PCR of genomic DNA isolated from tail, utilizing trkB.flox forward primers 5′ TGG TGT ACT GAG CCT TCT CCA G 3′ and trkB.flox reverse primers 5′ CGA GGC TTG GAG TTG ACT GGT 3′. All animal procedures of this study were approved by the IACUC of Weill Cornell Medical College and all efforts were made to minimize animal suffering.Heterozygous Following sacrifice, embryos were immediately fixed in 3% paraformaldehyde in PBS for 18 hours. Dissected tissues were embedded in paraffin for histological analysis for hematoxylin-eosin staining, or infiltrated with 30% sucrose in PBS prior to cryoprotection in 30% sucrose:OCT  for immunohistochemical analysis. For electron microscopic analysis, embryos and newborn mice were sacrificed by decapitation and hearts were immediately removed from the chest cavity and fixed in Karnovsky’s fixative solution for 18 hours prior to embedding in Epon. One µm sections were stained with toluidine blue for initial evaluation, followed by ultra-thin sections that were counterstained with lead citrate and viewed with a JEOL electron microscope. To analyze vascular permeability, FITC-dextran  was injected as a bolus through the tail vein of 3-month-old mice. After perfusion for 10 minutes, the animals were sacrificed and hearts harvested and processed for fluorescence microscopy. FITC-dextran intensity was quantified by densitometry using ImageJ software.trkb gene was performed using PerfeCTa SYBR Green FastMix ROX (Quanta Biosciences) and the thermocycler ABI Prism 7900 HT (Applied Biosystems). At least 5 mice of each genotype were evaluated using primers that annealed to sequences in exon 5 (5′-ACTGCCTCAATGAGAGCAGC-3′) and exon 6 (5′-GGAAAGGGTCACAGACTTTCC-3′), present in all trkb transcripts. The level of expression of trkb mRNA in gene-targeted animals was calculated as the fold change relative to the expression level of trkb in the corresponding wild type littermate. Relative mRNA expression level for each sample was normalized to the actin gene and 18S ribosomal RNA as reference genes.RNA from the hearts of 3 to 6 week old mice was extracted using RNeasy Plus Mini Kit (Qiagen) according to the manufacturer’s instructions. cDNA from total RNA was generated using random hexamers and Superscript III (Invitrogen). qPCR for the mouse http://rsb.info.nih.gov/ij/) particle analysis plug-in was used to set the lower and higher size of pixels to be considered as a countable object. Both selection thresholds were arbitrarily defined, but maintained rigorously for all the images of different conditions. Finally, objects were counted in each image using the ImageJ cell counter plug-in. NG2, PDGFRβ and PECAM intensity was analyzed by densitometry. To further ensure absence of bias in object counting or densitometry analysis, sections were coded, and the operator was blind to treatment groups. Negative controls without primary antibodies were performed for each immunodetection.For postnatal tissue, 10 micron serial frozen sections were stained with NG2 antibody  or CD31/PECAM  to detect pericytes/SMCs or endothelial cells, respectively. We used isolectin B4  to identify endothelial cells and PDGFRβ  for pericytes/SMCs in embryos. The TrkB antibody was purchased from Millipore (Cat# 07–225). The collagen IV antibody was from Abcam (Cat# Ab6586). Collagen intensity was quantified by densitometry using ImageJ software. For double-immunofluorescence, frozen sections were incubated with primary antibodies overnight, followed by incubation with either Alexa 488 or 568 conjugated secondary antibodies  at room temperature and visualized using a Zeiss microscope. Fluorescence field images were captured digitally . Counts of NG2 or PDGFRβ and PECAM objects in the heart were performed after unbiased intensity and size selection criteria. A gray level threshold was selected for all the microphotographs of all different treatments and staining. After this selection, ImageJ  was added to fresh media without FBS and cells were photographed at 6, 13 and 24 h followed by area analysis of the scratch using ImageJ. The width and area of each scratch in each condition was analyzed in 7–10 random and separate fields .Human umbilical smooth muscle cells (HuSMCs) were isolated as described previously HuSMCs were plated on glass bottom dishes (MatTek Corporation) and cultured in DMEM containing 10% FBS for 24 hours. Medium was changed to serum-free DMEM overnight. Cells were then imaged in phenol red-free DMEM (Invitrogen) supplemented with 30 mM HEPES-NaOH (pH 7.4) using an Olympus IX71 inverted microscope driven by IPLab software (BD Biosciences) and equipped with a 60X PlanApoN objective (NA 1.42), a Hamamatsu EM-CCD camera and a heated stage maintained at 37°C. Images were captured every 15 seconds. Cells were treated with BDNF (100 ng/ml). For inhibitor studies, cells were pre-treated with 10 µM Y-27632  or 10 nM K-252a  for 45 minutes, or pre-incubated with 1 µg/ml C3 transferase  for 4 hours. All movies were exported and processed using IPLab (BD Biosciences) and ImageJ (NIH) software.HuSMCs were lysed in TNE buffer  and phosphatase inhibitors (Sigma)) for 30 minutes at 4°C. 100 µg of total cell lysate protein was loaded on SDS-polyacrylamide gel and proteins were resolved and transferred to PVDF membrane. The following primary antibodies were used: TrkB , AKT , phospho-AKT , ERK , phospho-ERK , MLC , and phospho-MLC  antibodies. Anti β-actin  was used as a loading control. The respective secondary antibodies (anti-rabbit or anti-mouse IgG) were conjugated with HRP. Chemiluminescence of the blots was detected using ECL Advance Western Blotting Detection kit.AATGCTCGCAAGGACTTTCAT; a kind gift of Dr. Simon S. Murray, The University of Melbourne, Australia) and a scramble control were obtained from Open Biosystems Thermo Scientific . Lentivirus was packaged by co-transfection of shRNA constructs with packaging plasmids pMD2.G and pCMV-dR8.9 using TransIT-LT transfection reagent  into HEK293FT cells. Media was changed 24 h later, and the media supernatant was collected 48 h after transfection. Supernatants were filtered through a 0.45-mm filter and pelleted by centrifugation with PEG-it . Pellets were resuspended in medium. HuSMCs were infected, 72 h later media was changed to serum free, followed by BDNF treatment (100 ng/ml). The efficacy of TrkB knockdown was assessed by Western blot.Lentiviral plasmids containing shRNA against human TrkB (targeting sequence: −/−), consistent with a prior report in utero lethality of the embryo and early fetus −/−trkb or wild type littermates during late gestation. Histological analysis of the hearts from E15.5 to E16.5 embryos showed reduced ventricular wall thickness from the −/−trkb embryos as compared to wild type littermates, most prominently in the left ventricular wall  in mice with bdnf deficiency BDNF and TrkB are expressed in the developing cardiac vascular bed with BDNF expressed by endothelial cells and TrkB expressed prominently by pericytes and vascular smooth muscle cells (SMCs) lar wall . There wlar wall . Moreove−/−trkb embryos to survive until late gestation and to form identifiable cardiac arteries, capillaries, and veins suggest that the earlier processes of vasculogenesis and sprouting angiogenesis are not greatly impaired. To establish the onset of intramyocardial vessel defects, −/−trkb embryos and wild type littermates were examined from E14.5 to P0. Intramyocardial hemorrhage in −/−trkb embryos could be detected first at E16.5  and PDGFRβ antibodies, respectively. At E17.5, PDGFRβ-positivity was significantly reduced in the left ventricular wall of −/−trkb embryos as compared to wild type littermate controls  f/fTrkb-SMCCre+ allele demonstrated 20% reduction in Mendelian inheritance  and PECAM  immunodetection to determine whether deletion of TrkB results in loss of pericytes/SMCs. At postnatal day 21, rol mice . Thus, tstaining , despitestaining .f/f-trkbSMCCre+ mice, we examined the cardiac vasculature at the ultrastructural level at postnatal day 21. In wild type mice, the endothelial abluminal surface was free of cytoplasmic extensions, and the basement membrane between endothelial cells and pericytes was intact , but we detected a significant reduction in collagen IV deposition as compared with wild type-SMCCre+ mice  in 3-month-old mice, demonstrated an increase in extracellular accumulation of the dye around the pericyte/SMC-deficient vasculature of the f/f-trkbSMCCre+ hearts . HuSMCs express full length TrkB, as shown by immunohistochemical staining  have reported that the interaction of cells with growth factors in the extracellular matrix initiates ERK1/2 signaling leading to the activation of the motility machinery by enhancing phosphorylation of myosin light chain (MLC) To characterize the mechanisms by which BDNF/TrkB activation in pericytes/SMCs modulates their morphological changes and possible fast migration, we first evaluated the potential mediators of cell migration downstream of TrkB by examining the phosphoprotein profile in SMCs using the 4G10 antibody (pan-anti-phosphotyrosine). We observed phosphorylated proteins migrating at ∼42 kDda in the BDNF-treated HuSMCs as compared to untreated cells (data not shown). Reprobing of the membrane with specific antibodies, revealed that BDNF treatment induces activation of Ras/mitogen-activated protein kinases (ERK1/2) . Klemke To determine whether BDNF regulates actomyosin contractility in HuSMCs, we investigated the phosphorylation of MLC. Immunofluorescence analyses of phospho-MLC from unstimulated cells showed uniform and diffuse immunoreactivity, whereas HuSMCs treated with BDNF exhibited significant increase in phospho-MLC detection . Analysif/f-trkbSMCCre+ mice may therefore be a consequence of impaired migration of pericytes/SMCs, leading to inadequate vascular ensheathment.Together, these findings suggest that BDNF-TrkB can promote pericyte/SMC migration via activation of actomyosin contractility involving ERK1/2 and Rho/ROCK pathways . The sigin vivo and SMCs migration in vitro. The lack of pericyte investment around endothelial cells in the cardiac capillaries and arterioles of TrkB deficient mice resulted in vascular leakage and ultimately in embryonic lethality. These studies identify a novel mechanism by which a growth factor:receptor system regulates in a paracrine manner the pericyte coverage, stability and homeostasis of the cardiac microvasculature.Coronary blood vessel development requires BDNF, as mice deficient in this growth factor exhibit apoptosis of endothelial cells in the heart, leading to vessel destabilization and myocardial hemorrhage. These data raised the hypothesis that BDNF may act as an autocrine factor on endothelial cells or in a paracrine manner to induce activation of TrkB on neighboring pericytes, to stabilize nascent blood vessels. Here we establish the dependence of the cardiac vasculature on TrkB-mediated pericyte/SMC coverage of microvessels during development pdgfb and pdgfrβ knockout mice which lack desmin-positive perivascular cells −/−trkb mice may also have a role. Indeed, a previous report showed increased Tunnel labeling in the subepicardium of TrkB deficient embryos as compared to their wild type littermates at E12.5, although the identity of the apoptotic cells was not determined −/−trkb  in trkb mRNA in the f/ftrkb-SMCre+ mice heart , Erk1/2 and MLC phosphorylation and the consequently migration of the cells. Rho GTPases and MLC have been described as key regulators of pericytes/SMC shape and contractility Dysfunction in pericyte/SMC migration has been reported in many microvascular diseases, such as hypertension and diabetic microangiopathy. Therefore, it is of great importance to better understand the mechanisms by which pericytes regulate microvessel function.Figure S1TrkB mRNA analysis in f/ftrkb-SMCCre+ animals. TrkB mRNA is markedly decreased in homozygous mice lacking trkb gene specifically in smooth muscle cells (f/ftrkb-SMCCre+) compared to wild type-SMCCre+ control littermates. Bars represent mean ± s.e.m. n = 5 animals for each genotype, including mice from 3 different litters between the ages of 3 to 6 weeks old. Statistical comparisons were made by one-way analysis of variance test. *P<0.05.(DOCX)Click here for additional data file.Table S1f/fTrkb-SMCCre+ genotype analysis. Genotype analysis of 137 offsprings from f/wtrkb-SMCCre+ heterozygous intercrosses at postnatal day 21. Numbers of animals of each genotype are shown with the correspondent percentage of the total mice alive at P21 in brackets.(DOCX)Click here for additional data file.Movie S1Effect of BDNF on HuSMCs analyzed by live imaging. Increased membrane protrusions in HuSMCs in the presence of BDNF.(AVI)Click here for additional data file.Movie S2The effect of BDNF on HuSMCs was prevented by K252a. BDNF-induced membrane protrusion in HuSMCs was inhibited by K252a.(AVI)Click here for additional data file.