Substrate mechanical properties have emerged as potent determinants of cell functions and fate. We here tested the hypothesis that different forms of endocytosis are regulated by the elasticity of the synthetic hydrogels cells are cultured on. Towards this objective, we quantified cell-associated fluorescence of the established endocytosis markers transferrin (Tf) and cholera toxin subunit B (CTb) using a flow-cytometry based protocol, and imaged marker internalization using microscopy techniques. Our results demonstrated that clathrin-mediated endocytosis of Tf following a 10-minute incubation with a fibroblast cell line was lower on the softer substrates studied (5 kPa) compared to those with elasticities of 40 and 85 kPa. This effect was cancelled after 1-hour incubation revealing that intracellular accumulation of Tf at this time point did not depend on substrate elasticity. Lipid-raft mediated endocytosis of CTb, on the other hand, was not affected by substrate elasticity in the studied range of time and substrate elasticity. The use of pharmacologic contractility inhibitors revealed inhibition of endocytosis for both Tf and CTb after a 10-minute incubation and a dissimilar effect after 1 hour depending on the inhibitor type. Further, the internalization of fluorescent NPs, used as model drug delivery systems, showed a dependence on substrate elasticity, while transfection efficiency was unaffected by it. Finally, an independence on substrate elasticity of Tf and CTb association with HeLa cells indicated that there are cell-type differences in this respect. Overall, our results suggest that clathrin-mediated but not lipid-raft mediated endocytosis is potentially influenced by substrate mechanics at the cellular level, while intracellular trafficking and accumulation show a more complex dependence. Our findings are discussed in the context of previous work on how substrate mechanics affect the fundamental process of endocytosis and highlight important considerations for future studies. Cells respond to the mechanics of their microenvironment by transducing mechanical cues to biochemical information that contributes to the regulation of their adhesion, migration and differentiation Endocytosis is the process by which cells internalize extracellular material along with surrounding fluid by engulfing part of their plasma membrane. Endocytosis can be subdivided in several categories, according to the cell machinery and components necessary Beyond providing cells with essential nutrients, endocytosis has numerous additional physiological functions. Cells exploit this process to regulate membrane receptor presentation, trafficking and control spatiotemporally signaling and thus sense their environment in vitro studies performed on cells cultured on very rigid substrates, like plastic or glass. The focus has been on the influence of biochemical parameters while biomechanical factors originating from the substrate have been neglected, even though indications exist that they could be important in the process. In particular, studies have implicated several proteins that are involved in cell adhesion and that depend on cytoskeleton contractility, as endocytosis regulators The majority of our knowledge on endocytosis to date stems from in vitroet al. concluded that endothelial cells internalized carboxylated polystyrene nanoparticles (NPs) with a diameter of 100 nm at greater numbers when adhered on stiffer gels 6 kPa but the amount of NPs/ unit cell area decreased with stiffness Very few studies to date have addressed potential effects of substrate mechanics on endocytosis regulation in vitroin vivo situation; differences in tissue elasticity, especially between healthy and pathological sites, such as in injured arteries Accordingly, we here set out to investigate whether distinct forms of endocytosis are regulated by the elasticity of the cell substrate. Beyond providing insight on cell mechanosensing, a possible correlation would provide a convenient means to regulate endocytosis and associated gene and drug delivery Previous studies were conducted with engineered nanomaterials for which the internalization pathways were not specified. They did not therefore provide insight on the intriguing possibility that some forms of endocytosis are regulated by substrate elasticity, while others not. Moreover, the analysis was based on fluorescence microscopy imaging, which is limited in respect to the number of cells analyzed and prone to biased selection during acquisition. In addition, only 2-dimensional fluorescent images of cells were analyzed, neglecting cell volume effects.In order to overcome the aforementioned shortcomings, we investigated the dependence of substrate elasticity on different forms of endocytosis by using established endocytosis markers and flow cytometry to analyze cell-associated fluorescence. This technique has the advantage of analyzing a much higher number of cells/experiment  in an unbiased fashion. Our results with specific endocytosis markers revealed that substrate elasticity in the range between 5 and 85 kPa affected initial (10 minute) clathrin mediated but not lipid-raft mediated internalization by a fibroblast cell line. At the same time, endocytosis marker accumulation over a longer incubation period of 1 hour did not show significant dependence on substrate elasticity. Interestingly, pharmacological manipulation of actomyosin contractility on cells cultured on plastic had a similar effect on both clathrin-mediated and lipid-raft mediated endocytosis after a 10-minute incubation time, but showed differential effects after 1-hour incubation, depending on the inhibitor used. The experimental protocol was next extended to different model compounds, including nanoparticles, which exhibited a elasticity-dependent cell association. Finally, experiments with HeLa cells demonstrated cell-type differences in respect to the impact of substrate elasticity on endocytosis.4×5×104 cells/gel; we therefore adapted our preparation protocol in order to form hydrogels on circular supports (PDMS or glass) ranging from 20 to 30 mm in diameter, corresponding to surface areas of 1.6 to 2.4 cm2, respectively. This way, we ensured sub-confluent conditions and minimized possible effects of mechanical influence coming from neighboring cells, directly or through the substrate We utilized fibronectin (FN)-coated, PEG-based hydrogels as substrates with tunable elasticity as recently reported from our group The REF52 fibroblast cell line was initially used to study the effect of substrate elasticity on different forms of endocytosis. We selected this cell line because we recently demonstrated that REF52 cells are responsive in the studied range of substrate elasticity in terms of adhesion and motility Tf is a marker of receptor-mediated endocytosis that requires clathrin-coated vesicle formation and internalization Cholera toxin subunit B (CTb) binds ganglioside GM1, which is present in lipid rafts on the cell membrane and is internalized in a cholesterol-dependent manner by cells In order to estimate the fraction of marker internalization, we quantified the amount of accessible Tf or CTb on the cell surface prior to flow cytometry analysis by using an antibody against AF488 that quenches its fluorescence. We first verified that the antibody quenched >90% of AF488 fluorescence at concentrations that largely exceeded the ones expected to be present on cells in our experiments . AdditioOur experimental data so far indicated that substrate elasticity in the examined range did not influence the amount of internalized Tf or CTb by REF52 cells after 1-hour incubation. Tf uptake and recycling occurs within a few minutes following its association with the TfR Overall, our results so far indicate that the kinetics of Tf internalization by REF52 cells is regulated by substrate elasticity in the examined range of elasticity values, but cellular association of Tf is equalized after 1-hour. On the other hand, lipid-raft mediated endocytosis does not appear to depend on substrate elasticity in REF52 cells under the experimental conditions studied.Substrate elasticity regulates stress fiber formation and associated cell contractility, with cells on softer substrates being less contractile Association of Tf and CTb after a 10- or 60-minute incubation, in the presence or absence of inhibitors was quantified using flow cytometry as above. After 10 minutes, a significant decrease of Tf association was observed in both Y27632-treated cells (15%) and blebbistatin-treated cells (22%) . Tf uptaIt is important to note that the distinct, time-dependent effect for the two inhibitors point towards discrete mechanisms of regulation of endocytosis and intracellular trafficking. Altered Tf recycling induced by blebbistatin could explain the higher accumulation of Tf within cells after 1 hour, since under control conditions TfRs are expected to recycle several times to the plasma membrane in this time frame versus cells treated with Y27632 or blebbistatin -DNA complexes by mMSCs after 2-hour incubation was inhibited by Y27632 to a small but significant extent (approximately 8%) bistatin . There wbistatin . TherefoStudies investigating the effect of blebbistatin on different forms of endocytosis reported contradictory results. Samaniego et al. showed a decrease in Tf uptake (5 and 10 min incubation) by blebbistatin-treated T lymphocytes, similar to the one observed after Y27632 treatment as discussed above Interestingly, the scattering properties of cells treated with contractility inhibitors as determined by flow cytometry were significantly different from those of untreated cells. Both signals from the forward scatter detector (FSC) and the side scatter detector (SSC) significantly increased for Y27632-and blebbistatin-treated REF52 cells . The FSCIt is interesting to note that the value of SSC increased for cells cultured on the intermediate and stiff substrates compared to the soft ones . This re1) a high molecular weight  fluorescent dextran that does not have a specific extracellular receptor and therefore served as a non-specific pinocytosis marker and 2) carboxylate-modified, fluorescent, latex nanoparticles (NPs) with a diameter of 100 nm that served as model drug delivery carriers.We next decided to extend our experimental procedure to study cell association of two different model compounds by REF52 cells. We studied cell association after 1-hour incubation of The amount of associated dextran was low compared to association of specific markers and therefore a high concentration (250 µg/ml) of dextran was required for the uptake experiments. Flow cytometry analysis revealed a lack of dependence on substrate elasticity after 1-hour incubation, indicating that non-stimulated extracellular fluid uptake was not regulated by this parameter under our experimental conditions .On the other hand, uptake of NPs depended on substrate elasticity as indicated by the lower mean fluorescent intensity (MFI) of cells cultured on the softer gels . This fiNext, we asked whether transfection efficiency using a commercially available transfection agent (Promofectin) was dependent on substrate elasticity. Previously, it was suggested that transfection efficiency is regulated by substrate elasticity Finally, we asked whether our results on the association of the specific markers Tf and CTb as a function of substrate elasticity on endocytosis were valid for other cell types. HeLa is a human cancer cell line, widely used in pharmacological and drug delivery studies, and was used here for this purpose First, the analysis of a large number of cells using flow cytometry revealed major differences in homogeneity of uptake depending on the marker type. Consequently, it is important to analyze many cells in order to avoid artifacts associated with small sample sizes. Second, in order to distinguish between internalization and accumulation of a particular endocytosis marker, which can depend on additional processes such as intracellular trafficking and exocytosis, early time points for analysis should be selected. Third, the use of quenching antibodies provided in a reproducible manner an estimation of the surface-exposed fluorescent markers, and therefore the internalized fraction. Fourth, flow cytometry allowed us to examine the correlation between cell-associated fluorescence and cell size, as expressed by the FSC signal. We observed a weak correlation between these two parameters for both Tf and CTb association  more efficiently on stiffer FN-coated hydrogels (40 and 85 kPa) compared to softer ones (5 kPa). However, intracellular accumulation of Tf after longer exposure (1 hour) did not depend on substrate elasticity. Accordingly, internalization of fluorescent NPs, previously shown to be internalized through the clathrin-mediated pathway, showed a similar dependence on substrate elasticity. On the other hand, association of CTb or high MW dextran molecules was unaffected by substrate elasticity in the examined range, indicating that different forms of endocytosis are not regulated in the same manner by the substrate. Pharmacological inhibition of actomyosin contractility had significant effects on uptake of Tf, depending on inhibitor type; treatment with blebbistatin initially inhibited its uptake, while at longer time points an accumulation of Tf inside blebbistatin-treated cells was observed compared to control cells. This finding suggested that myosin II activity affects both internalization and recycling kinetics and it remains to be seen whether the phenotype recorded on the softer gels is linked to a decrease in myosin II activity. Finally, the experimental data obtained with HeLa cells showed independence of Tf association on substrate elasticity, indicating that cell type specific differences exist in this respect. Overall, the results presented here suggest that substrate elasticity can potentially influence distinct endocytosis pathways and cargo internalization and that the approach we have introduced is suitable for future studies on the endocytosis of more specialized drug delivery systems or specific receptors as a function of substrate mechanics.PEG-based hydrogels were prepared via Michael-type addition, cross-linking reaction of 4-arm PEG precursors functionalized with vinyl sulfone groups (PEGVS) or thiol groups (PEGSH) as previously described 2 in a humidified atmosphere. REF52 cells were serum-starved overnight (12–16 hours) prior to seeding on FN-coated gels. For cell seeding, cells were detached with accutase for 5 minutes, centrifuged and the cell pellet was resuspended in non-supplemented DMEM at the desired cell concentration. After 30 minutes of incubation on top of substrates, non-adhered cells were removed by aspiration and supplemented DMEM added. HeLa cells were seeded in the same manner but were incubated in serum-free medium only 1 hour prior to seeding.A rat fibroblast cell line (REF52) 1) 10 µg/ml transferrin (Tf) from human serum, alexa fluor 488 conjugate (Life Technologies) or 2) 1 ng/ml recombinant cholera toxin subunit B, alexa fluor 488 conjugate (Life Technologies). For non-specific pinocytosis cells were incubated for 1 hour with 250 µg/ml dextran , Oregon Green 488 conjugate (Life Technologies). Nanoparticle association studies were performed using commercially-available, fluorescent, carboxylate-modified poly(styrene) nanoparticles (NPs)  of 100 nm in diameter. Before addition to cells, NPs were sonicated for 5 minutes and diluted 1∶1000 to a concentration of 0.02 mg/ml. Cells were incubated for 1 hour with the NPs. For inhibition studies, the following inhibitors were added 30 minutes prior to marker addition: 1) 10 µM Y27632 (Sigma), 2) 50 µM blebbistatin (Sigma).The amount of associated fluorescence per cell was quantified using flow cytometry. Cells seeded on FN-coated gels or TCPS were washed once and incubated in DMEM without serum 30 minutes before each experiment. For measuring association of specific markers, cells were incubated for 10 minutes or 1 hour in non-supplemented DMEM with After incubation with markers or NPs, cells were washed three times with ice-cold PBS and detached from gels using trypsin-EDTA (5-minute incubation at 37°C). Cells were centrifuged  and the cell pellet resuspended in 0.25 ml PBS; cell suspension was kept under ice until flow cytometry analysis (20–60 min post-trypsinization). For quenching of extracellular fluorescence, an anti-Alexa Fluor 488 quenching antibody  was added to the cell suspension for at least 15 min prior to flow cytometry analysis.Flow cytometry was performed on a BD FACSCanto flow cytometer. A minimum of 5,000 events in a gate of live cells was recorded per sample . Due to variations in absolute values between different experiments we normalized values as following: first, the mean fluorescent intenstity (MFI) of non-treated cells was subtracted from the MFI of the sample; then the average MFI value of all samples for a given experiment was calculated and the value of each sample was normalized to this average value. Polystyrene beads of different sizes (Polysciences Inc.) were used to correlate the forward scatter signal (FSC) to particle diameter.Phase contrast and epifluorescence imaging were performed using a Delta Vision system (Applied Precision Inc.) on an Olympus IX inverted microscope equipped with a cooled CCD camera. For high magnification images a 60×/1.3 NA (Olympus) oil-immersion objective was used. Epifluorescence microscopy was additionally performed using a Leica DM6000B upright microscope equipped with a CCD camera. A water immersion objective 40×/0.8 NA (Leica) was used. Laser scanning confocal microscopy (LCSM) was performed on a Zeiss LSM 5 microscope using a 40×/1.2 NA (Zeiss) water immersion objective.Cell Outliner plugin of ImageJ software (NIH) from fluorescence microscopy images of WGA-stained cells.Cells were fixed with 4% paraformaldehyde in PBS (15 minutes in PBS) and stained with fluorescent agents. Wheat germ agglutinin, AlexaFluo 488 conjugate  was used to stain plasma membrane, phalloidin-tetramethyl rhodamine B isothiocyanate  stained filamentous actin and 4′,6-diamidino-2-phenylindole  labeled cell nuclei. Projected cell area was calculated using the Cells were cultured on top of FN-coated gels or glass for 24 h prior to transfection. A plasmid for transient transgene expression of green fluorescent protein (GFP) was purchased from Promokine [pPK-CMV-R4 Receptor Vector (GFP)]. Promofectin (PromoKine) was used to transfect cells according to manufacturers' protocol. Briefly, the plasmid (1∶20 in DMEM) was mixed with promofectin (1∶10 in DMEM) and incubated for 15 min at room temperature. Next the transfection complex was added (1∶70) to a glass-bottom dish containing multiple gels and a control glass surface in supplemented DMEM. In this way, the amount of available DNA/cell was the same for the different conditions in each experiment. The percentage of transfected cells was analyzed by immunofluorescence microscopy by dividing the number of GFP-positive cells to the total number of cells. Results of transfection efficiency on gels are normalized in respect to that on glass, since absolute values varied considerably between experiments.All statistical analyses were performed using the software Prism (GraphPad Inc.). Experiments of cell association on hydrogels of differing elasticity were statistically analyzed using the Tukey test, which compares all pairs of columns. Comparisons between control and samples treated with contractility inhibitors or quenching antibody were analyzed using an unpaired t-test. Only statistical significant differences are presented in graphs with p values <0.01, <0.05 and <0.001 represented as *, ** and ***, respectively.Figure S1Mechanical characterization of PEG hydrogels using AFM. (A) Young's moduli of three different gel formulations determined by AFM force spectroscopy using a spherical glass tip (diameter 10 µm) and the Hertz model to analyze the force-distance curves. The average values of 3 independent experiments  and the standard deviation are presented. (B) A surface area of 100 µm ×100 µm on gels was probed to evaluate homogeneity of elasticity. 100 points were analyzed  and 5 force curves/point were analyzed to yield the Young's modulus of that point. Data are presented as color-coded surface plots and reveal variations 5–10% among values.(PDF)Click here for additional data file.Figure S2Internalization of Tf by REF52 cells is uniform and qualitatively similar between gels of differing elasticity. Epifluorescence microscopy images (multiple stitched fields) of REF52 cells on PEG hydrogels of varying stiffness, incubated for 1 h with Alexa Fluor 568-conjugated Tf and plasma membrane stained with WGA. Homogeneous uptake of Tf by REF52 cells and intracellular localization was noted for all values of elasticity investigated. Scale bars: 50 µm.(TIFF)Click here for additional data file.Figure S3Internalization of CTb by REF52 cells is heterogeneous among the cell population. Epifluorescence microscopy images (multiple stitched fields) of REF52 cells on PEG hydrogels of varying stiffness, incubated for 1 h with Alexa Fluor 568-conjugated CTb and plasma membrane stained with WGA. The extent of CTb association with REF52 cells varied considerably between cells. However, the pattern of association was similar between hydrogels of differing elasticity for all values of elasticity investigated. Scale bars: 100 µm.(TIFF)Click here for additional data file.Figure S4Estimation of extracellular marker fraction using anti-alexa fluor 488 (anti-AF488) quenching antibody. (A) Quenching kinetics and efficiency of the anti-AF488 antibody (10 µg/ml) on a 5 nM (0.4 µg/ml) solution of AF488-Tf showed a maximal 90% quenching of fluorescence within 10 minutes of mixing. The antibody concentration used is the same as that used in cell experiments while AF488-Tf concentration is much higher compared to that on cell-associated Tf or CTb, as estimated by fluorescence measurements. (B) MFI of REF52 cells treated with anti-AF488 normalized to MFI in its absence. Incubation of REF52 cells with the quenching antibody on cells cultured on FN-coated TCPS revealed a 11% decrease in Tf MFI and 38% decrease in CTb MFI, indicating that approximately 90% of Tf and 60% of CTb are internalized (mean and standard deviations of at least 3 samples and 2 independent experiments). Substrate elasticity did not affect the fraction of internalized markers (n = 1). (C) The effect of Y27632 and blebbistatin treatment on the extracellular fraction of Tf was evaluated using anti-AF488. The same fraction of extracellular Tf was recorded independent of cell treatment. Mean and standard deviations are shown of 3 samples.(TIFF)Click here for additional data file.Figure S5Rho kinase inhibition with Y27632 did not alter intracellular fluorescence pattern of Tf or CTb on REF52 cells. Confocal microscopy images of REF52 cells on FN-coated glass after 1-hour incubation with AF568-labeled markers, fixation and staining with WGA-AF488. Tf was internalized at similar numbers by cells and mainly localized at a perinuclear site, independently of Y27632 treatment (upper row), while CTb showed heterogeneous uptake efficiency among the cell population that was also independent of Y27632 treatment (lower row).(TIFF)Click here for additional data file.Figure S6Blebbistatin treatment of REF52 cells affects Tf internalization and recycling kinetics. MFI of REF52 cells incubated with Tf at different time points on FN-coated plastic in the presence or absence of 50 µM blebbistatin. At short incubations blebbistatin inhibits Tf association by cells, while at longer time points the amount of Tf is enhanced compared to control conditions. The quasi-linear increase of MFI per cell indicates that blebbistatin has an effect of intracellular trafficking and recycling of Tf. Each data point represents the average of two experiments.(TIFF)Click here for additional data file.Figure S7The SSC signal but not the FSC signal of REF52 cells depends on the elasticity of the substrate they were cultured on. Flow cytometry analysis of REF52 cells cultured on gels did not show a dependence of their FSC signal (A), while cells on soft gels showed a significantly lower SSC signal compared to cells cultured on intermediate or stiff hydrogels (B). Values from at least 4 independent experiments are presented with the value from each gel represented by a single dot and the mean value a solid line.(TIFF)Click here for additional data file.Figure S8Transfection efficiency of HeLa or REF52 cells is not significantly affected by substrate elasticity. (A) Typical overlaid optical and fluorescence microscopy images of HeLa cells on FN-coated hydrogels of different elasticity used to calculate transfection efficiency. Insets show micrographs of the fluorescence channel (GFP). Scale bars: 50 µm. Transfection efficiency of (B) HeLa or (C) REF52 cells normalized to the value obtained on FN-coated glass. A non-significant decrease of the fraction of transfected cells with increasing stiffness was noted. Mean and SEM values from at least 3 gels and two independent experiments are presented .(TIFF)Click here for additional data file.Figure S9Cell density of HeLa cells does not depend on substrate elasticity. Inverted fluorescence microscopy images (stitched tiles) of HeLa cells on soft, intermediate and stiff hydrogels, stained with WGA. The amount of cells adhered was similar on all hydrogels, independent of their elasticity. Scale bars: 200 µm.(TIFF)Click here for additional data file.Figure S10Tf or CTb association with cells is very weakly correlated to cell size. Dot plots of cell-associated fluorescence as a function of apparent cell diameter for Tf (left) or CTb (right) association. Data from 5 random experiments performed on cells on top of gels or glass are presented, along with the calculated Pearson's correlation coefficient.(TIFF)Click here for additional data file.


Accumulating evidence suggests that the efficacy of ACT in melanoma is mediated by T cells that target somatic mutations expressed by the patients' tumors. Moreover, in one patient with metastatic epithelial (bile duct) cancer, we recently identified tumor-infiltrating Th1 cells that specifically recognized a mutation in ERBB2IP expressed by the patient's tumors and observed tumor regression after adoptive transfer of a highly enriched population of ERBB2IP mutation-specific Th1 cells. Thus, we hypothesize that mutation-specific T cells may be frequently elicited in patients with metastatic gastrointestinal (GI) cancers and may be harnessed for ACT. To test this, we first performed whole-exome sequencing on metastatic lesions from GI cancer patients to identify the mutations. Next, we generated mingene constructs that encoded each mutation and transfected these minigenes into autologous antigen presenting cells (APCs) to allow for the processing and presentation of all the mutations expressed by the tumor. These APCs were then co-cultured with tumor infiltrating lymphocytes (TIL) and T cell reactivity against the mutations was determined by IFN-γ ELISPOT and 4-1BB and OX40 upregulation by flow cytometry. In one patient with colon cancer, 119 mutations were evaluated for mutation reactivity. Several, but not all, TIL cultures were found to contain highly variable proportions of CD8+ T cells that specifically recognized a mutation in CASP8 (67 F→V). Intriguingly, upon further expansion in vitro, these mutation-reactive CD8+ T cells were markedly outgrown by other cells in culture. In another patient with rectal cancer, 155 mutations were evaluated for mutation reactivity. We found at least 3 different mutation reactivities, two comprising CD8+ T cell responses and one CD4+ T cell response. In a third patient (cholangiocarcinoma), we did not detect T cells reactive against 38 mutations tested. For this patient, the "mutation call" threshold has been lowered and an additional 125 putative mutations will be evaluated. Thus, our preliminary data suggests that the ability of the human immune system to mount a T cell response against somatic mutations in metastatic GI cancers may not be a rare event. Current efforts are focused on evaluating more patients for mutation-specific T cell responses and on developing methods to enrich mutation-specific T cells or T cell receptors for therapy, which may provide a path to extend cell transfer immunotherapy to almost all common solid cancers.Written informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.


In conclusion, our study proves that the paravascular pathway dynamically mediates the pathological complications following acute SAH independently of glymphatic control.Subarachnoid hemorrhage (SAH) is a devastating disease with high mortality. The mechanisms underlying its pathological complications have not been fully identified. Here, we investigate the potential involvement of the glymphatic system in the neuropathology of SAH. We demonstrate that blood components rapidly enter the paravascular space following SAH and penetrate into the perivascular parenchyma throughout the brain, causing disastrous events such as cerebral vasospasm, delayed cerebral ischemia, microcirculation dysfunction and widespread perivascular neuroinflammation. Clearance of the paravascular pathway with tissue-type plasminogen activator ameliorates the behavioral deficits and alleviates histological injury of SAH. Interestingly, AQP4 Recently, several studies have shown that the brain has in fact the proper lymphatic system, including sinus-associated lymphatic vessels and the glymphatic system (GS).15 Sinus-associated lymphatic vessels express all of the molecular hallmarks of lymphatic endothelial cells, contain cerebrospinal fluid (CSF) and immune cells, and drain into the deep cervical lymph nodes.13Previous studies have shown that the blockade of cerebral lymphatic drainage deteriorated the secondary cerebral ischemia after SAH, suggesting that the cerebral lymphatic drainage pathway could be involved in the pathological mechanism of SAH.16 The GS is a specialized brain-wide anatomic structure locating at the PVS surrounding the brain vasculature, which is ensheathed with the astroglial endfeet and astroglial water channel aquaporin-4 (AQP4).15 The GS facilitates the efficient lymphatic clearance of extracellular solutes and fluid in the brain through astroglial-mediated interstitial fluid bulk flow.14There is a histologically defined space in the brain, the Virchow–Robin space, where the subarachnoid space meets the paravascular space .17 ischemic stroke18 and aged brain.19 Interestingly, brain imaging study with magnetic resonance imaging reported weakened GS perfusion following acute stroke or SAH.20 However, little is known about whether the GS is involved in the secondary complications of SAH. Here, we examined the potential involvement of GS in SAH-associated pathology progression with in vivo two-photon microscopy and CLARITY technique.22 Our data showed that subarachnoid blood flowed into the brain parenchyma rapidly through the PVS, causing CVS, vasculitis, widespread microinfraction and neuroinflammation in the animal model of SAH and SAH patients. Prevention of CVS with Fasudil23 did not improve the neurological impairment nor alleviated the pathology, while the PVS clearance with tissue-type plasminogen activator (tPA) infusion improved the behavioral recovery and reduced neuroinflammation in the brain. Interestingly, AQP4−/− mice showed no improvements in neurological deficits and neuroinflammation at day 7 after SAH compared with WT control mice. Our study therefore suggested that the paravacular pathway dynamically mediates the pathological complications following acute SAH independently of glymphatic control.Impairment of GS involves neurological conditions including traumatic brain injuries,2 region over the target hemisphere), the decompressive thinned-skull window was generated over both hemispheres and much larger in area (4 × 4 mm2). The injection of blood or artificial CSF (aCSF) resulted in an immediate increase in ICP  was considered closely related to early brain injury, decrease of cerebral blood flow (CBF) and global cerebral ischemia after SAH. To control the SAH-induced elevated ICP, we performed a decompressive (DC) thinned-skull window over both hemispheres before SAH as shown in in vivo imaging, physiological parameters  did not differ between SAH and sham-operated animals, indicating comparable physiological conditions in all investigated animals  into cisterna magna . We thenTo confirm that natural serum proteins could also enter the parenchyma through the paravascular pathway, we labeled fresh arterial blood with albumin-FITC for injection into the cisterna magna. Intensive FITC signal was found in the parenchyma within 30 min after injection of albumin-FITC  and its To histologically confirm the entrance of blood components into brain parenchyma through PVS, we applied the CLARITY technique to the mouse brain after SAH to reconstruct a 3D pattern of PVS-mediated diffusion and found that at 1 h after SAH, a large quantity of fibrinogen was deposited on the outer wall of penetrating vessels and their collaterals . Immunosα, IL-1β and MCP-1 were remarkably increased in the perivascular parenchyma at day 7 after SAH , but showed no effects on the arterioles with the diameter less than 30 μm , we confirmed that SAH could induce pial arterioles spasm . A previtriction . To evalan 30 μm . Moreovean 30 μm . These rin vivo.The live imaging showed that blood cells aggregated to form blood clots within the paravascular space at 2–3 min after SAH . We ther−/− mice, when compared with WT control mice  scan images of SAH patients who received the diagnosis and treatment in the First Affiliated Hospital of Sun Yat-Sen University in the past 2 years. Using unenhanced CT scan, CT angiography (CTA) and contrast-enhanced CT scan, we found the high-density areas were almost overlapped on these CT scan images in a set of SAH patients , suggestWhen we retrospectively analyzed the CT scan images of SAH patients, we have identified some SAH patients with no early CVS , however, showing infraction sites on day 6  . These d26 Among these devastating complications, DCI is an important cause for poor prognosis of SAH.2 It is generally believed that the toxic effects of blood in the subarachnoid space after SAH are the key factor leading to DCI.32 In the present study, we found that, in SAH animal models, subarachnoid space blood quickly enters the paravascular pathway, with its components efficiently diffusing into the brain parenchyma. The rapid paravascular pathway of blood components entry into the brain parenchyma resulted in extensive perivascular neuroinflammation and microcirculation dysfunction throughout the brain. We also confirmed the distribution of blood components in the PVS in SAH patients by CT scan images and histological examinations. Thus, paravascular pathway plays a key role in mediating both the acute and delayed pathological complications following SAH.SAH can cause focal or generalized brain dysfunction, including increased ICP, cerebral edema and swelling, blood–brain barrier damage, reduction in CBF, CVS, acute cerebral ischemia, acute and chronic hydrocephalus and DCI.7 However, clinical trials targeting CVS over the past decades fail to show the desired effect.7 We found that Fasudil administration was effective in reducing the incidence and severity of large-artery (diameter of 40–80 μm) vasospasm, but failed to the arterioles diameter less than 30 μm, meanwhile failed to improve the neurological deficits and alleviate the neuroinflammation in SAH animals. In addition, it has been proposed that DCI probably results from microcirculation dysfunction after SAH.9 SAH-induced elevated ICP often caused intracranial circulatory decrease and was considered closely relation to microcirculation dysfunction.9 In present study, we performed the bilateral decompressive thinned-skull window over both hemispheres to relatively control the SAH-induced elevated ICP. However, we still observed disrupted microcirculation and even the formation of microthrombi in the capillary network in the brain as early as 6 h after SAH, suggesting that a more complex mechanism of pathogenesis may be involved in microcirculation dysfunction.It is commonly believed that CVS occurring at 4–10 days after SAH is the main reason for DCI, and that the reversal of CVS can improve prognosis of DCI.33 In our SAH animal study we detected extensive perivascular neuroinflammation throughout the brain as revealed by pronounced activation of microglial cells and upregulation of TLR4, TNF-α, IL-1β and MCP-1 in the perivascular parenchyma. Consistently, other studies reported that there was an increased expression of TLR4 in both SAH patients and animals.35 TLR4 is a pattern recognition receptor and can be activated by exogenous pathogenic microorganisms and foreign matters when passing through the blood–brain barrier.38 Its activation subsequently triggers the activation of nuclear factor-κB and induces the expression of inflammatory factor.39 We therefore propose that blood components and their degradation products entering the perivascular parenchyma via the paravascular pathway activate TLR4 of microglial cells, which triggers the subsequent inflammation cascade after SAH.Microcirculation dysfunction could result from perivascular neuroinflammation in the brain.41 Under normal circumstances, macromolecules are unable to pass through the AQP4 water channel expressed on the endfeet of astrocytes.14 In the present study, we found that FITC-d2000 that normally is confined in the PVS could enter the brain parenchyma after SAH, suggesting that SAH may change the PVS permeability and lead to more molecules to perfuse into the brain parenchyma. Using AQP4 knockout mice, we found that the genetic deletion of AQP4 significantly reduced and retarded blood moving into the brain parenchyma along the PVS. However, substantial decrease in blood diffusion from the PVS into the brain parenchyma did not alleviate neuroinflammation nor improve the neurological deficits after SAH. This indicates that the paravascular pathway may mediate vasculitis and neuroinflammation after subarachnoid hemorrhage independently of glymphatic control.AQP4 is the major water channel in the nervous system, and has been recognized as the critical functional component of the GS.42 Future work needs to further investigate whether sinus-associated lymphatic vessels are involved in vasculitis and neuroinflammation after SAH.The recently discovered sinus-associated lymphatic vessels provided a conventional path for immune cells to exit the CNS, which performed the function of immune surveillance and might involve in diseases as diverse as the CNS infection and immune demyelination.18 and ameliorate cortical circulation.20 Furthermore, clinical studies confirmed that the intrathecal injection of thrombolytic agents can reduce the incidence of DCI and improve its prognosis.43 Therefore, we propose that tPA application to CSF after SAH can improve the GS function and thus promote the clearance of unfavorable products in the brain tissue, thus significantly alleviating both the histological and the behavioral impairment after SAH.In the present study, we also found that the paravascular pathway would be blocked due to the formation of blood clots in the PVS after SAH. Administration of tPA after SAH significantly improved the GS clearance and alleviated neuroinflammation. In line with this, previous studies reported that application of tPA could significantly improve the GS dysfunction due to SAHIn conclusion, our study confirms that the paravacular pathway plays important roles in mediating pathological complications following SAH, including CVS, DCI, parenchymal arterial inflammation, microcirculation dysfunction and widespread perivascular neuroinflammation independently of glymphatic control . The stu−/− (Aqp4-null) breeders from Dr. Gang Hu  and raised them in the Laboratory Animal Center, Sun Yat-Sen University. Mice were housed in a temperature-controlled, 12 : 12 light/dark room and were allowed free access to water and food.All animal protocols were approved by the ethical committee of the University of Macau. Unless otherwise noted, C57BL/6 mice weighing between 20 and 25 g and aging between 8 and 12 weeks were used in this study. We obtained Aqp4in vivo two-photon microscopy, unless otherwise noted, the thinned-skull window over both hemispheres  was performed using a dental drill to a total skull thickness of approximately 20–30 μm in all experimental animals before SAH induction  and xylazine . To decrease the elevated ICP induced by SAH and image with nduction . ICP wasn=13, 5 mice for immunofluorescence analysis, 3 mice for CLARITY analysis and 5 mice for western-blot analysis); (2) SAH induced by injection of fresh unheparinized arterial blood ; (3) the PVS permeability group that received either TITC-d2000 (n=3) or albumin-FITC (n=3); (4) the tPA-treated group that was subdivided into control  and tPA treatment ; (5) the Fasudil group that was subdivided into control  and Fasudil treatment ; (6) the AQP4 group that was subdivided into WT (n=5) and AQP4−/− (n=5); and (7) the SAH model induced by femtosecond laser injury (n=3).Sixty-five animals were assigned randomly to the following seven experimental groups: (1) Sham-operated group  to rupture the pial arteriole. For details, see μm (single stacks) to a depth of 250 μm in a XYZ order (512 × 512 pixels). For details, see Thinned skull window was prepared, and a Leica SP5 two-photon imaging system  equipped with a Ti:Sapphire laser (Coherent Chameleon Ultra II), × 25/0.95 NA water-immersion objectives and Leica LAS X software was used to image the vasculature. Dynamic imaging was captured with 342 ms intervals using a XYT order for 1 h. Stacks of images were acquired using a step size of 1.0 Mice brain after perfused with formaldehyde acrylamide hydrogel were extracted for CLARITY processing. First, the brain was incubated in hydrogel monomer solution at 4 °C for 6 h and then in hydrogel monomer solution without 4% PFA at 4 °C for 3 days. The brain was then embedded in polymerized hydrogel at 37 °C for 3 h and cut into 2 mm-thick coronal sections with a mouse brain matrix. Clarification was completed by incubation in a solution of 8% (wt/vol) SDS (Sigma) in 0.1 M PBS (pH 7.5) at 37 °C for 2–3 weeks, followed by washing twice for 1 day in 0.1 M PBS+0.1% Triton X-100 . For immunostaining and other details, see We retrospectively analyzed 24 cases of hospitalized aneurysmal SAH patients who simultaneously underwent the examination of unenhanced cerebral CT scan, cerebral CTA and contrast-enhanced cerebral CT scan on admission from April 2013 to April 2015 in the First Affiliated Hospital of Sun Yat-Sen University. The diagnosis of SAH was based on the patient's medical history and clinical manifestation and verified by an examination of unenhanced cerebral CT scan. Among these 24 cases, there were 13 males and 11 females, with ages ranging from 19 to 68 years old. For details, see For human brain tissue sample data, the brain sample of SAH was isolated from a SAH patient  who received intracranial aneurysm clipping with right pterional approach on day 1 after SAH at Tangdu Hospital, Xi'An. The brain sample of control was isolated from a drug-resistant mesial epilepsy patient  who received temporal lobectomy on 27 March 2015 at Tangdu Hospital, Xi'An. This part of the study complied with the guidelines of the Declaration of Helsinki and was approved by the Human Ethics Committee of Tangdu Hospital, Xi'An, China. The written informed consent was obtained from the subject. The brain tissue sample was then proceeded for HE staining.t-test. Differences in neurological scoring and western-blot analysis were evaluated by one-way ANOVA followed with Tukey's post hoc test for multiple comparisons. P<0.05 was considered to be statistically significant.All the data were presented as the mean±S.E.M. Statistical analysis was performed with SPSS 17.0 . Differences in perivascular Iba-1 and GFAP immunofluorescence were compared by an unpaired


These analyses quantify temporal dynamics of algal-derived LCEFA and food quality in a freshwater ecosystem that has undergone large community changes as a result of shifting resource management practices, highlighting diatoms, cryptophytes and dinoflagellates as key sources of LCEFA. Moreover, the analyses indicate that future shifts towards cyanobacteria-dominated communities will result in lower LCEFA content in aquatic ecosystems.Essential fatty acids (EFA), which are primarily generated by phytoplankton, limit growth and reproduction in diverse heterotrophs. The biochemical composition of phytoplankton is well-known to be governed both by phylogeny and environmental conditions. Nutrients, light, salinity, and temperature all affect both phytoplankton growth and fatty acid composition. However, the relative importance of taxonomy and environment on algal fatty acid content has yet to be comparatively quantified, thus inhibiting predictions of changes to phytoplankton food quality in response to global environmental change. We compiled 1145 published marine and freshwater phytoplankton fatty acid profiles, consisting of 208 species from six major taxonomic groups, cultured in a wide range of environmental conditions, and used a multivariate distance-based linear model to quantify the total variation explained by each variable. Our results show that taxonomic group accounts for 3-4 times more variation in phytoplankton fatty acids than the most important growth condition variables. The results underscore that environmental conditions clearly affect phytoplankton fatty acid profiles, but also show that conditions account for relatively low variation compared to phylogeny. This suggests that the underlying mechanism determining basal food quality in aquatic habitats is primarily phytoplankton community composition, and allows for prediction of environmental-scale EFA dynamics based on phytoplankton community data. We used the compiled dataset to calculate seasonal dynamics of long-chain EFA (LCEFA; ≥C We also identified additional potential datasets using a combination of prior knowledge of the literature and through tracing the citations of other key studies in the field. The justification for this varied approach is that phytoplankton fatty acid profiles are reported throughout a diverse literature with very different original study goals. As a result of this literature search we screened 399 papers, including phycological surveys and experimental trophic studies. We built a new fatty acid database from the published data from 58 of these studies , which f-2 s-1), hours of light per day, temperature (°C), salinity , and a single binary indicator of nutrient status or growth phase . We therefore assumed that cultures in exponential growth were not nutrient limited and that ‘stationary’ cultures were nutrient limited. We standardized the various units for each measurement or environmental condition used by the original authors into consistent units , and fatty acids as a percentage of algal dry weight (FA % DW). The majority of the fatty acid profiles (n = 730) analyzed here were either originally reported as % FA, or were converted to this format from the original concentration units used by the original authors. Because the absolute quantity of particular fatty acids may be the ideal format to consider from a food quality perspective ,52,53, oWe used principal components analysis (PCA) to visualize arcsine-square root transformed multivariate fatty acid signatures of the six major algal taxonomic groups see  in the lX matrix) on a multivariate dataset , in the form of a Euclidean distance matrix. The explanatory matrix consisted of the raw continuous variables  and two groups of categorical variables (taxonomic group and nutrient status), which were coded as a series of individual binary variables and grouped as common indicator variable ‘sets’ prior to running DISTLM in PERMANOVA+ for Primer [We used a distance-based linear model (DISTLM ), an extr Primer .R2 as the selection criterion, and report results of marginal tests as well as total variation explained by each variable. The full model is visualized with a dbRDA, which shows results of fitting the model first to the most important variable and sequentially partitioning the remaining unexplained variation among remaining variables.The DISTLM was run on each of two distinct arcsine-square root transformed fatty acid datasets (% FA and FA % DW), using the same six major algal taxonomic groups . Only prWe calculated the sum of the long-chain essential ɷ-3 and ɷ-6 fatty acids (LCEFA); defined here as docosahexaenoic acid , eicosapentaenoic acid , and arachidonic acid  for both the FA % DW and % FA datasets. We performed post-hoc correlations  between this univariate fatty acid summary category and each of continuous culture condition variables that explained more than 5% of the variation in the full fatty acid dataset DISTLM marginal tests.i compared to the maximal LCEFA content of all AG:We defined a fatty acid-based food quality index (FQI) based on the composition of ɷ LCEFA at the algal group level . We firsi) is the average Σ LCEFA of AGi divided by the maximum Σ LCEFA of all groups. We applied this calculation to both % FA and FA % DW datasets to compare the relative LCEFA ranking among phytoplankton groups between the two fatty acid data types that are most commonly reported in the literature.Where the FQI for each algal group  of the total biomass (μg C L-1) of AGi in the field multiplied by the average fatty acid content of AGi. The AGi FA % DW data is first divided by 100 to express it as a proportion before it is multiplied by the AGi biomass.Where, for each fatty acid category , the total calculated μg FA L2, mean depth ca. 33 m), seasonally stratified lake [-1) was calculated from the original reported biovolume data (μm3 mL-1) following equations for diatoms and non-diatom phytoplankton reported in ref. [-1 divided by the total calculated μg ɷ-6 L-1 from Daphnia galeata), and ≥5 (Daphnia magna)  , as a cied lake , which hied lake ,65,66). ied lake , indicatied lake . Average in ref. . The moniewed in ).F5 = 95.16, p = 0.0001) by their multivariate fatty acid profiles . There was slight negative but non-significant relationship between LCEFA % DW and temperature  and the most important culture condition variables from the DISTLM marginal tests, identified negative correlations between LCEFA % DW and both hours light  compared with the clear-water years (~10–40 μg C L-1). The analyses of calculated LCEFA show that despite major differences in community biomass and peak biomass timing between these phases, diatoms and cryptophytes are the primary drivers of LCEFA attributable to algae in both phases (-1) relative to the eutrophic phase (0.195 μg LCEFA L-1), and the timing of maximal LCEFA content was ca. 1 month earlier during the clear-water phase (April) compared to the eutrophic phase  or ‘nurture’ (environment) for algal fatty acids at an unprecedented scale, encompassing many permutations of culture conditions for many variables and more than 200 unique species. Our results clarify that the primary underlying mechanism determining fatty acid contents in aquatic habitats in nature is the phytoplankton community composition. Thus, changes to environmental conditions in aquatic ecosystems are likely to drive basal food quality in aquatic ecosystems indirectly through changes in phytoplankton community composition due to stratification, algal competition, or trophic pressures  rather tTaxonomic group explained the greatest proportion of the total variation in fatty acid profiles in this study ~36–44%), but was lower than what was reported for marine phytoplankton , freshw%, but wa2 [Our results also corroborate previous research on individual algal strains for all major phytoplankton groups, confirming that light, temperature, nutrient status, and salinity do clearly affect phytoplankton fatty acids , that PUFA-rich galactolipids (particularly high in EPA and ARA) accumulate at lower irradiances as a result of increased growth of chloroplasts and thylakoid membranes for higher photosynthetic capacity [-1 and total LCEFA % DW. Taken together, these novel findings imply that for diverse algal groups, relatively shorter light duration  at the lower end of intermediate light intensities where light saturation is achieved , might be ideal for maximizing LCEFA content in algal cultures. Interestingly, temperature explained only little variation in the full fatty acid profiles in each dataset ; medium (haptophytes and chlorophytes); and low (cyanobacteria). This hierarchy follows the food quality rankings, for a subset of these algal groups to freshwater zooplankton, described by Brett & Müller-Navarra [et al. [Our results show both the extent of variation and overall group hierarchy of LCEFA content among the different algal groups . The med-Navarra , based o [et al. , based o [et al. . DespiteOscillatoria, were a large contributor to total annual average phytoplankton biomass in Lake Washington until the early 1970s, but were generally rare after 1972  [et al. [Long-term LCEFA dynamics we show here should be interpreted with caution and treated as hypotheses. These hypotheses offer a few interesting perspectives and potential interpretations of the Lake Washington data. Our analyses indicate that optimal ɷ-3:ɷ-6 fatty acid ratios for a (≥3–5)  were rara (≥3–5)  and larg [et al.  measured18 ɷ-3 and ɷ-6 fatty acids such as 18:2ɷ-6 (LIN) or 18:3ɷ-3 (ALA) that are also often considered as essential fatty acids for consumers [18 EFA to LCEFA, primary consumers are generally not efficient at these conversions and this trophic modification varies substantially among taxa and depending upon conditions. Thus, our analysis is conservative in the estimation of total algal-derived EFA. The choice of these three conditionally essential LCEFA for broad analyses is not unprecedented [de novo [20 ɷ-3 from precursor C18 ɷ-3 fatty acids Click here for additional data file.S1 FileDetailed description of the criteria used for dataset inclusion in the literature search, the different kinds of data , detection limits, and the environmental variables in each of the published studies.(PDF)Click here for additional data file.S1 Fig(a) The PCA includes the six dominant phytoplankton groups in all culture conditions Click here for additional data file.S2 FigThe FQI is based on Σ LCEFA, as described in (TIF)Click here for additional data file.S1 TableA total of 58 studies of the 399 screened had sufficient data on the culture conditions  and consistent FA variables for analysis beyond initial screening see .(PDF)Click here for additional data file.S2 TableTable is organized by FA data type and indicating the six major algal groups that are the focus of analyses. The FA observation (n profiles) in the ‘included’ category is the number of raw profiles that were used in the PCA  or eligi(PDF)Click here for additional data file.S3 TableResults of DISTLM marginal tests, quantifying the relative contribution of algal group affiliation and culture condition variables (following abbreviations of (PDF)Click here for additional data file.


Plasmodium vivax (PvTRAP) exhibits sequence heterogeneity among isolates, an issue relevant to vaccine development. To gain insights into variation in the complete PvTRAP sequences of parasites in Thailand, 114 vivax malaria patients were recruited in 2006–2007 from 4 major endemic provinces bordering Myanmar , Cambodia  and Malaysia . In total, 26 amino acid substitutions were detected and 9 of which were novel, resulting in 44 distinct haplotypes. Haplotype and nucleotide diversities were lowest in southern P. vivax population while higher levels of diversities were observed in other populations. Evidences of positive selection on PvTRAP were demonstrated in domains II and IV and purifying selection in domains I, II and VI. Genetic differentiation was significant between each population except that between populations bordering Myanmar where transmigration was common. Regression analysis of pairwise linearized Fst and geographic distance suggests that P. vivax populations in Thailand have been isolated by distance. Sequence diversity of PvTRAP seems to be temporally stable over one decade in Tak province based on comparison of isolates collected in 1996 (n = 36) and 2006–2007. Besides natural selection, evidences of intragenic recombination have been supported in this study that could maintain and further generate diversity in this locus. It remains to be investigated whether amino acid substitutions in PvTRAP could influence host immune responses although several predicted variant T cell epitopes drastically altered the epitope scores. Knowledge on geographic diversity in PvTRAP constitutes an important basis for vaccine design provided that vaccination largely confers variant-specific immunity.Thrombospondin-related adhesive protein (TRAP) of malaria parasites is essential for sporozoite motility and invasions into mosquito’s salivary gland and vertebrate’s hepatocyte; thereby, it is a promising target for pre-erythrocytic vaccine. TRAP of  Plasmodium falciparum is the most pernicious and prevalent species, the significance of P. vivax should not be underappreciated because it can cause chronic relapsing illness due to reactivation of hypnozoites and it can potentially lead to severe complications similar to those caused by P. falciparumP. vivax are of particular concern if they would be wide-spreading as chloroquine-resistant P. falciparumP. vivax and P. falciparum co-circulate in several endemic areas outside Africa where co-infections of both species are not uncommon In low- and middle-income countries in tropical areas, malaria remains one of the leading ten causes of morbidity and mortality, resulting in an estimated economic loss of nearly 40 million disability-adjusted life-years (DALYs) P. falciparum has resulted in limited clinical efficacy in field studies To date, malarial circumsporozoite (CS) protein is a prime candidate for pre-erythrocytic vaccine development P. falciparum (PfTRAP) and of P. vivax (PvTRAP) from clinical isolates revealed microheterogeneity of sequence that has been maintained by positive selective pressure P. falciparumTRAP has been shown to mediate gliding motility and invasion processes of malarial sporozoites into vertebrate’s hepatocyte and mosquito’s salivary gland P. vivax and P. falciparum have been circulating in Thailand with almost comparable prevalence since the past 2 decades. However, regional prevalence of P. vivax relative to P. falciparum seems to differ across major endemic areas of Thailand P. vivax populations in this country exhibited spatial variation in the extent of sequence diversity of major malaria vaccine candidate loci Both The study protocol was approved by the Institutional Review Board on Human Research of Faculty of Medicine, Chulalongkorn University (IRB303/56). Written informed consent was obtained from all participants.P. vivax-infected patients in northwestern , southwestern , eastern  and southern  Thailand during 2006 and 2007. Venous blood samples were taken from each patient and were preserved in EDTA anticoagulant. These isolates have been previously determined to contain single clone infection based on sequence analysis of the merozoite surface protein-1 gene (Pvmsp-1) as described previously Blood samples were obtained from 114 PvTRAP sequence was amplified by nested PCR using a forward outer primer  and a reverse outer primer . The inner pairs of primers were PvTRAP-F1  and PvTRAP-R1 . PCR amplification was performed in a total volume of 30 µl of the reaction mixture containing template DNA, 2.5 mM MgCl2, 300 mM each deoxynucleoside triphosphate, 3 µl of 10x ExTaq PCR buffer, 0.3 µM of each primer and 1.25 units of ExTaq DNA polymerase . Thermal cycling profile for primary PCR included the preamplification denaturation at 94°C for 2 min followed by 35 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 2 min, and a final extension at 72°C for 5 min. Nested PCR was done using the same thermal profile except that amplification was done for 25 cycles. DNA amplification was carried out by using a GeneAmp 9700 PCR thermal cycler . ExTaq DNA polymerase reportedly possesses efficient 5′→3′ exonuclease activity to increase fidelity and no strand displacement . The PCR product was examined by electrophoresis in a 1% agarose gel and was purified by using QIAquick PCR purification kit . The PvTRAP sequences were determined directly and bi-directionally from PCR-purified templates and were performed on an ABI3100 Genetic Analyzer using the Big Dye Terminator v3.1 Cycle Sequencing Kit . Whenever singleton substitution occurred, sequence was re-determined using PCR products from two independent amplifications from the same DNA template. Sequences in this study have been deposited in the GenBank Database under the accession numbers KJ807186 to KJ807299.DNA was prepared from blood samples using QIAamp kit . The complete PvTRAP sequence of the Salvador I strain as reference. The PvTRAP sequences of our previous Thai and Brazilian isolates previously published PvTRAP sequences were aligned with the TRAP sequences of P. knowlesi (PkTRAP) and P. cynomolgi (PcyTRAP) . Haplotype diversity (h) and its sampling variance were computed according to equations 8.4 and 8.12 but replacing 2n by n Sequence alignment with the Clustal X program dS) and nonsynonymous substitutions per nonsynonymous site (dN) were computed by using Nei and Gojobori’s method dS and dN was set at 5%.The rates of synonymous substitutions per synonymous site  and the differences between the number of singletons and the average number of nucleotide differences between pairs of sequences (F* test) PvTRAP, a sliding window analysis of Tajima’s D across the coding sequences was done using a window size of 250 bp and a step size of 10 bp Tests for departure from neutral evolution based on intraspecific comparisons were done by Tajima’s D and F tests, and McDonald-Kreitman tests. Fu and Li’s D statistics measure the differences between the number of mutations in external branches of the genealogy and the number of mutations while their F statistic compares the differences between the number of mutations in external branches of the genealogy and the average number of nucleotide differences between pairs of sequences Evidence of departure from neutrality based on interspecific comparsion included Fu and Li’s PvTRAP gene. The single-likelihood ancestor counting (SLAC), fixed effects likelihood (FEL), random effects likelihood (REL) and mixed effects model of evolution (MEME) methods implemented in the HyPhy package a priori distribution of rates across sites whereas REL model first fits a distribution of rates across sites and then infers the substitution rate for individual sites. MEM algorithm detects codons under episodic positive selection unmasked by the abundance of purifying selection along the lineages p values<0.1 and Bayes Factor>1000 for REL followed the default values available on the Datamonkey Web Server Tests of departure from neutrality at specific codons were performed based on estimation of the global ratio of the rate of non-synonymous to synonymous substitutions (dN/dS) across the FST identical to the weighted average F-statistic over loci, θw Fst was calculated based on Fst/(1-Fst) to address the relationship between the magnitude of population differentiation and distance between endemic areas The population genetic structure was analyzed by molecular variance approach (AMOVA) using the Arlequin 3.5 software which is similar to the Weir and Cockerham’s method but it takes into account the number of mutations between haplotypes The genetic structure was also determined by STRUCTURE 2.3.4 program that deploys the Bayesian model–based clustering approach PvTRAP haplotypes was constructed by using the Maximum Likelihood method based on the model with the lowest Bayesian Information Criterion (BIC) scores The phylogenetic tree of the Amino acid properties were characterized based on polarity and charge. Polarity of each residue is categorized as polar  or nonpolar (the remainder). Charge property includes positive , negative (D and E) or neutral (the remainder). Prediction of HLA-I binding peptides was based on the method taken into account proteasomal C terminal cleavage and transporter associated with antigen processing (TAP) transport efficiency. The predicted scores were determined by the NetCTL program PvTRAP sequences, 109 isolates contained 1668 bp and 5 isolates from Tak province whose sequences were identical had 1677 bp. Sequence alignment with the Salvador I strain revealed 36 nucleotide substitutions within 33 codons, resulting in 26 nonsynonymous and 7 synonymous changes. Comparing with previous data from Brazilian, Thai Of 114 complete PvTRAP haplotypes in southern Thailand  was rather skewed towards few haplotypes as shown by a relatively lower value of haplotype diversity (h = 0.653±0.063) whereas parasite populations from Tak, Prachuap Khirikhan and Chanthaburi provinces had significantly higher haplotype diversity values, suggesting a more even distribution of haplotype frequencies in these latter endemic regions  to 0.00272±0.00032 (isolates from Prachuap Khirikhan province). The magnitudes of nucleotide diversity in this locus did not significantly differ among isolates from Tak, Prachuap Khirikhan and Chanthaburi provinces but these were significantly greater than that from Yala and Narathiwat provinces (p<10−4) (The distribution of  regions . It is n regions . The nuc(p<10−4) .STF values did not deviate significantly from zero, implying that they shared a common origin or a high genetic admixture. However, P. vivax populations from Tak and Prachuap Khirikhan provinces displayed the STF values that were significantly greater than zero when compared with populations from Chanthaburi province (p<10−5) as well as that from Yala and Narathiwat provinces (p<10−5). A significant departure from neutrality was also noted when isolates from Yala and Narathiwat provinces were compared with those from Chanthaburi province (p<10−5), implying genetic differentiation between populations with minimum or absence of gene flow between these areas. Furthermore, the Fst values between Tak isolates in the present study (collected in 2006–2007) and those collected in 1996 P. vivax population in this area . Likewise, positive correlation was found when geographic linear distance between endemic areas was considered , implying that the majority of P. vivax populations in Thailand were genetically isolated by distance  except P. vivax isolates from Yala and Narathiwat provinces . Closer looks into the rate of nucleotide substitutions in each domain of PvTRAP has revealed that Nd significantly outnumbered Sd in domains II and IV of isolates from Tak, Prachuap Khirikhan and Chanthaburi provinces. Although no significant difference in Sd and Nd for the entire coding region of the PvTRAP gene was observed when all parasite populations were considered, Nd in domain IV was obviously greater than Sd , reflecting a signature of positive or balancing selection. Consistent results were obtained when the TRAP gene of P. knowlesi was used as the outgroup sequence  for P. vivax populations in Thailand except those from Yala and Narathiwat provinces (0.05<p<0.1). Although these recombination breakpoints occur at different positions, i.e. between nucleotides 1008 and 1009, 1170 and 1171, and 1173 and 1174 for Chanthaburi, Prachuap Khirikhan and Tak provinces, respectively, it is noteworthy that all were located in domain IV of the PvTRAP gene. Meanwhile, estimation of the parameter Rm reveals 3 and 5 recombination sites in parasite populations from Tak and Prachuap Khirikhan provinces whereas Chanthaburi isolates and southern Thai P. vivax populations had equal number of potential recombination sites (Rm = 2). Locations of potential recombination sites for each population are listed in PvTRAP locus.Evidence of recombination in the PvTRAP haplotypes from worldwide origins  reveals that the majority of Brazilian isolates were placed in a separate lineage while the majority of Tak isolates collected in 1996, in 2006–2007 and most Prachuap Khirikhan isolates were clustered together. Several isolates from Chanthaburi, Yala and Narathiwat provinces seem to be located in separate branches from the remaining Thai isolates. However, there was no clear lineage for each parasite population because several isolates from different endemic areas shared or was placed in closely related branches. More importantly, all branches in this phylogenetic inference received bootstrap values less than 80%, suggesting no distinct lineage of the  origins .Host adhesion and motility are fundamental properties of malarial sporozoites to establish infection that involves sporozoite-specific proteins, one of which is TRAP. It has been shown that TRAP-deficient sporozoites were severely impaired in their host cell adherence property and gliding motility; thereby, invasion of mosquito’s salivary glands and hepatocytes was interrupted PvTRAP locus among parasite populations in Thailand seems to be comparable among endemic areas except that from southern region  than that from Thai-Cambodia border (Chanthaburi isolates) seems to be in line with Rm in each population. It is likely that transmigration of people could maintain and spread of P. vivax harboring variant PvTRAP haplotypes while recombination could further generate novel alleles. Intriguingly, it seems that reduction in genetic diversity of southern parasite population may not be simply explained by clonal population structure of P. vivax in this region because recombination event could be traced despite the paucity of potential recombination sites observed (Rm = 2). In contrast, several decades of extensive malaria control in this region and a lack of remarkable re-introduction of malaria cases from Malaysia could have resulted in population bottleneck as previously suggested PvTRAP haplotypes may gradually increase the level of genetic diversity in this population.The extent of sequence diversity of the n region . The resPvTRAP locus had significant difference in the rate of nonsynonymous substitutions than that of synonymous substitutions. Evidence of positive or balancing selection was also reaffirmed by the McDonald-Kreitman test. Although departure from neutrality in the entire coding PvTRAP sequence was not apparent by using Tajima’s D and related statistics, sliding window analysis has identified significant negative Tajima’s D values in domains II of P. vivax population from Prachuap Khirikhan and Chanthaburi provinces while significant positive values occurred in domain IV among isolates from Yala and Narathiwat provinces. Differential patterns of departure from neutrality among populations could reflect different evolutionary forces exerted on PvTRAP of each population. Alternatively, intrinsic statistical property of Tajima’s D statistics can be confounded by demographic processes D observed in southern Thai isolates could imply balancing selection or a recent bottleneck because an excess of intermediate frequency alleles as rare alleles are lost from the population due to genetic drift can be found immediately after the bottleneck. Meanwhile, significant negative Tajima’s D value in domains II could imply purifying selection and selective sweep while migration and population growth may be the confounders PvMSP4 and PvMSP5 in which frequency of recombination seems to correlate with endemicity of each area Our analyses have shown that domains II and IV of the PvTRAP locus among P. vivax populations could be deployed for analysis of population structure. Pairwise comparison of populations revealed that South Korean isolates displayed high and significant Fst indices when compared with malaria populations in other endemic areas. Likewise, slightly lower but significant Fst indices were observed when isolates from Brazil were compared with populations elsewhere  having low and non-significant Fst indices. Therefore, diversity in the PvTRAP locus of parasites in Thailand seems to exhibit spatial but not temporal variation. It is noteworthy that regression analysis of linear geographic as well as normal logarithm of geographic distances and pairwise linearized Fst values yielded significant correlation , indicating that P. vivax populations in these 4 endemic areas have been isolated by distance Click here for additional data file.Table S2Characteristics of amino acid substitutions in PvTRAP of worldwide and Thai isolates.(DOC)Click here for additional data file.Table S3Minimum number of recombination events (Rm) and recombination sites in the PvTRAP gene of each parasite population in Thailand.(DOC)Click here for additional data file.


At 12 wk of age, the offspring ovarian reserve was depleted following exposure to maternal obesogenic diet (P < 0.05), but not postweaning obesogenic diet. Maternal obesogenic diet was associated with increased mitochondrial DNA biogenesis , increased mitochondrial antioxidant defenses  and increased lipoxygenase expression  in the ovary. There was also significantly increased expression of the transcriptional regulator NF-κB (P < 0.05). There was no effect of postweaning diet on any measured ovarian parameters. Maternal diet thus plays a central role in determining follicular reserve in adult female offspring. Our observations suggest that lipid peroxidation and mitochondrial biogenesis are the key intracellular pathways involved in programming of ovarian reserve.—Aiken, C. E., Tarry-Adkins, J. L., Penfold, N. C., Dearden, L., Ozanne, S. E. Decreased ovarian reserve, dysregulation of mitochondrial biogenesis, and increased lipid peroxidation in female mouse offspring exposed to an obesogenic maternal diet.Maternal diet during pregnancy influences the later life reproductive potential of female offspring. We investigate the molecular mechanisms underlying the depletion of ovarian follicular reserve in young adult females following exposure to obesogenic diet in early life. Furthermore, we explore the interaction between adverse maternal diet and postweaning diet in generating reduced ovarian reserve. Female mice were exposed to either maternal obesogenic (high fat/high sugar) or maternal control diet  It is well established that the early life environment plays an important role in programming many aspects of physiology and metabolism in the developing organism. Developmental programming persists throughout life and may be transmitted to subsequent generations , 2. SuboThe reproductive system appears to be exquisitely sensitive to early life influences. Maternal diet during pregnancy affects numerous parameters of offspring reproductive function  includinThe interplay between maternal diet during pregnancy and postweaning offspring diet in generating adverse programming effects has been investigated by numerous studies –17. An oIn this study, we utilize a mouse model of maternal diet-induced obesity using an obesogenic diet that is rich in simple sugars and saturated fat to better understand the effects of a typical Western diet during pregnancy on reproductive outcomes in female offspring. The primary aim of this study is to determine which, if any, of the numerous intracellular pathways previously implicated in developmental programming are involved in generating an adverse phenotype in the ovary. The second aim of this study is to examine the combined effects of adverse maternal and offspring diets on ovarian reserve.ad libitum either a standard laboratory chow diet  or an obesogenic diet . The obesogenic diet was supplemented with a separate pot of sweetened condensed milk  available to the animals within the cage. A detailed description of the dietary regimen has been published previously .All animal experiments underwent ethical review by the University of Cambridge Animal Welfare and Ethical Review Board and were carried out under the UK Home Office Animals (Scientific Procedures) Act . Female C57BL/6J mice were randomized to be fed Fixed ovaries were embedded and serially sectioned at 5 μm. Every third section was stained with hematoxylin and eosin for morphometric analysis  . Only fo1) previous work on reproductive programming (2) knowledge of programming mechanisms in other organ systems (3) relevant literature review. The genes forming the initial screen are listed with their q values in An initial screen of 31 candidate genes was developed to test which molecular pathways might be involved in generating adverse reproductive system effects , which was absent in all samples.RNA was extracted using a miRNeasy mini Kit . The kit was used according to the manufacturer’s instructions, with the addition of DNaseI digestion to ensure that the samples were free from genomic DNA contamination. The extracted RNA was quantified using a Nanodrop spectrophotometer . cDNA was synthesized from 1 μg RNA using oligo-dT primers and M-MLV reverse transcriptase. Gene expression was quantified via RT-PCR  using custom-designed primers , against a single-copy nuclear gene, nth endonuclease III-like 1 (Nthl1), was used to estimate the average copy number of mitochondrial DNA (mtDNA)/nuclear DNA (y) reflects the efficiency of the reaction, with an optimum value of 3.333 (100% efficiency). tC values for Nthl1 were subtracted from those for Cox1 to give ΔtC. A gradient of ΔtC across the dilution series < 0.1 was accepted. Average mtDNA copy number per nuclear genome (2 Nthl1 copies) was calculated as 2 × 2(ΔCt).Total DNA  was extracted using a DNeasy Blood and Tissue kit (Qiagen) according to the manufacturer’s instructions. A ratiometric assay of the levels of a single-copy mitochondrial gene, cytochrome c oxidase . This adjustment was designed for this study to take account of the specific number of genes that were tested within the initial screen . In all cases, the number of litters = 8 for all groups. Study power was determined based on effect sizes for primordial follicle counts and mtDNA copy number observed in our previous studies of follicular reserve in other dietary models   , the master regulator of mtDNA biogenesis, were significantly up-regulated by the maternal obesogenic diet compared with controls (P < 0.05). There was also an increase in the expression of nuclear respiratory factor 2 (Nrf2), an important DNA-binding transcription factor that regulates Tfam and mtDNA biogenesis, but this did not reach statistical significance (P = 0.08). Peroxisome proliferator-activated receptor γ coactivator-1-α (Pgc-1α) was not significantly altered by maternal diet . Expression levels of glutathione peroxidase 4 (Gpx4), which in mice is spliced into a number of variants including a mitochondrial isoform (P < 0.01). Copper/zinc superoxide dismutase (CuZnSOD), which localizes to the mitochondrial intermembrane space as well as the cytoplasm, was significantly up-regulated with exposure to maternal obesogenic diet (P < 0.05). There was an increased expression of the copper chaperone (Ccs) of CuZnSOD, but this did not reach statistical significance (P = 0.09).Expression levels of  isoform , were hiNf-kb) (P < 0.01). There was also significant up-regulation of both arachidonate 12-lipoxygenase (Alox12) and arachidonate 15-lipoxygenase (Alox15) expression in the maternal obesogenic diet group, reflecting increased lipid peroxidation (P < 0.05). There was no significant difference in the expression levels of peroxisome proliferator-activated receptor γ (Ppar-γ) between the 2 maternal diet groups , and collagenase 1-α 1 (Col1A1) (data not shown).None of the molecular markers of cellular ageing measured at the mRNA expression level analyzed in this study showed significant expression differences in response to maternal diet. Measured genes included Generation of an adverse phenotype in the reproductive system of young female offspring after exposure to a suboptimal maternal diet has been shown in a number of animal models –11 and hin utero by maternal diet , implies a compensated deficit in mitochondrial function. Mitochondria occupy a unique and critical position in the intracellular redox balance, in that they are both the primary generators of oxidative stress, yet particularly vulnerable to its effects (MnSOD and Gpx4, in keeping with the need for increased protection from reactive oxygen species.We observed significant effects of a maternal obesogenic diet on mitochondrial biogenesis in the ovaries of young female offspring. The observed increase in mtDNA copy number, combined with increased gene expression of the main regulators of mtDNA biogenesis ( effects , 31. An Alox12 and Alox15. Lipid peroxidation is implicated in the pathogenesis of many oxidative stress-related disease states, including ageing and metabolic diseases (Alox12 expression on both ovulation and follicular reserve: Alox12 is expressed in granulosa cells, thecal cells, and follicular fluid at the time of ovulation in the rat (Alox12 gene have been linked with early age at natural menopause in various human populations (In addition to dysregulation of mitochondrial biogenesis, we also observed an increase in the key enzymes involved in lipid peroxidation: diseases . Understdiseases , includidiseases . Aside fdiseases . Such im the rat , 36, andulations , 38, impNf-kb was significantly up-regulated in the ovaries of offspring exposed to an obesogenic maternal diet, even at a young age. Nf-kb expression is induced in response to increased levels of reactive oxygen species (Nf-kb plays a role in regulating granulosa cell development during the formation of ovarian follicles (Nf-kb expression has previously been demonstrated in the ovaries of young obese female mice prior to the onset of ovarian dysfunction (Nf-kb (Our observations of increased levels of intracellular redox regulators are strengthened by the associated up-regulation of downstream transcriptional pathways. In particular, we observed that the central transcriptional regulator  species  and has  species . In the ollicles , 42, whifunction . The resn (Nf-kb –46.We have previously observed that the end products of lipid peroxidation are significantly increased in the ovary in older rat offspring exposed to a maternal low-protein diet . Unfortu10 at physiologic doses (Our conclusion that a maternal obesogenic diet reduces ovarian follicular reserve, dysregulates ovarian mitochondrial biogenesis, and increases lipid peroxidation in young female offspring has important implications for maintaining reproductive potential at a population level, particularly in light of trends in many populations toward increasing maternal obesity , 5. Impoic doses  as a can


Nestin, a kind of intermediate filament protein initially identified in neuroepithelial stem cells, was recently reported as a credible criteria for MSCs in bone marrow. In this study, we used a green fluorescent protein (GFP) labeled bone marrow replacement model to trace the nestin positive bone marrow derived cells (BMDCs) of skin defected-mice. We found that after skin injured, numbers of nestin+ cells in peripheral blood and bone marrow both increased. A remarkable concentration of nestin+ BMDCs around skin wound was detected, while few of these cells could be observed in uninjured skin or other organs. This recruitment effect could not be promoted by granulocyte colony-stimulating factor (G-CSF), suggests a different mobilization mechanism from ones G-CSF takes effect on hematopoietic cells. Our results proposed nestin+ BMDCs as mobilized candidates in skin injury repair, which provide a new insight of endogenous MSCs therapy.Exogenously infused mesenchymal stem cells (MSCs) are thought to migrate to injury site through peripheral blood stream and participate in tissue repair. However, whether and how endogenous bone marrow MSCs mobilized to circulating and targeted to tissue injury has raised some controversy, and related studies were restricted by the difficulty of MSCs identifying  Mesenchymal stem cells (MSCs) are greatly potential in tissue injury repair due to their multilineage differentiation ability, immunomodulatory capacity and trophic function –2. AlthoGrowing evidence has shown that systemic infused MSCs migrate to host organs including heart, liver, spleen and bone marrow, where they mostly reside in initially –12. More+ BMDCs are confirmed to be MSCs, and this finding facilitates tracing MSCs in vivo by providing a reliable phenotypic marker.One of the challenges in studying of mobilized MSCs is the lack of a universally accepted marker for defining the MSCs phenotype, which makes identifying MSCs in vivo quite difficult and might be the reason for the conflicting results of previous studies , 5, 28. + MSCs are components of hematopoietic stem cells (HSCs) niches that are important for HSC maintaining [The nestinntaining , 30. Thentaining . It is kntaining , 32. Intntaining –36 by G-ntaining , but to + BMDCs after skin defect, analyzed their distribution in peripheral blood and injured skin, and observed G-CSF’s effect on mobilization of these cells.In this study, we used the GFP-positive bone marrow chimeric mice as models to trace the migration of nestin+/+ transgenic female C57BL/6 mice were obtained from the Laboratory Animal Research Centre of the Fourth Military Medical University (FMMU). This study was approved by Fourth Military Medical University Intramural Animal Use and Care Committee and all interventions were performed in according with the committee guidelines for the use and care of experimental animals. All efforts were made to minimize suffering. Anesthetics were used as indicated.8-weeks old wild type and GFP2 narcosis.Mice were anesthetized by intraperitoneal administering 1% pentobarbital. Once adequate anesthesia was obtained, a 6 mm or 20 mm wound was punched in the depilated dorsal skin. Wounds were left uncovered, and mice were housed individually with sterile paper bedding. Mice were given analgesic and antibiotics and allowed to recover. Animals (4–7/group) were sacrificed 4 and 7 days after wounding. Biopsies containing the wounded tissue and the surrounding skin were harvested. At the conclusion of the study period or prior to the harvesting of mouse wounds, peripheral blood or bone marrow, all animals were euthanized by CO60 ionizing radiation of 8.5 Gy in dosage. The donor GFP transgenic C57BL/6 mice were sacrificed and the femurs and tibias were isolated. Then the epiphyseal sides of the bones were cut off and the whole bone marrow tissue was flushed out with PBS by a 1ml injector. Red blood cell lysis buffer  was used to remove the erythrocytes. Then 2×106 of bone marrow cells were suspended in 100 μl PBS and injected through tail vein to each recipient mouse within 6 hours after the irradiation. Then 2 x 106 GFP+ bone marrow cells were suspended into 100 ml PBS and injected intravenously by tail vein into wild-type C57BL/6 mice within 24 h after 8.5 Gray of whole body irradiation. The successful creation of GFP chimera was confirmed by analysis of the GFP+ peripheral blood mononuclear cells (PBMCs) by flow cytometry . The percentage of GFP+ PBMCs in recipients quickly increased and reached 85% by 30 days (data was not shown), and then the chimera mice were used in the study.Wild type C57BL/6 mice were used as recipients and received a disposable whole body Co24 chimeric mice received an intravenous injection of G-CSF  through tail veins for continuous 5 days after the skin surgery (10ug/kg weight/dose), while 12 chimeric mice received same volume of normal saline as control.+/nestin+ cells distribution.To assess the healing process of skin defect, samples of injured skin were collected on day 7 after surgery for H&E staining was performed as described before . Samples+ cells of the chimeric mice 1 hour or 4 days after surgery following the instruction manual. A region of interest (ROI) of 1cm in diameter for each mouse was drawn to contain the whole area of radiation signal around the skin injury, and the results were expressed as total fluorescence signal efficiency .A Xenogen In Vivo Imaging System  was used to image and analyze the distribution of GFPOn day 7 after the skin defect was made, the full thickness of injured skin tissue together with 3mm width of healthy skin around was isolated from each sample, and total RNA was isolated using Trizol reagent . Reverse transcription of mRNA was possessed with a PrimeScript RT reagent kit  and RT-PCR was performed using the QuantiTect SYBR Green PCR Kit  and detected on the ABI Prism 7500 HT sequence detection system . GAPDH was used as internal control gene and the primer sequences were as follow: CXCL12, Forward, 5’-GAGCCAACGTCAAGCATCTG-3’, Reverse, 5’- CGGGTCAATGCACACTTGTC-3’; GAPDH, Forward, 5’-AGCAGTCCCGTACACTGGCAAAC-3’, Reverse, 5’- TCTGTGGTGATGTAAATGTCCTCT -3’.Data are presented as means ± SD from at least three independent experiments. Statistical comparison of two groups was carried out by two-tailed unpaired Student's t test using SPSS 17.0 software.To investigate the change in quantity of nestin positive cells upon acute injury, a circular full thickness skin defect of 6 mm in diameter was made on the back of C57BL/6 mice, and 4 days later the bone marrow tissue and peripheral blood samples were gathered, lysised of red cells and detected nestin positive ratio by flow cytometry. The results showed that quantity of nestin positive cells in peripheral blood was at a basal level lower than 1%, while skin injury raised this ratio for 4 times or more. Number of nestin positive cells in bone marrow tissue also increased after the operation .+ BMDCs were mobilized when injury happens, a chimeric mouse model with GFP positive bone marrow derived cells was used. Wild type C57BL/6 mice received a lethal dose of ionizing radiation, in which case their own bone marrow tissues were destroyed and could be replaced with ones derived from GFP transgenic mice. After the bone marrow replacement, bone marrow derived cells of these chimeric mice were GFP positive while all of other cells were GFP negative , then the WBMC were lysised of red cells and immediately intravenous injected to the recipient mice within 6 hours after the radiation. One month later, the veinal blood of the recipient mice was gathered, lysised of red cells and detected GFP positive ratio by cytometry. The chimeric mice model was considered successfully constructed if the GFP positive ratio was over 85%. (B) The GFP positive ratio of the chimeric mice and the negative control.(A) Recipient mice received a disposable Co(TIF)Click here for additional data file.S2 Fig+/CD31+ double positive cells. All scale bars, 100μm.Arrows indicate the GFP(TIF)Click here for additional data file.


Various inflammatory neurologic diseases are associated with viral infections. These agents may cause direct damage of infected cells associated with immunological alterations such as chronic activation, immunodeficiency and infiltration of inflammatory cells into the central nervous system that underlie the pathogenesis of inflammatory neurologic diseases. Therefore, characterization of local immune responses that are associated with the inflammatory milieu may provide evidence or pathogenic “signature” of an immunopathogenic process in virus-associated neurologic diseases. Multicolor flow cytometry is a powerful technology to provide a large amount of cellular information for each sample and allow identification and characterization of novel cell subsets, even rare cell population. Using the technique, we characterized the cellular immunophenotypes  in cerebrospinal fluid (CSF) and blood of patients with virus-associated neurologic diseases including HTLV-1 associated myelopathy/tropical spastic paraparesis , HIV adequately treated with antiretroviral drugs (n=28), multiple sclerosis  and progressive multifocal leukoencephalopathy  compared to healthy volunteers (n=8). CD4+ and CD8+ T cells were significantly altered among the groups, and the CD4/CD8 ratio reduced in HIV, HAM/TSP and a subset of PML, but elevated in MS. In patients with HAM/TSP, activated CD4+ T cells were significantly increased in the CSF as well as the blood and correlated with HTLV-1 proviral DNA loads. HAM/TSP patients had an increased frequency of effector CD4+ and CD8+ T cells in the blood, but not in the CSF. Antibody secreting B cells were elevated in the CSF of patients with HAM/TSP, MS and PML and were significantly correlated with intrathecal HTLV-1 Gag-specific antibody responses in HAM/TSP patients. Immunophenotyping of CSF cells may reflect immune pathology in inflammatory neurologic diseases and can serve to highlight differential diagnoses that may lead to a better understanding of disease pathogenesis and clinical treatment.


To date, there are no simple and minimally invasive methods to diagnose MDR. Extracellular vesicles (EVs) are shed by all cells, carry a specific cargo from the donor cells and are present in several body fluids, which means that they can potentially be easily collected from cancer patients and become the source of biomarkers to diagnose cancer. This data article contains a full list of the proteins identified in the EVs shed by an isogenic pair of chronic myeloid leukaemia cells (MDR cells and their drug-sensitive counterparts) by LC/MS/MS analysis, together with their GeneOntology analysis. In addition, it also contains data from protein content analysis and Dynamic light scattering count-rate events of the referred EVs as well as of the EVs shed from an isogenic pair of non-small cell lung cancer cells (MDR cells and their drug-sensitive counterparts). The interpretation of the data presented in this article and further extensive insights can be found in “Multidrug resistant tumour cells shed more microvesicles-like EVs and less exosomes than their drug-sensitive counterpart cells”  Specifications Table•These data describe the quantification of EVs isolated from MDR cells and from their drug sensitive counterpart cells.•Data regarding the use of LC–MS/MS analysis to compare EVs isolated from MDR and drug-sensitive counterpart cells.•EVs isolated from MDR and their drug-sensitive counterpart cells could be valuable for future research on the acquisition of MDR phenotype and improving knowledge in the diagnosis of MDR.K562 – drug-sensitive cells and K562Dox – MDR cells) have been mentioned. Moreover, The protein content and DLS count-rate events of EVs isolated from MDR and drug-sensitive cells have been shown . In Tabl1(H460 – drug-sensitive cells and RH460 – MDR cells)K562 – drug-sensitive cells and K562Dox – MDR cells) .EVs were isolated from two pairs of isogenic cell lines (MDR and the drug-sensitive counterpart) from two different cancer models, non-small cell lung cancer-NSCLC 1.1EVs were collected from the supernatant of equivalent amounts of cells, cultured in EVs-depleted culture medium. EVs were isolated from these culture media as previously published 1.2Protein amount of the isolated EVs was quantified as previous published 1.3EVs count rate was measured by dynamic light scattering (DLS), using a Nano series Malvern Zetasizer Instrument (Prager Instruments) as previously published 1.4Pellets of EVs were prepared using previously established methods 1.5http://www.pantherdb.org/).Proteins identified in the samples of EVs were analysed using GeneOntology (GO) in the PANTHER database to identify biological processes, molecular functions and pathways (