The relationship between colony numbers and concentration of cells plated is an important parameter of clonogenic assay systems. The cloning efficiency for an ideal sample should be independent of cell concentration, thus giving a straight line through the origin when colony numbers are plotted against cell concentration. A simple statistical method has been developed to test if this is the case for individual tumour samples. Colony data from 51 freshly obtained tumour samples, which had sufficient cells to plate 3 or more dilutions and gave at least 20 colonies per plate at one or more of the dilutions, were tested. The results indicated that colony formation was linear for 27 (53%) of the samples. The remaining 24 samples could be classified into 2 groups: type I, in which cloning efficiencies increased with increasing cell concentration and type II, which had reduced cloning efficiencies at high cell concentrations. Fifteen (29%) of the samples had type I non-linearity and 9 (18%) exhibited non-linearity of type II. These findings indicate that the relationship between colonies and cells plated should be examined for each biopsy sample particularly in each experiment where the effects of cytotoxic drugs are tested.


Interestingly, DJ-1 expression was increased in brains of zebrafish under conditions of oxidative stress, indicating that DJ-1 is a part of stress-responsive machinery. Since oxidative stress is one of the major contributors to the development of Alzheimer's disease (AD), we also examined DJ-1 expression in AD brains. Using DJ-1 specific antibodies, we failed to detect a robust staining of DJ-1 in brain tissues from control subjects. However, DJ-1 immunoreactivity was detected in hippocampal pyramidal neurons and astrocytes of AD brains. Therefore, our results strongly suggest that DJ-1 expression is not necessary during zebrafish development but can be induced in zebrafish exposed to oxidative stress and is present in human AD brains.Mutations in the DJ-1 gene have been linked to autosomal recessive familial Parkinson's disease. To understand the function of DJ-1, we determined the DJ-1 expression in both zebrafish and post mortem human brains. We found that DJ-1 was expressed early during zebrafish development and throughout adulthood. Knock down (KD) of DJ-1 by injection of morpholino did not cause dramatic morphologic alterations during development, and no loss of dopaminergic neurons was observed in embryos lacking DJ-1. However, DJ-1 KD embryos were more susceptible to programmed cell death. While a slight reduction in staining for islet-1 positive neurons was observed in both DJ-1 KD and H Parkinson's disease (PD) and Alzheimer's disease (AD) are the two most common neurodegenerative disorders. PD is characterized by loss of dopaminergic (DA) neurons in the substantia nigra and accumulation of intraneuronal inclusions known as Lewy bodies. AD is characterized by two major hallmarks, neurofibrillary tangles and neuritic plaques. Genetic studies of PD have demonstrated that two autosomal dominant genes (α-synuclein and LRRK2) -3 and thin situ hybridization [2O2 significantly increases the immunoreactivity of DJ-1 [High levels of DJ-1 mRNA have been detected in neuronal and non-neuronal populations of several regions in mouse brain by dization . A recen of DJ-1 . This up of DJ-1 .Pharmacological studies have shown that DJ-1 is needed for the protective effects of certain compounds which are known to inhibit the production of reactive oxygen species . DJ-1 prL166P in addition to E64D, M26I, A104T, and D149A is one of known mutations within DJ-1 that has been identified from familial PD cases. These mutant forms of DJ-1 show different levels of structural alteration (except for E64D due to similar residue replacement), which lead to reduced stability of DJ-1 ,21. The 2O2, and DJ-1 appeared to function as a peroxiredoxin-like peroxidase in these animals [DJ-1 knockout (KO) mice do not differ from wild type mice with respect to morphology or dopaminergic cell loss -24, with animals . In zebr animals , and KD  animals . In Dros animals .Zebrafish are beginning to be used for aging research, e.g., brief exposure of zebrafish embryos with Dichlorodihydrofluorescein diacetate allows repeated measurement of the level of reactive oxygen species generation in live fish . For neu2O2. We found that KD of DJ-1 and H2O2 had similar effects on islet-1 positive neurons  homeodomain protein), with both groups of embryos showing an increased number of apoptotic cells. Since the oxidative stress is one of the major contributors to the AD pathogenesis [In this report, we compared KD of DJ-1 to oxidative stress in zebrafish embryos. In addition, we examined DJ-1 expression in the adult fish brain by treating live animals with Hogenesis ,39, we eEmbryos were obtained from natural spawning of wild-type (Tubingen longfin strain) adults and were raised and staged according to Kimmel et al. .DJ-1 MO, directed against the 5' untranslated region (UTR) of DJ-1  was injected into fertilized zebrafish eggs at the one-cell stage at which time morpholinos can rapidly spread into all cells. The morpholino sequences were as follows: DJ-1 MO, 5'-GCGTCTAATAACCTGTGCGTGTCTG-3' control MO, 5'-CCTCCTACCTCAGTTACAATTTATA-3'.The above standard control MO was synthesized against the zebrafish β-globin intron. MO oligonucleotides were dissolved in 1× Danieu solution at 5 mM, diluted to 0.1 mM prior to injection, and ~4 ng was injected into each embryo. After injection, embryos were incubated at 28.5°C in embryo medium  containiTUNEL assay was performed using the TMR-RED in situ cell death detection kit  accordinIn situ hybridization was carried out according to standard protocols  using a Human postmortem cortical tissue was collected in accordance with Institutional Review Board-approved guidelines at Brigham and Women's hospital. Blocks of cortex were fixed for ~2 hrs in 10% neutral buffered formalin, as previously described . ParaffiHuman, mouse and zebrafish brain samples were homogenized in lysis buffer containing 0.2% NP-40. The lysates were collected after 1 hr of centrifugation at 100 000 × g (human and mouse) or after 5 min of centrifuge at 18,000 × g (zebrafish). Individual de-chorionated and de-yolked embryos were lysed, and all samples were heated in 1 × sample buffer  and then separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Antibodies KAM-SA100  and anti-DJ-1-N (1:500) and an enhanced chemiluminescence system  were used to detect the western blots.Wildtype CHO cells and APP stably transfected CHO cells (CHO+APP) were grown for 24 hrs before the conditioned media were collected for the measurement of Aβ levels by ELISA, as previously described . The samWe generated an affinity purified polyclonal antibody DJ-1-N against the N-terminus of DJ-1 that recognizes a single band at 23 kDa corresponding to endogenous DJ-1 from human cortex homogenate; this band was absent when the antibody was preabsorbed with its cognate peptide Fig. . The samUsing the human DJ-1 sequence, we identified multiple zebrafish ESTs and obtained the complete zebrafish DJ-1 sequence by the alignment of these ESTs with genomic zebrafish DJ-1 sequences from the Sanger Institute. We only found one DJ-1 homologue in zebrafish. Zebrafish DJ-1 has 4 exons that encode 189 amino acids, and the protein is 87% homologous to human DJ-1. cDNA from zebrafish embryos at the 1-somite stage was prepared and the full-length zebrafish DJ-1 cloned. The sequence of cDNA was confirmed and found to be identical to the one previously reported .A morphant technology that uses morpholino phosphodiamidate anti-sense oligonucleotides  to target genes in vivo -49 was uBoth control and DJ-1 MO injected embryos shared an almost identical morphology Fig. , and no 2O2 to induce an environment of oxidative stress. We found that control MO injected embryos showed well defined islet-1 positive neurons along the spinal cord  also showed DJ-1 immunoreactivity in scattered astrocytes (data not shown), but they showed weak or no DJ-1 staining in pyramidal neurons of the CA1, CA2, CA3 sub-regions and the hilus of the dentate gyrus (CA4) Fig. , except To search for any mechanistic cause for DJ-1 expression in AD brains, we examined the effect of Aβ on the regulation of DJ-1 expression. We have used two cell lines, CHO that expresses endogenous levels of APP, and CHO+APP that expresses high levels of APP and generates a large amount of Aβ. When we measured the conditioned media from CHO and CHO+APP cells, we found elevated levels of Aβ40 Fig.  and 7B. It is known that oxidative stress and mitochondrial dysfunction contribute to the pathogenesis of AD and PD. In AD, Aβ may directly interact with mitochondria and lead to increased free radical production; in PD, DJ-1, parkin and PINK1 are all linked to oxidative stress and/or mitochondrial dysfunction {Reviewed by ,53}. In Consistent with earlier studies in DJ-1 KO mice -24, KD oWe found that DJ-1 KD embryos have an increased number of apoptotic cells, consistent with an earlier report showing a p53 dependent apoptosis in DJ-1 KD zebrafish embryos . In cult2O2 on DJ-1 expression in the brains of adult zebrafish in vivo. For the first time, we found an increase in DJ-1 protein in brains of adult fish subjected to a stressful environment such as oxidative stress. While it is difficult to discern whether the increase in DJ-1 was derived from neuronal or glial sources, we predict that it was a global response from exposure to H2O2 in the environment.Recent studies have shown that DJ-1 expression is up-regulated in cultured cells in an environment that promotes the formation of intracellular reactive oxygen species ,14. We hTo further address this relationship, we utilized brain sections from post mortem AD and control subjects. We found that the avidity of different antibodies and the methods for preserving brain tissues were critical for revealing DJ-1 immunostaining. Whereas antibodies raised against the C-terminus of DJ-1 Park7) ,9 or ful ,9 or fuOur comparison staining with DJ-1-N and other DJ-1 antibodies  reveals an important finding on the potential binding region of DJ-1 when it dimerizes. Earlier reports, including ours, have demonstrated that DJ-1 dimerizes and forms a high molecular weight complex in cultured cells and human brains ,59. It s2O2. Therefore, the similarity of increased DJ-1 staining in H2O2 treated zebrafish and AD brains suggest that a common pathway might be elicited under damaging conditions. As previously reported, DJ-1 is needed to avoid oxidative stress-induced cell death in cultured neuroblastomas, dopaminergic cells and primary neuronal cells [The intriguing finding of positive staining of DJ-1 in AD brains but not control brains has important implications for both PD and AD pathogenesis. In brains of AD patients, the neuritic plaque composed of Aβ is one of the major characteristics of AD pathology. A lack of change in DJ-1 expression in cultured cells exposed to high levels of Aβ suggests that Aβ has no direct effect on steady state levels of DJ-1. During the neurodegenerative process, accumulation of reactive oxygen species renders neurons, such as pyramidal neurons around hippocampal area, extremely vulnerable, and gradual neuronal loss is inevitable when the brain is exposed to insults. The fact that DJ-1 itself is subjected to cysteine and methionine oxidation  as well al cells . Therefo2O2 [Furthermore, DJ-1 is a non-traditional peroxiredoxin-like peroxidase , and enh2O2 .2O2 illustrates a new plate form that allows us to examine the role of DJ-1 involved in response to oxidative stress. Investigation of the function/dysfunction of DJ-1 in a whole vertebrate animal exposed to a stressful environment has a direct physiological relevance to human diseases. Therefore, exploring mechanisms of DJ-1 associated with PD and AD pathogenesis will provide insight into common pathways involved in both neurodegenerative diseases. Any newly identified pathways will offer novel drug targets and exciting opportunities for therapeutic intervention.Future studies are necessary to characterize the role of DJ-1 in PD and AD pathogenesis such as whether the DJ-1 protein we found in pyramidal neurons in AD hippocampus is an acidic isoform , basic iAD: Alzheimer's disease; Aβ: amyloid β-protein; DA: dopaminergic; hpf: hours post fertilization; KD: knock down; KO: knock out; MO: morpholino; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; PD: Parkinson's disease; TH: tyrosine hydroxylase.The authors declare that they have no competing interests.SB and JS carried out the biochemical and immunohistochemical analyses, HL carried out zebrafish experiments, TY carried out cell culture experiments, MS, JS, MGS, CAL, and QL participated in the design of the study, SB and WX conceived of the study and draft the manuscript. All authors read and approved the final manuscript.


The appropriate control of mitotic entry and exit is reliant on a series of interlocking signaling events that coordinately drive the biological processes required for accurate cell division. Overlaid onto these signals that promote orchestrated cell division are checkpoints that ensure appropriate mitotic spindle formation, a lack of DNA damage, kinetochore attachment, and that each daughter cell has the appropriate complement of DNA. We recently discovered that AMP-activated protein kinase (AMPK) modulates the G2/M phase of cell cycle progression in part through its suppression of mammalian target of rapamycin (mTOR) signaling. AMPK directly phosphorylates the critical mTOR binding partner raptor inhibiting mTORC1 (mTOR-raptor rapamycin sensitive mTOR kinase complex 1). As mTOR has been previously tied to mitotic control, we examined further how raptor may contribute to this process.We have discovered that raptor becomes highly phosphorylated in cells in mitosis. Utilizing tandem mass spectrometry, we identified a number of novel phosphorylation sites in raptor, and using phospho-specific antibodies demonstrated that raptor becomes phosphorylated on phospho-serine/threonine-proline sites in mitosis. A combination of site-directed mutagenesis in a tagged raptor cDNA and analysis with a series of new phospho-specific antibodies generated against different sites in raptor revealed that Serine 696 and Threonine 706 represent two key sites in raptor phosphorylated in mitosis. We demonstrate that the mitotic cyclin-dependent kinase cdc2/CDK1 is the kinase responsible for phosphorylating these sites, and its mitotic partner Cyclin B efficiently coimmunoprecipitates with raptor in mitotic cells.This study demonstrates that the key mTOR binding partner raptor is directly phosphorylated during mitosis by cdc2. This reinforces previous studies suggesting that mTOR activity is highly regulated and important for mitotic progression, and points to a direct modulation of the mTORC1 complex during mitosis. The serine/threonine protein kinase mammalian target of rapamycin (mTOR) is a key mediator of the cellular response to nutrient status through its regulation of translation, ribosome biogenesis, mitochondrial metabolism, and autophagy The best-established substrates of mTORC1 demonstrate the importance of mTOR in translational control. mTOR phosphorylates S6K1 at T389 to enhance S6K1 activity, which amongst other things phosphorylates the S6 subunit of the ribosome to promote translation. mTOR also phosphorylates 4EBP1, causing its dissociation from its binding partner eIF4E, which is then free to associate with the cap-complex to promote cap-dependent translation The activity of mTORC1 is dependent on the small Ras-like GTPase, Rheb, whose GTP-loaded state is regulated by a GTPase-accelerating protein (GAP) complex composed of the TSC1 and TSC2 tumor suppressors. Inputs from a variety of pathways converge on the TSC1/2 complex to regulate mTORC1 signaling regulatory associated protein of TOR) is thought to act as the key mTORC1 scaffolding protein that binds mTOR substrates via the TOR signaling (TOS) motif, facilitating their phosphorylation by mTOR. A handful of recent studies have demonstrated the importance of phosphorylation of raptor on various sites in the regulation of mTOR signaling by pro- and anti-proliferative signals. Phosphorylation by Rsk at S721 as well as by mTOR at S863 have been shown to enhance mTORC1 activity In addition to the hub of signaling at TSC2, phosphorylation of components of mTORC1 have recently been shown to have important regulatory roles in mTOR signaling We have shown previously that under energy stress conditions, fewer cells proceed into G2/M and that this cell cycle arrest is dependent on AMPK phosphorylation of raptor and inhibition of mTORC1 activity. This suggested that perhaps mTOR signaling might play a role in mitosis, as suppression of mTOR blocks entry into G2/M and inappropriate activation of mTOR signaling drives cells into G2/M. In our investigations into the regulation of mTOR signaling in mitosis, we identified several sites in raptor phosphorylated by Cdc2 that may play a role in mitotic progression.Myc-raptor, AU1-mTOR, HA-GbL and Flag-PRAS40 originated in Dr. David Sabatini's Lab  and were obtained from Addgene.org . Ha-tagged 4ebp1 and S6K1 were obtained from Dr. John Blenis . Myc-raptor was subcloned into pENTR3C (Invitrogen), and serine to alanine point mutations were made using QuikChange II XL (Stratagene). Mutant alleles were then put into an FBneo DEST vector by LR reaction (Invitrogen). All constructs were fully sequence verified. Phospho-Plk1 T210 was from BD Pharmingen (#558400). Phospho-raptor  and Phospho-histone H3 antibodies were obtained from Millipore. Anti-raptor used for endogenous raptor immunoprecipitations was from Invitrogen (#42-4000). The 9E10 anti-myc antibody was used for immunoprecipitations (Roche). Antibodies against raptor (#2280), mTOR (#2983), GbL (#3274), PRAS40 (#2610), phospho-S6K T389 (#9234), pAurora A, B, C (T288/T232/T198) (#2914), Cyclin B1 (#4138), phospho-4EBP1 (T37/46) (#2855), phospho-4EBP1 (S65) (#9451), myc-tag (#2272), phospho-threonine-proline (#2321), and GST-tag (#2622) were from Cell Signaling Technologies.2. Cells were transfected with Lipofectamine 2000 (Invitrogen) as per manufacturer's instruction for 32–36 hours. Nocodazole (1 ug/mL) (SIGMA) or taxol (1 uM)  treatment was administered for 16–18 hours prior to lysis . Replacement of endogenous raptor with myc-tagged raptor was achieved by infecting Hela cells with a retrovirus expressing myc-wt or myc-S696/T706/S711AAA raptor in the FBneo vector, and selection in neomycin. These stables were subsequently infected with a lentivirus expressing a short-hairpin RNA that targets the 3′ UTR of human raptor in the pLKO vector and selected in puromycin and distributed by Addgene (<http://www.addgene.org/pgvec1?f=c&identifier=1858&atqx=raptor&cmd=findpl>). A549 cells were synchronized in G1/S by double thymidine block as follows: 2 mM thymidine was added to the media for 14–16 hours, plates were washed twice with PBS, then complete thymidine-free media was added. Eight to ten hours later, 2 mM thymidine was added again for 14–16 hours, cells were washed twice with PBS, then released into thymidine-free media. 50 uM roscovitine was administered for 6 hours following 16 hours nocodazole treatment in A549 cells. Torin1 (50 nM)  was added for 1 h.Hela, A549 and HEK293T cells were grown in DMEM with 10% FBS at 37° with 5% COFor immunoprecipitations, cells were washed with ice cold PBS and collected in lysis buffer 1  as per manufacturer's directions) for experiments in Anti-myc immunoprecipitations were performed on cell lysates from HEK293T cells transiently transfected with myc-raptor treated with or without nocodazole. After washing the beads twice in lysis buffer 1, they were washed twice in CIAP buffer  then incubated with 5 uL calf-intestinal alkaline phosphatase (CIAP) (NEB) with constant agitation at 37°C for 30 minutes.2, 0.1 mM Na3VO4, 5 mM b-glycerophosphate, 2 mM DTT). Immunoprecipitates were then incubated with 20 uL kinase reaction mix  with or without 175 ng recombinant Cdc2/cyclin B  at 30° for 30 minutes with constant shaking. Reaction was quenched by addition of sample buffer to 1X and boiling at 95° for 5 minutes.Anti-myc immunoprecipitations from HEK293T cells transiently transfected with myc-raptor for 16 hours followed by 12 hours of 2 uM hydroxyurea treatment were washed three times with lysis buffer 1, then three times with kinase buffer  in the data-dependent acquisition and positive ion mode at 300 nL/min. MS/MS spectra collected via collision induced dissociation in the ion trap were searched against the concatenated target and decoy (reversed) single entry Raptor and full Swiss-Prot protein databases using Sequest  with differential modifications for Ser/Thr/Tyr phosphorylation (+79.97) and the sample processing artifacts Met oxidation (+15.99), deamidation of Asn and Gln (+0.984) and Cys alkylation (+57.02). Phosphorylated and unphosphorylated peptide sequences were identified if they initially passed the following Sequest scoring thresholds against the target database: 1+ ions, Xcorr ≥2.0 Sf ≥0.4, P≥5; 2+ ions, Xcorr ≥2.0, Sf ≥0.4, P≥5; 3+ ions, Xcorr ≥2.60, Sf ≥0.4, P≥5 against the target protein database. Passing MS/MS spectra were manually inspected to be sure that all b- and y- fragment ions aligned with the assigned sequence and modification sites. Determination of the exact sites of phosphorylation was aided using FuzzyIons and GraphMod and phosphorylation site maps were created using ProteinReport software . False discovery rates (FDR) of peptide hits (phosphorylated and unphosphorylated) were estimated below 1.5% based on reversed database hits.For all mass spectrometry (MS) experiments, myc-Raptor immunoprecipitates were separated using SDS-PAGE, the gel was stained with Coomassie blue, and the myc-Raptor band was excised. Samples were subjected to reduction with dithiothreitol, alkylation with iodoacetamide, and in-gel digestion with trypsin or chymotrypsin overnight at pH 8.3, followed by reversed-phase microcapillary/tandem mass spectrometry (LC/MS/MS). LC/MS/MS was performed using an Easy-nLC nanoflow HPLC (Proxeon Biosciences) with a self-packed 75 µm id x 15 cm CWe have shown previously that cells undergoing energy stress arrest in G2/M and that if they lack the ability to downregulate mTORC1 signaling during energy stress, they proceed inappropriately into mitosis www.phosphosite.org; In attempts to identify the residues of raptor responsible for the nocodazole-induced bandshift, microcapillary liquid-chromatography/tandem mass spectrometry (LC/MS/MS) was performed on raptor immunoprecipitated from cells with or without nocodazole treatment. We identified several novel phosphorylation sites from this analysis, including Ser696, Thr706, Ser738, Ser771 and Ser877 . Of thesNext we examined whether mutation of any of the identified serine/threonine-proline sites to alanine would alter the mobility shift induced in raptor upon nocodazole or taxol treatment. From this analysis, we discovered that an allele of raptor with mutation of Ser696 and Thr706 to alanine, showed reduced band-shifting . MutatioTo directly determine the residues of raptor up-regulated following nocodazole treatment, phospho-specific-antibodies against Ser696, Thr706, Ser877, and Ser863 were generated and verified as recognizing only wild-type myc-tagged raptor immunoprecipitated from HEK293T cells, but not raptor mutated at the specified phospho-acceptor residue . TreatmeHaving identified several residues of raptor phosphorylated in cells blocked in mitosis, we next sought to identify the upstream kinase for Ser696 and Thr706. Knowing the upstream kinase was active following nocodazole treatment, and was proline-directed, we decided to examine the mitotic CDK family member Cdc2. First we tested whether recombinant Cdc2-cyclin B kinase complexes were capable of in vitro phosphorylation of raptor immunoprecipitated from hydroxyurea treated HEK293T cells (and hence derived from cells where Cdc2 would be inactive) . Indeed To examine whether Phospho-Thr706 can be detected during natural mitotic progression and is not simply due to kinases activated by microtubule stress, we synchronized A549 cells using double thymidine block and endogenous raptor was immunoprecipitated at various timepoints following thymidine release and immunoblotted with the phospho-raptor Thr706 antibody . MitoticFinally, we examined whether we could detect and in vivo association between raptor and the cdc2/cyclin B kinase complex. Utilizing Hela cells stably expressing low levels of tagged raptor, raptor immunoprecipitates from cycling or taxol-arrested cells revealed the presence of endogenous cyclin B .mTORC1 is a multi-protein complex whose function requires proper association of all components It has been demonstrated that raptor acts as a scaffolding protein between mTOR and its substrates To confirm these results in a more physiological system, cell lines stably over-expressing a low level of myc-tagged raptor alleles with stable knockdown of endogenous raptor with short hairpin RNA that targets the 3′ UTR of raptor How signaling pathways coupled to nutrient uptake and expenditure couple to the cell cycle machinery and proliferation control has been an area of increasing investigation. The mTORC1 signaling pathway is a critical integrator of environmental inputs into protein translation and cell growth. However, the precise role of mTORC1 signaling in mitotic progression remains enigmatic Our previous studies indicated the presence of G2/M metabolic checkpoint enforced by AMPK in a manner dependent on its ability to phosphorylate raptor and suppress mTORC1 activity. Cells expressing non-phosphorylatable alleles of raptor continued to progress through mitosis unabated unlike those expressing wild-type raptor, and ultimately displayed increased rates of apoptosis. Consistent with an AMPK/mTORC1 dependent checkpoint, AMPKα2 and its upstream kinase LKB1 were isolated in an RNAi screen for modulators of G2/M in mammalian cells Previous studies also suggest that mTOR signaling plays a positive role in the progression through mitosis in a variety of species. In budding yeast, a temperature sensitive allele of raptor or rapamycin-treatment of cells both induce mitotic delay and a prolonged G2 We demonstrate here that the mitotic kinase cdc2 directly phosphorylates raptor during mitosis, though we have been unable to demonstrate the contribution those phosphorylations play to overall mitotic progression or mTORC1 signaling during mitosis in the tumor cell settings we have examined thus far. Importantly, our data suggests there may be additional cdc2 sites beyond Ser696, Thr706, and Ser711 in raptor and until these sites are fully identified, the phenotype of a fully cdc2 non-phosphorylatable raptor remains unknown. Nontheless, the cdc2 sites in raptor may be more critical for growth control in non-tumorigenic settings, which is an area requiring further investigation.One additional complicating factor is these analyses is the fact that cdc2 has been reported to directly phosphorylate both S6K1 A complication of much of the previous literature studying the effect of mTOR on G2/M progression utilizing rapamycin is that recent finding from several labs that rapamycin does not fully inhibit mTORC1 kinase activity. Kinase inhibitors directed at mTOR itself yield changes in mTORC1 signaling and growth arrest phenotypes more similar to RNAi for raptor Additional tools including phospho-specific antibodies which can work for immunolocalization may better reveal where the population of cdc2-phosphorylated raptor and 4ebp1 are during the different stages of mitosis. Understanding how AMPK activity and mTOR activity are controlled spatially and temporally during mitosis will undoubtedly lead to fundamental insights into how nutrients control cell division as well as to how protein translation is coupled to timely cell cycle exit during differentiation or stem cell renewal. A deeper understanding of how mTORC1 controls cell cycle progression is essential for use of targeted mTOR inhibitors in the treatment of cancer and many other mTOR-related pathologies Table S1All identified in vivo phosphorylation sites in raptor. Conservation is indicated and predicted kinases from Scansite are listed in black. Reported in vivo kinases are indicated in red.(0.76 MB TIF)Click here for additional data file.


We observed the growth of 2 sublines of leukaemia L1210 in histocompatible DBA2 mice given 10(3) cells i.p. and studied the protective effect of Corynebacterium parvum (CP). The growth of subline L1210-M was unaffected by pretreatment with CP or admixture with 10(5) peritoneal cells (PC) from CP-treated mice. In contrast, the growth of subline L1210-C was inhibited; CP pretreatment increased the proportion of long-term survivors (70% vs 20%) and admixture with CP-PC prolonged the survival time . In vitro experiments indicated that Sublines M and C were equally sensitive to cytostasis by CP-PC, as measured in a terminal labelling assay (greater than 90% inhibition of proliferation). However, subline C was much more sensitive to cytolysis (18h 125IUDR-release assay) by CP-PC; percentage specific release from L1210-C was at least 90%, whilst from L1210-M it was generally less than 25%. The differential susceptibility of the 2 sublines to cytolytic PC was maintained through 75 passages in culture. The effector cells were considered to be macrophages, because they were adherent, phagocytic, and sensitive to silica. Cytolysis was unrelated to endotoxin contamination, because it was not inhibited by polymyxin B, and was inhibited by pre-incubating PC in culture medium for 24 or 48 h before adding target cells. Thus the relevance of nonspecific macrophage-mediated cytotoxicity in vitro to tumour resistance in vivo may depend on the strength of the cytotoxic reaction.


Fifteen of the 31 animals infused with LPS and not treated with BN 52021, a PAF receptor antagonist, died within 30 min after the commencement of LPS infusion (non-survivors), while the other 16 survived the experimental period of 3 h, though in a state of shock (survivors). No alterations were observed in plasma concentrations of eicosanoids in the non-survivors. A significant, though transient, increase in eicosanoid concentrations occurred only in the survivors. Treatment with BN 52021  injected 5 min prior to LPS infusion, failed to exert any effect on the survival rate. However, pretreatment with BN 52021 prevented circulatory collapse in the survivors and reduced the concentration of cyclooxygenase enzyme products, without affecting LTB4 release. Exogenous administration of PAF (0.01 μg kg−1) caused hypotension and increased TXB2 levels although 6-keto PGF1α and LTB4 concentrations were unchanged. The data suggest that prostanoid formation may be secondary to PAF release in circulatory collapse evoked by LPS infusion in survivors, and give further support to the suggestion that PAF prostanoid interaction is important during endotoxic shock. However, their role in early death seems to be negligible, indicating the importance of other mediators.The effects of platelet activating factor (PAF) on eicosanoid release during endotoxic shock was investigated in anaesthetized pigs receiving 5 μg kg


Nipah virus targets human endothelial cells via NiV-F and NiV-G envelope glycoproteins, resulting in endothelial syncytia formation and vascular compromise. Endothelial cells respond to viral infection by releasing innate immune effectors, including galectins, which are secreted proteins that bind to specific glycan ligands on cell surface glycoproteins. We demonstrate that galectin-1 reduces NiV-F mediated fusion of endothelial cells, and that endogenous galectin-1 in endothelial cells is sufficient to inhibit syncytia formation. Galectin-1 regulates NiV-F mediated cell fusion at three distinct points, including retarding maturation of nascent NiV-F, reducing NiV-F lateral mobility on the plasma membrane, and directly inhibiting the conformational change in NiV-F required for triggering fusion. Characterization of the NiV-F N-glycome showed that the critical site for galectin-1 inhibition is rich in glycan structures known to bind galectin-1. These studies identify a unique set of mechanisms for regulating pathophysiology of NiV infection at the level of the target cell. in vivo, and demonstrate that endogenous galectin-1 made by endothelial cells contributes to limiting cell-cell fusion caused by NiV-F. As endothelial syncytia formation is one of the primary pathophysiologic events in Nipah virus infection, contributing to the hemorrhagic diathesis seen in infected patients, understanding the mechanism of endothelial cell fusion and the ability of galectin-1 to ameliorate cell fusion are critical for development of new approaches to mitigate these events.Nipah virus (NiV) is classified as a “priority pathogen” by the NIH. NiV infection of humans results in multi-organ hemorrhage due to endothelial syncytia formation, and also causes fatal encephalitis in up to 70% of patients. As there are no effective vaccines or therapeutics for NiV, understanding the mechanism of endothelial damage by NiV is a critical goal. Our present work defines the interaction between galectin-1, an innate immune lectin that is secreted by human endothelial cells, with the fusion glycoprotein of NiV. We demonstrate that galectin-1 can block the function of the NiV-F protein via three distinct mechanisms, and thus reduce the ability of NiV-F to cause endothelial cell-cell fusion. Importantly, in this study, we use human endothelial cells, the primary target of Nipah virus  Nipah virus (NiV) is a lethal emerging virus that infects agricultural livestock and humans. In 1999–2000, NiV infection of agricultural workers in Malaysia and Singapore resulted in a 40% mortality rate, and subsequent outbreaks in Bangladesh resulted in an average case-fatality ratio greater than 70% Paramyxoviridae, is an enveloped virus with two viral envelope glycoproteins, NiV-G and NiV-F, that mediate viral entry NiV, a member of a new genus of 0, is initially expressed at the cell surface as a single glycosylated polypeptide precursor, but subsequently undergoes endocytosis and endosomal proteolytic cleavage by cathepsin L into F1 and F2 subunits that are disulfide linked to form the mature fusion protein NiV-F1/2The NiV-F fusion glycoprotein, NiV-Fin vitro and in vivo activation of human endothelial cells increased synthesis as well as secretion and cell surface localization of galectin-1 Infection of endothelial cells by viruses results in the release of innate immune effectors, including galectins, a family of mammalian lectins We have previously shown that recombinant human galectin-1 inhibits cell fusion and syncytia formation caused by NiV-F in vivo. Since endothelial cell syncytia formation is a pathognomic feature of NiV infection, we investigated the mechanisms involved in galectin-1 modulation of cell-cell fusion. We found that galectin-1 regulates cell fusion at three distinct points in the process; galectin-1 retains immature NiV-F0 on the cell surface to reduce production of the mature NiV-F fusion protein, galectin-1 reduces lateral movement of NiV-F on the plasma membrane that is required for cell-cell fusion, and galectin-1 directly inhibits the fusogenic activity of NiV-F by preventing fusion-peptide exposure and pre-hairpin intermediate (PHI) formation. The biological significance of our results is underscored by our demonstration that endogenous galectin-1 on endothelial cells is sufficient to reduce NiV-F mediated fusion. These studies identify a unique set of mechanisms for regulating the pathophysiology of NiV induced syncytia formation at the target cell level, and contribute to our understanding of the interaction between galectin-1 and glycoproteins of microbial pathogens.In the present study, we demonstrate that galectin-1 regulates NiV-F mediated fusion of endothelial cells and neural cells, the targets of NiV infection In vivo, endothelial cells and neuronal cells are the main targets of Nipah virus We previously found that galectin-1 inhibits syncytia formation in Vero cells mediated by NiV-F and NiV-G Nipah virus infection results in extensive damage to endothelial cells as a result of syncytia formation, culminating in multi-organ hemorrhage and death. During viral infection, endothelial cells become activated and release immune mediators, including galectin-1 Conversely, in order to determine if endogenous galectin-1, even on resting HUVECs, was sufficient to affect NiV-F and G mediated syncytia formation, we reduced expression of galectin-1 in HUVECs using lentiviral vectors expressing siRNAs targeted against galectin-1. A combination of three siRNAs reduced galectin-1 protein approximately 70% (data not shown), and reduced cell surface galectin-1 approximately two-fold . ReductiTo confirm that cell surface galectin-1 was responsible for the effect on syncytia formation, we added exogenous galectin-1 to HUVECs in which galectin-1 expression was decreased by siRNA. We observed the expected increase of cell surface galectin-1 , as wellGFP was expressed on transfected Vero cells  in the presence or absence of galectin-1  to total NiV-F protein. Addition of galectin-1 decreased NiV-F processing by approximately 50% at the 6 hour time point . The PHI then undergoes six-helix bundle formation, which is the conformational change that physically drives fusion of opposing membranes 0 maturation, contributes to the mechanism of galectin-1 mediated inhibition of NiV-F mediated cell fusion.To detect triggering, wild type CHO cells or CHO cells stably expressing ephrinB2 (CHOB2) were mixed with CHO cells transfected with NiV-F and G at 4°C for 1.5 hrs; NiV-G binding to ephrinB2 is an energy independent process. The biotinylated HR2 peptide was added to the cells in the presence or absence of galectin-1, and fusion was induced by incubation at 37°C for an additional 1.5 hours, because PHI formation is an energy dependent process. Binding of the HR2 peptide indicates that triggering has occurred. We observed no triggering of NiV-F at 4°C, regardless of the presence of galectin-1 . In the in vivo (F2–F5) 0 and NiV-F1, glycomic screening was performed using MALDI-TOF mass spectrometry. The N-glycans were released by PNGase F and analyzed as their permethylated derivatives. The resulting spectra exhibit a series of singly charged sodiated molecular ions ([M+Na]+) to which putative structures are assigned based on the molecular compositions and knowledge of the N-glycan biosynthetic pathway. The profile for the complete propeptide, NiV-F0, can be seen in 0 include high mannose , complex  and hybrid structures . The complex and hybrid structures contain lactosamine  moieties that can be recognized by galectin-1. The MALDI-TOF mass spectrum of permethylated N-glycans from NiV-F1 is displayed in Supplementary 0 and NiV-F1 suggests that the glycosylation sites on the NiV-F2 subunit (F2 and F3 glycans) are modified with larger complex structures than those on NiV-F1 subunit (F4 and F5 glycans).The NiV-F fusion protein contains 5 consensus sites for N-glycosylation, labeled F1–F5; four of these predicted sites have been found to be glycosylated NNTHDLVGDVR) was observed at an elution time of ∼50 min (4+). Previous research has shown that alpha 2,6-linked sialic acid caps block galectin-1 binding while alpha 2,3 sialic acid caps do not . Treatment of the tryptic glycopeptides prior to the nano-LC ES-MS experiment with Sialidase S  revealed that both alpha 2,3 and alpha 2,6-linked sialic acid are present, with 2,3-linked sialic acid being the more abundant  This report defines several molecular mechanisms by which galectin-1 binding to NiV-F interferes with maturation and function of NiV-F to reduce cell fusion, and also demonstrates that endogenous endothelial galectin-1 can influence the extent of syncytia formation. Since syncytia formation is the pathognomonic hallmark of NiV infection, we predict that, 2 at 37°C. PK-13 porcine fibroblast cells and 293T cells were maintained in DMEM (Invitrogen) with 10% FBS (Hyclone) and 2mM Glutamax. BSR cells stably transfected with T7 polymerase were maintained in DMEM with 10% FBS (Hyclone), 2mM Glutamax, and 0.5mg/ml G418 (Sigma). U87 glioblastoma cells  were maintained in DMEM with 10% FBS (heat inactivated at 55°C for 30 min), 2mM Glutamax, and 50 units/ml penicillin/streptomycin. mVECs Vero cells and CHO cells (ATCC) were maintained in MEM alpha (Invitrogen) with 10% FBS (Hyclone) and 2mM Glutamax in 5%COE. coli and purified by affinity chromatography on lactosyl-Sepharose, as in Codon-optimized NiV-F and G plasmids tagged with AU1 were previously described GFP and HA-tagged NiV-G at 15ug per plasmid using Lipofectamine 2000 (Invitrogen). Cells were cultured overnight, lifted with 5mM EDTA (ethylenediaminetetraacetic acid) and overlayed in the absence or presence of galectin-1 onto ephrinB2 positive cells . After 2 hrs (Vero/U87) or 6 hrs (HUVEC/mVEC), cells were fixed with 2% paraformaldehyde (EMS). After 4′,6′-diamidino-2-phenylindole (DAPI) staining (Invitrogen), nuclei inside syncytia per ×100 field were counted by fluorescence microscopy as previously described PK-13 cells were transfected with codon-optimized, AU1-tagged NiV-F, NiV-F3, or NiV-FHUVEC cells were incubated with or without 20µM galectin-1 for 30 min at 37°C. Cells were fixed in DTSSP (Thermo Scientific) at 0.2mg/ml for 10 min at room temperature, and quenched by addition of 100µl of 1M Tris pH 7.5 for 15 min at room temperature. Cells were washed with PBS and lifted with 5mM EDTA at 37°C for 10 min. Cells were stained with a rabbit galectin-1 antibody (Strategic) for 1 hr at 4°C. Cells were washed with PBS and stained with FITC-conjugated AffiniPure goat anti-rabbit IgG (H+L) antibody (Jackson ImmunoResearch) at 8µg/ml for 1 hour at 4°C. Cells were washed in PBS and resuspended in PBS with 1% BSA (Gemini BioProducts) for analysis by flow cytometry. Flow cytometric analysis of HUVEC cell surface galectin-1 was performed on a Becton-Dickinson FACScan, using CellQuest software (Becton-Dickinson).5 cells per well in a 6 well tissue culture dish (Corning). A single well of HUVECs was treated with each virus  in PBS with 1% heat inactivated FBS (Hyclone) and cells were spinoculated at 2000 rpm at 37°C for 2 hrs. Infected cells were grown in full media for 4 days and knockdown was assessed by western blot and flow cytometry.3 different siRNA constructs directed toward human galectin-1 mRNA in lentivectors (pSIH1-H1-copGFP plasmid) (System Biosciences) were packaged into VSV using pPACKH1 Lentivector Packaging Kit (System Bioscience). Lentivirus was produced in 293T cells; three different viruses corresponding to the 3 different siRNAs were isolated, as well as a virus containing only the GFP infection marker, and a virus containing siRNA to an irrelevant protein, CD43. HUVECs were plated at 1.4×10GFP plasmid was performed in 35mm glass bottom culture dishes (MatTek) on a 37°C heated stage. Cells were treated with 20µM galectin-1 or buffer control for 10 min prior to photobleaching. Images were acquired on a confocal microscope (Leica SP2 1P-FCS) with a HCX PL APO 63.0×1.40 oil objective and fully opened pinhole. Photobleaching of NiV-F-GFP was performed using 5 scans with the 488-nm laser at full power. Recovery data (six cells per time point from each of two independent experiments) was collected every 2.2 seconds for a total of 25 time points. Images were acquired with equivalent acquisition settings including pre-bleach, bleach, and post-bleach measurements. Bleaching and recovery were measured in a fixed area and compared to an area absent of fluorescence outside of the cell.Fluorescence Recovery After Photobleaching analysis of Vero cells transfected with NiV-F2, 1.25mM MgSO4, 5mM Na2HPO4, 20mM Hepes pH 7.4) and cell surface biotinylated using the EZ-Link sulfo-NHS-SS-Biotin (Pierce). Biotinylation was quenched in 20mM glycine and cells were washed again in KRPH buffer. Cells were incubated in media with or without galectin-1 or buffer control for the designated times at 37°C to allow endocytosis. After timepoints, cells were washed and remaining biotin cleaved twice with cleavage buffer  and quenched in 20mM glycine for 15 min. Cells were lysed in lysis buffer . Biotinylated NiV-F in cell lysates was quantified by ELISA using mouse anti-AU1 (1∶1000) coated Reacti-Bind goat anti-mouse plates (Pierce) to capture AU1-tagged NiV-F, and detected with streptavidin-HRP (Biorad).PK-13 cells were transfected with NiV-F or NiV-F3. After overnight incubation, cells were washed twice with KRPH buffer  for 30 min, and then in chase (non-radioactive) media for 4 to 6 hrs, in the presence or absence of 20µM galectin-1. Cells were lysed in 200µl RIPA buffer . NiV-F was immunoprecipitated from cleared cell lysates using a combination of anti-NiV-F polysera 293T cells grown in 6-well plates were transfected with 0.25µg of NiV-F plasmid DNA and 1.75µg pcDNA3 per well. 24 hrs post transfection, cells were incubated in media lacking methionine and cysteine for 45 min followed by labeling with media containing Fusion-nonpermissive PK-13 target cells were transfected with codon optimized NiV-F, NiV-G, and a plasmid containing a T7 promoter driven luciferase at a 3∶3∶1 ratio with 30µg of total plasmid DNA and grown overnight. BSR cells stably transfected with a T7 polymerase (BSRT7) were lifted with 5mM EDTA at 37°C for 10 min. BSRT7 cells were co-cultured with transfected PK-13 cells for 6 hrs with or without galectin-1. After 6 hrs, the cells were lysed in 0.3% Triton-X 100 by two rounds of freeze/thaw at −80°C. Luciferase expression was quantified using a Luciferase assay system (Promega). Briefly, lysates were transferred to a 96 well opaque black plate, luciferase assay substrate was added, and light production was measured by luminometry (Turner Biosystems).PK-13 cells were transfected with NiV-F and simultaneously treated with chlorpromazine (Sigma) for 16 hrs. Cells were lysed in 50mM Tris-HCl (pH 7.4), 1% Nonidet P-40, 5mM EDTA, 150mM NaCl, 1mM PMSF, 10mg/ml aprotinin, 10mg/ml leupeptin, and 10mM sodium orthovanadate with scraping. Lysates were microfuged for 15 min at 10,000 rpm. Samples were denatured in NuPAGE reducing agent and NuPAGE Sample Buffer (Invitrogen) before loading. Lysates (10 µg) were separated on a 12% Bis-Tris gel (Invitrogen NuPAGE Electrophoresis System) and electroblotted onto nitrocellulose (Whatman). The membrane was blocked and probed as previously described NiV-F triggering was measured essentially as in Fusion kinetics were determined in a beta-lactamase reporter cell-cell fusion assay, as previously described 0 and NiV-F1 were destained using 100% acetonitrile, incubated with 10mM DTT for 30 min at 56°C, followed by 55 mM iodoacetic acid for 30 min at RT. Reduced and carboxymethylated NiV-F was digested with 0.5µg sequencing grade-modified trypsin  at 37°C for 14 hrs. Tryptic peptides and glycopeptides were extracted from the gel pieces by incubating with 0.1% trifluoroacetic acid (TFA) and 100% acetonitrile; the supernatant was pooled and reduced in volume on a rotary evaporator. For glycomic screening the supernatant was lyophilised before being dissolved in 200µl ammonium bicarbonate  and incubated with 5U of Peptide-N-glycosidase F (PNGase F)  for 24 hrs at 37°C to release the N-glycans. The reaction was terminated by lyophilisation. Glycans were separated from peptides by C18 Sep-Pak purification and permethylated as previously described Glycan analysis was performed on NiV-F protein pseudotyped onto VSV viral-like particles produced in 293T cells. Gel bands containing purified NiV-F+ using a 4800 MALDI-TOF/TOF  mass spectrometer as previously described MALDI-TOF MS and MS/MS data on permethylated N-glycan samples were acquired in positive ion mode [M+Na]Sample was dried, resuspended in 50µl of 50mM ammonium acetate (pH 5.5) and incubated with 10mU of Sialidase S at 37°C for 14 hrs. After digestion, NiV-F peptides and desialylated glycopeptides were desalted and separated using a C18-microtrap peptide cartridge . The sample was loaded directly onto the microtrap cartridge with a 25µl gastight syringe. The microtrap was first solvated with methanol, washed off with acetonitrile and conditioned with 0.1% TFA. The sample was loaded onto the column and washed with 0.1% TFA prior to eluting with 15µl 30 and 60% acetonitrile in 0.1% TFA, respectively. Eluted fractions were combined and dried down gently under nitrogen before online LC-ES-MS and MS/MS analysis.Figure S1A, Galectin-1 inhibits heterologous fusion in a dimer dependent manner. EphrinB2 positive cells stably expressing the T7 polymerase were added to a monolayer of ephrinB2 negative cells transfected with NiV-F, NiV-G and a luciferase construct with a T7 dependent promoter. Luciferase expression correlates with cell fusion. Data are shown as percent fusion based on the total fusion without any treatment (Lane 2). Lane 1 is a negative control and lane 3 is heterologous fusion with buffer alone. Lane 4, 30µM galectin-1. Lane 5, 10µM forced galectin-1 dimer (GG). Lane 6, 30µM monomeric galectin-1 mutant, N-Gal-1. B, Galectin inhibits heterologous fusion in a carbohydrate dependent manner. The galectin-1 effect of fusion inhibition was abrogated by addition of the cognate disaccharide lactose, but not by sucrose. In both panels, data are the mean + S.D. of a representative experiment performed in triplicate.Heterologous fusion is inhibited by galectin-1. Galectin-1 inhibits function of the mature fusion protein. (0.05 MB TIF)Click here for additional data file.Figure S21. Data were acquired in the positive ion mode and all molecular ions are [M+Na]+. Peaks labeled with * represent contaminating hexose polymers. Peak assignments are based on theoretical compositions together with knowledge of the biosynthetic pathways. Symbol nomenclature is that used employed by the Consortium for Functional Glycomics (CFG) for the representation of glycan structures. Key: Galactose (yellow circle), Mannose (green circle), GlcNAc (blue square), Fucose (red triangle), NeuAc, (purple diamond).MALDI-TOF mass spectrum of permethylated N-glycans from NiV-F(0.32 MB TIF)Click here for additional data file.Figure S3NQSLQQSK, observed as doubly, triply and quadruply charged molecular ion signals in the MS data summed between the ion retention times 51.8–52.9 min (data not shown). The m/z values in the table correspond to the smallest isotope in each cluster and the Mr values are calculated accordingly. Potential structures of the glycan compositions are given, deduced by taking into account prior glycomic experimental data and knowledge of the biosynthetic pathways. Key: Galactose (yellow circle), Mannose (green circle), GlcNAc (blue square), Fucose (red triangle), NeuAc, (purple diamond).The structure of the glycans found at the F5 glycosylation site. The table shows the glycan compositions of the glycopeptide, VDISSQISSM(0.65 MB TIF)Click here for additional data file.Figure S4NTTCPTAVLGNVIISLGK, observed as doubly, triply and quadruply charged molecular ion signals in the MS data summed between the ion retention times 84.7–86.8 min (data not shown). Key: Galactose (yellow circle), Mannose (green circle), GlcNAc (blue square), Fucose (red triangle), NeuAc, (purple diamond).The structure of the glycans found at glycosylation site, F4. The glycan compositions of the glycopeptide, AISQSGTLLMID(0.81 MB TIF)Click here for additional data file.


Cell-free Human T-cell Leukemia Virus type I (HTLV-I) virions are poorly infectious and cell-to-cell contact is often required to achieve infection. Other factors might thus importantly contribute in increasing infection by HTLV-I. Galectin-1 is a galactoside-binding lectin which is secreted by activated T lymphocytes. Several functions have been attributed to this protein including its capacity to increase cell-to-cell adhesion. Based on previous studies, we postulated that this protein could also accentuate HTLV-I infection.Herein, we demonstrate that galectin-1 expression and release are higher in HTLV-I-infected T cells in comparison to uninfected T cells. Furthermore, galectin-1 expression was activated in various cell lines expressing the wild type viral Tax protein while this induction was minimal upon expression of NF-κB activation-defective TaxM22. Cotransfection of these Tax expression vectors with galectin-1 promoter-driven luciferase constructs confirmed that Tax upregulated galectin-1 promoter activity. However, a NF-κB-independent mechanism was strongly favoured in this induction of galectin-1 expression as no activation of the promoter was apparent in Jurkat cells treated with known NF-κB activators. Using HTLV-I envelope pseudotyped HIV-1 virions, galectin-1 was shown to increase infectivity. In addition, a co-culture assay with HTLV-I-infected cells also indicated an increase in cell fusion upon addition of galectin-1. This effect was not mediated by factors present in the supernatant of the HTLV-I-infected cells.These data suggest that HTLV-I Tax increases galectin-1 expression and that this modulation could play an important role in HTLV-I infection by stabilizing both cell-to-cell and virus-cell interactions. The 0.5 kb and 1.2 kb galectin-1 promoter fragments were cut out of pBSK with SacI and NdeI enzymes and ligated into pGL3-Basic  digested by SacI and SmaI.A PCR-based approach was used to insert the luciferase gene under the control of the galectin-1 promoter. Genomic DNA was isolated from 293T cells with the QIAamp DNA Blood Mini Kit . Two fragments of the galectin-1 promoter region (0.5 kb or 1.2 kb) were amplified from 200 ng of genomic DNA by PCR with the forward primers gal-0.5 kb (5'-GTTAAGTCAGTGGCCCTCTGCAG-3') or gal-1.2 kb (5'-CAGAGGAGATGTTAAGAGAGCAGAC-3') and the reverse primer gal-as1 (5'-CGCACCAGCTGTCAGAAGACTCC-3'). PCR amplifications were then performed in the presence of 0.2 mM dNTPs, 1 μM of each primer, 1 U of Vent polymerase  through 35 cycles . The PCR products were purified with the QIAquick PCR purification kit  and ligated into the pBluescript SK (pBSK) vector in SmaI. Positive clones were sequenced and compared to the human galectin-1 promoter sequence , Molt-4, CEM-T4, PM1, Sup T1, C8166-45, C91-PL, MJ, MT2 and S1T cell lines or from transfected 293T cells was extracted with the TRIzol reagent . Extracted RNA (5 μg) was then reverse transcripted with the M-MLV reverse transcriptase (1 U)  and oligo dT primers. Next, PCR amplification was performed on the resulting cDNA with primers act-s (5'-CGTGACATTAAGGAGAAGCTGTGC-3') and act-as (5'-TCTAGGAGGAGCAATGATCTTGAT-3') for β-actin mRNA; gal-s (5'-GACTCAATCATGGCTTGTGGTCTG-3') and gal-as (5'-GCTGATTTCAGTCAAAGGCCACAC-3') for galectin-1 mRNA; or tax-s (5'-ATGGCCCACTTCCCAGGGTTTGGAC-3') and tax-as (5'-TCAGACTTCTGTTTCGAGGAAATG-3') for Tax mRNA. PCR amplifications were performed in the presence of 0.2 mM dNTPs, 1 μM of each primer, 1 U Vent polymerase and 30 amplification cycles . The PCR products were then migrated on a 1.5% agarose gel.® Plus mini Kit  according to the manufacturer's instructions. Real-time RT-PCR reactions were then performed in the presence of each specific primer. Briefly, RNA (5 μg) was reverse transcripted with the M-MLV reverse transcriptase (1 U) (Invitrogen) and oligo dT primers. PCR reactions were then initiated in a final volume of 10 μl containing 1 μl of cDNA, 0.5 μM of each primer, and 1× reaction mix, including Taq DNA polymerase, the reaction buffer, and SYBR green . All primer sequences were generated using the Light Cycler Probe Design Software 2.0  and checked for specificity using GenBank Blast analysis. The galectin-1 primers were the following: 5'-GACTCAATCATGGCTTGTGGTCTG-3' (reverse) and 5'-GCTGATTTCAGTCAAAGGCCACAC-3' (forward). In all PCR reactions, negative controls consisting of a RT-like reaction step with no added reverse transcriptase in addition to a blank sample were carried out and showed no PCR amplification (data not shown). Thermal cycling for quantification of both transcripts was initiated with a denaturation step of 95°C for 10 seconds, followed by 50 cycles . Amplification of the human HPRT-1 (Hypoxanthine Phosphoribosyl Transferase 1) cDNA with forward and reverse primers  was used as a reference gene for normalisation. To verify the amplification of each single product with its suitable melting temperature, and to provide an accurate quantification with the Rel Quant Software, dissociation curves were run for all reactions and amplified products were visualized by electrophoresis on a 1.5% agarose gel.RNA was first isolated from 293T transfected cells, by the RNeasy7) were transiently transfected by electroporation as previously described [Jurkat, CEM-T4 and SupT1 cells . Galectin-1 concentration was determined by an in house ELISA assay specific for galectin-1.A2.01, HSB-2, Jurkat (clone E6.1), Molt-4, PM1, CEM-T4, SupT1, C8166-45, C91-PL, MJ, MT2 and S1T cell lines were seeded at 5 × 105 cells) for 48 hours at 37°C before lysis. In certain experiments, 24 hours after transfection, TNF-α was added at a concentration of 10 ng/ml. Luciferase activity was next measured as previously described [HIV-1-based viruses pseudotyped with the HTLV-I envelope protein were prepared as previously described . Brieflyescribed . Experim5) were then added to an equal number of transfected Jurkat cells in a flat-bottom 96-well plate. Galectin-1 was added in various concentrations (ranging from 0 to 4 μM) in the absence or presence of 50 mM lactose for 24 hours at 37°C before lysis and quantification of luciferase activity. As a control, transfected cells were similarly incubated with supernatant of C91-PL cells harvested after a 24 hour incubation at a concentration of 1 × 106 cells/ml and filtered through a 0.22 μM filter. Values are expressed as the average luciferase activity +/- standard deviation calculated from triplicates.Jurkat cells were transfected with pHTLV-Luc by electroporation as described above. HTLV-I-infected C91-PL cells  . HomoscePrevious studies have suggested that expression of various genes are positively modulated in HTLV-I-infected cells ,57. In oAs some of the tested HTLV-I-infected cells have been reported to only express the viral Tax protein, we then looked if Tax expression indeed could modulate galectin mRNA levels. 293T cells were transfected with either a vector containing a complete HTLV-I proviral genome (i.e. K30), or expression vectors coding for Tax WT or Tax mutants defective in their ability to activate transcription factors NF-κB, SRF and/or CREB. Galectin-1 expression was then analyzed by RT-PCR. As shown in Figure Next, RT-PCR analyses were performed in a more representative context, i.e T cell lines. Hence, the wild-type Tax expression vector was transfected in CEM-T4 and SupT1 T cell lines and analysed by RT-PCR for galectin-1 expression. As denoted in Figure As the data suggest that HTLV-I Tax induces the expression of galectin-1 in non-T and T cell lines, it is likely that Tax plays a role in the modulation of galectin-1 mRNA levels in HTLV-I-infected cell lines.To determine whether the effect of Tax on galectin-1 expression resulted from direct activation of transcription from the galectin-1 promoter, two different luciferase-encoding vectors driven by the human galectin-1 promoter were constructed. Two fragments of 0.5 kbp and 1.2 kbp containing the transcription initiation site deduced from sequence homology with the mouse galectin-1 gene were derived from the human galectin-1 promoter region. Both fragments were cloned upstream of the luciferase reporter gene of the pGL3-Basic vector. Before determining the effect of Tax on these constructs, the Tax M22 expression vector was first tested in the context of Jurkat cells to see if it was specifically deficient in activating NF-κB Figure . These rLower induction of the galectin-1 promoter by TaxM22, which is deficient for NF-κB activation, raised the possibility that this transcription factor was crucial for Tax-mediated increase in galectin-1 expression. However, Jurkat cells transfected with the 1.2 kb galectin-1 promoter construct did not show higher luciferase activity upon stimulation with two known potent NF-κB activating agents, PMA and TNF-α, thereby strongly suggesting that NF-κB was not involved in the modulation of galectin-1 promoter activity by Tax Figure . As no kAs we have demonstrated that HTLV-I-infected cell lines express higher levels of galectin-1 mRNA, we next studied whether these cells produced more extracellular galectin-1. Figure Together, the data suggest that mRNA and secretion of galectin-1 were both upregulated in cells chronically infected with HTLV-I.As galectin-1 can stabilize cell-to-cell and cell-virus interactions by cross-linking different entities, we studied whether extracellular galectin-1 could facilitate HTLV-I infection. To initiate this study, Jurkat E6.1 cells were first infected with luciferase-expressing HIV virions pseudotyped with the HTLV-I gp46 envelope in the presence of various concentrations of purified galectin-1 (0–4 μM) for 48 hours; luciferase activity was then measured. The use of HTLV-I gp46-pseudotyped virions that can express luciferase allows us to detect a single round of infection and although different from wild-type HTLV-I virions, it should be representative of the type of interactions and fusogenic activities of gp46 occurring on the surface of HTLV-I virions upon infection. Infection of Jurkat E6.1 cells by the pseudotyped virions was increased by 1.6 fold in the presence of 2 μM of galectin-1, an increase which was statistically significant  Figure . LactoseA more physiological model was also used to study the impact of soluble galectin-1 on infection by HTLV-I pseudotyped virus. PBMCs isolated from a healthy donor were stimulated with IL-2 and PHA-L for 72 hours and, after washing, were then similarly treated upon infection by the HTLV-I gp46-pseudotyped virions. The infection of PBMCs by pseudotyped virions was increased by 1.8 fold in the presence of 4 μM of galectin-1 Figure . The posTo eliminate the possibility that galectin-1 was positively modulating LTR activity of the integrated proviral DNA of our gp46-pseudotyped virions, Jurkat cells were transfected with a vector containing the luciferase reporter gene under the control of the HIV-1 LTR, after which different concentrations of galectin-1 (0–4 μM) was added. Measurement of luciferase activity demonstrated that the presence of galectin-1 had no impact on the transcription levels dependent on the HIV-1 LTR (data not shown).Hence, these results show that extracellular galectin-1 increases infection of a T cell line and PBMCs by free HTLV-I gp46-pseudotyped viruses and that this increase relies on the binding of cell/virus surface carbohydrates by the galectin-1 CRD.To study whether galectin-1 can possibly facilitate cell fusion events, a co-culture system allowing a quantitative evaluation of cell fusion by luciferase assay was used . This ceThese results show that soluble galectin-1 can also increase cytoplasmic cell exchange likely occurring though gp46-dependent cell fusion events between an HTLV-I-infected cells and uninfected T cells, again being inhibited by the addition of lactose.HTLV-I is a poorly infectious virus and, in this regard, the presence of various molecules that facilitate infection may be important for viral transmission. Several studies have been conducted on the implication of adhesion molecules incorporated by retroviruses  and their positive impact on viral replication . SimilarIn this study, we have demonstrated that galectin-1 is more strongly expressed and secreted in chronically HTLV-I-infected T cell lines compared to uninfected T cells. These results agree with the study of Pise-Masison and colleagues, which showed through DNA microarray experiments that galectin-1 gene expression is upregulated in HTLV-I-transformed and immortalized cell lines . FurtherAs cell-free virus has very low infectivity, it is assumed that HTLV-I infection is mediated by the interaction between non-infected and infected cells, although recent evidence has demonstrated that cell-free virus can infect isolated dendritic cells -74. We hin vivo conditions are likely to differ from our cell culture settings. Previous studies have demonstrated that lymphoid organ tissues are sites where an important number of infected T lymphocytes are located [Although galectin-1 concentrations in our infection experiments were higher than the measured levels from the supernatant of infected cells,  located -77. Give located . MoreoveIn summary, our study demonstrates a bidirectional interaction between HTLV-I and galectin-1. The data demonstrated that expression of galectin-1 was increased in chronically HTLV-I-infected cells and that this modulation of galectin-1 expression was largely attributed to the viral transactivator Tax in NF-κB- and CREB-independent manners. In addition, this study showed that HTLV-I-infected cells secrete galectin-1 at a higher level than the uninfected cells and that extracellular galectin-1 facilitates HTLV-I infection and promotes higher levels of gp46-dependent cell fusion. Given that our previous studies had demonstrated that HIV-1 is also enhanced in its infectivity by galectin-1 ,36, otheThe authors declare that they have no competing interests.SG carried all RT-PCR analyses, transfection experiments and infection and syncytium formation assay and has drafted the manuscript. IP has conducted the ELISA assay. MO has participated in the design of the study. AV has performed the real-time RT-PCR experiments and has helped in drafting the manuscript. MJT and SS have helped in drafting and finalizing the manuscript and have provided important input on the design of the study. BB conceived the study, participated in its coordination and helped in drafting and finalizing the manuscript.


Cell Titer 96® AQueous One Solution cell proliferation assay was used to investigate the cytotoxicity of this cationic copolymer. Second, siRNAs targeting IKKβ (IKKΒ-siRNAs) were delivered into the HTFs using CS-g-(PEI-b-mPEG) as the vehicle. Real-time reverse transcription polymerase chain reaction (RT–PCR) subsequently assessed the mRNA level of IKKβ, and western blot assay was used to determine protein expression. After IKKB-siRNA transfection, Cell Titer 96® AQueous One Solution cell proliferation assay was used to evaluate the proliferation of HTFs.First, a novel cationic copolymer composed of low molecular weight, linear poly(ethyleneimine) [PEI] blocked with polyethylene glycol (PEG) and grafted onto a chitosan (CS) molecule was synthesized. CS-g-(PEI-b-mPEG)/siRNA complexes tended to decrease whereas their zeta potential tended to increase as the N/P ratio increased. The CS-g-(PEI-b-mPEG) copolymer showed good siRNA binding ability and high siRNA protection capacity. Furthermore, the copolymer presented remarkable transfection efficiency and showed much less cytotoxicity than 25 kDa PEI. IKKB-siRNAs were successfully delivered into HTFs using CS-g-(PEI-b-mPEG) as a vector. As a result, the expression of IKKβ was downregulated at both the mRNA and protein levels, and the activation of nuclear factor-κB (NF-κB) in the HTFs was subsequently inhibited. Most impressively, the proliferation of HTFs was also effectively suppressed through the blocking of the NF-κB pathway.The diameter of the CS-g-(PEI-b-mPEG) is a promising candidate for siRNA delivery, featuring excellent biocompatibility, biodegradability, and transfection efficiency. The RNA interference (RNAi) strategy using cationic copolymers as siRNA carriers will be a safe and efficient anti-scarring method following glaucoma filtration surgery.All the results demonstrate that CS- RNA interference (RNAi) was originally recognized as an evolutionary conserved defense mechanism in higher eukaryotic cells, and this system can easily and effectively inhibit the expression of one specific gene . The RNAThe efficiency of RNAi mainly depends on the successful delivery of intact siRNA into mammalian cells. However, due to the low stability of siRNA against enzymatic degradation and low permeability across cell membranes, the efficacy of naked siRNA is insufficient. Therefore, it is necessary to develop efficient and convenient methods for siRNA delivery. Until now, viral delivery systems have been used as vectors for genes in many studies due to the advantage of high transfection efficacy, but the use of such delivery systems is limited by endogenous recombination and host immunity . MoreoveGlaucoma is an eye disease usually associated with increased intraocular pressure that leads to irreversible functional impairment of the optic nerve. Filtration surgery to enhance the drainage of aqueous humor is one of the most effective therapies for glaucoma , but theg-(PEI-b-mPEG), was synthesized. The properties of the CS-g-(PEI-b-mPEG)/siRNA complexes such as particle size, zeta potential, siRNA binding and protection capacity, transfection ability, and cytotoxicity were studied, and IKKΒ-siRNAs were then delivered into human Tenon’s capsule fibroblasts (HTFs) using CS-g-(PEI-b-mPEG) as the vehicle. The expression of IKKβ was detected at both the mRNA and protein levels after the transfection of IKKΒ-siRNAs. We also investigated the activation of NF-κB and the proliferation of HTFs after the RNA interference process targeting IKKβ.In the study reported here, absorbable, low molecular weight PEI was blocked with polyethylene glycol monomethyl ether (mPEG) and grafted onto chitosan. As a result, a novel biodegradable copolymer, CS-2, 95% humidified atmosphere.HeLa  cells were obtained from the American Type Culture Collection  and were maintained in Dulbecco’s modified Eagle’s medium  containing 10% fetal bovine serum , 2 mM of L-glutamine, 100 IU/ml of penicillin, 100 μg/ml of streptomycin, and 25 μg/ml of amphotericin B  at 37 °C with 5% CO2, 95% humidified atmosphere in DMEM supplemented with 10% FBS, 2 mM of L-glutamine, 100 IU/ml of penicillin, 100 μg/ml of streptomycin, and 25 μg/ml of amphotericin B. Cells between passages 3 and 6 were used for the following experiments.Tissue explants of human Tenon’s capsule were obtained from three male patients  who had undergone trauma or cataract surgery. Patients were informed of the nature and possible consequences of the tissue removal procedure, and written, informed consent was obtained. The tenets of the Declaration of Helsinki were followed, and approval by the Sun Yat-sen University Human Experimentation committee was granted. HTFs were cultured by a previously reported method  with som5 [Mw=3.50×105], which was measured by the viscosity method and the degree of deacetylation of chitosan was 88%, which was determined by proton nuclear magnetic resonance [1H NMR]), was purified by a solvent precipitation method. Polyethylene glycol monomethyl ether  was purchased from Sigma-Aldrich Chemie Gmbh . Linear PEI (Mn=600) and branched PEI  were obtained from Wako Pure Chemical Industries, Ltd. . CS-g-(PEI-b-mPEG) was synthesized by Jiang’s method with some modification [b-mPEG, was synthesized by an imine reaction, and then the periodate ion, IO4-, was used to oxidize CS to produce dialdehyde. A novel, comb-like copolymer, CS-g-(PEI-b-mPEG), was synthesized by an imine reaction between the amino groups of PEI-b-mPEG and the aldehyde groups of periodate-oxidized CS. The resultant product was purified by dialysis against double deionized (DD) water with the use of Spectra/Pro2 membrane  for 72 h to remove the unreacted PEI and mPEG and then freeze-dried for another 24 h. A schematic illustration of the synthesis process is shown in g-(PEI-b-mPEG) was proved by 1H NMR and gel permeation chromatography (GPC).Chitosan,  and incubated for 5 min at room temperature, and the CS-g-(PEI-b-mPEG) copolymer was dissolved in serum-free DMEM at a stock concentration of 1 mg/ml. Subsequently, different concentrations of the CS-g-(PEI-b-mPEG) solution were added into dissolved siRNA to make complexes of various charge ratios. The charge ratio of CS-g-(PEI-b-mPEG) and siRNA was expressed as the molar ratio of the amine groups of the copolymer (representing positive units) to the phosphates of siRNA (representing negative units) called the N/P ratio. The mixture was gently vortexed for 10 s and incubated at room temperature for 30 min to allow the formation of CS-g-(PEI-b-mPEG)/siRNA complexes.IKKΒ-siRNA, scrambled siRNA, and fluorescein isothiocyanate (FITC)-conjugated scrambled siRNA were all synthesized by Ribobio Co. Ltd. . Lyophilized siRNAs were dissolved in RNase-free Hg-(PEI-b-mPEG)/siRNA complexes with N/P ratios of 1, 3, 5, 10, and 20. The hydrodynamic diameter of the freshly prepared complexes was measured at 25 °C with a scattering angle of 90° , and the zeta potential was determined by the standard capillary electrophoresis cell of Zetasizer Nano ZS at position 17.0 and at 25 °C. All the average values were performed with the data from three separate measurements.Dynamic light scattering (DLS) with a Zetasizer Nano ZS instrument  was used to measure the diameter and zeta potential of the CS-g-(PEI-b-mPEG) and siRNA was determined by 4% agarose (low melting point) gel electrophoresis [g-(PEI-b-mPEG) were considered as N/P ratio=0), 1, 3, 5, 10, and 20 as described above, and samples, each containing 0.133 μg (1×10−2 nmol) of siRNAs, were loaded onto 4% agarose gel together with 1:6 dilution of the loading buffer. The electrophoresis was performed in TBE buffer  at 55 V for 1 h. To visualize the siRNA, the gel was immersed in 0.5 µg/ml of ethidium bromide solution (Sigma-Aldrich), and the fluorescence images were captured under ultraviolet (UV) illumination .The binding degree between CS-phoresis . The comg-(PEI-b-mPEG) to protect siRNA against enzymatic degradation was investigated according to a previously reported method with minor modifications [g-(PEI-b-mPEG)/siRNA complexes containing the same amount of siRNAs were prepared at an N/P ratio of 0 (naked siRNA), 1, 3, 5, 10, and 20. Subsequently, FBS was added as needed to achieve a final concentration of 10%. The mixtures were incubated at 37 °C, and at each determined time interval , a sample of each N/P ratio was removed and incubated at 70 °C for 5 min to inactivate the serum enzymes. Then, 5 µl of heparin (1000 IU/ml) was added to displace the siRNAs from the complexes before the mixtures were loaded onto a 15% polyacrylamide gel containing 7 M urea. Electrophoresis was performed in TBE buffer at 200 V for 1 h. Afterward, gels were stained in a 1:10,000 dilution of SYBR® Green II fluorescent RNA dye  for 40 min, and Bio-Capt version 10.0 software  was used to analyze the fluorescence intensity of each band. All experiments were performed in triplicate, and the fluorescence intensity of each band was compared with that of the non-FBS treated siRNAs (which served as the control) on the same gel.The ability of CS-ications . The CS-5 cells per well and incubated for 12 h or 24 h (reaching 60%–70% confluence). The culture media were then replaced with DMEM without serum or with antibiotics 2 h before transfection. The CS-g-(PEI-b-mPEG) and FITC-conjugated siRNA complexes were prepared as described above, and the N/P ratios were performed at 0 (naked FITC-conjugated siRNAs), 1, 3, 5, 10, and 20. A total volume of 2 ml of serum and antibiotics-free DMEM-containing complexes was added to each well, and the final concentration of siRNAs was 50 nM. After a 6 h incubation at 37 °C, the remaining media were discarded, and cells were trypsinized and resuspended in PBS at a density of 5×105 cells/ml for flow cytometry analysis. The transfection efficiency was calculated by measuring the percentage of FITC-labeled cells using a FACSAriaTM System .HeLa cells and HTFs were plated in six well plates with a density of 6×10® AQueous One Solution cell proliferation assay  was used to evaluate cell viability [3 cells per well and incubated for 24 h (reaching 80% confluence) before the copolymers were added. Then, serum-supplied DMEM were replaced by 200 µl of serum and antibiotic-free DMEM that contained various concentrations  of CS-g-(PEI-b-mPEG) or 25 kDa PEI. The CS-g-(PEI-b-mPEG) or 25 kDa PEI/siRNA complexes containing 100 nM of siRNAs were prepared at an N/P ratio of 0 (only scrambled siRNAs), 1, 3, 5, 10, and 20, and their cytotoxicity was also evaluated. After 24 h incubation at 37 °C, the media were replaced with fresh serum and antibiotic-free DMEM that contained 20 μl of Cell Titer 96® AQueous One Solution Reagent (MTS). Finally, after 4 h of additional incubation, a micro-plate reader  measured the absorbance of each well at 570 nm. Cell viability was calculated according to the following equation: Cell Titer 96iability . HTFs we570(sample) represents the average absorbance of cells treated with media that contain different concentrations of cationic polymers or cationic polymers/siRNA complexes, and OD570(control) represents the average absorbance of cells treated only with an equal volume of serum-free DMEM.where ODIKKβ (IKKΒ-siRNA) were derived from the coding sequence of the human IKKβ gene (GenBank NM_001556) and were designed using a siRNA Target Finder program. A BLAST search checked all the duplex sequences and target sequences of these siRNAs to preclude sequences with significant similarity to other genes in the human genome. The duplex sequences of IKKB-si1 were 5′-CCG ACA UUG UGG ACU UAC AdT dT, dTd TGG CUG UAA CAC CUG AAU GU-5′, and the duplex sequences of IKKB-si2 were 5′-GCU UAG AUA CCU UCA UGA AdT dT, dTd TCG AAU CUA UGG AAG UAC UU-5′. HTFs were plated in six well plates with a density of 6×105 cells per well and incubated for 12 h. Subsequently, the culture media were replaced with serum- and antibiotic-free DMEM 2 h before transfection. CS-g-(PEI-b-mPEG)/IKKΒ-si1 and CS-g-(PEI-b-mPEG)/IKKΒ-si2 complexes were prepared at an N/P ratio of 10 30 min before transfection, and cells were incubated with serum- and antibiotic-free DMEM that contained complexes corresponding to the determined final concentrations of IKKΒ-si1 or IKKΒ-si2  for 6 h. Then, the cells were maintained in serum-supplied DMEM for another 24 h or 48 h before the following assays were performed as described below. Non-transfected HTFs were regarded as the control, and cells were also transfected with 100 nM of scrambled siRNA.Two pairs of siRNA specifically targeting 5 to 2×105 HTFs 24 h after the transfection medium was removed using the RNeasy Micro Kit  according to the manufacturer’s protocol. The yield and purity of the RNA were spectrophotometrically determined, and the cDNA were prepared using the ReverAidTM First Strand cDNA Synthesis Kit . A real-time reverse transcription polymerase chain reaction (RT–PCR) procedure was conducted according to the manufacturer’s protocol for the SYBR® Premix Ex TaqTM Kit . Reaction participants were assembled in a 96 well optical reaction plate , and each well contained SYBR® Premix Ex TaqTM (2X), 200 nM of forward primer, 200 nM of reverse primer, ROX Reference Dye (50X), and cDNA solution with a total volume of 20 μl. For IKKβ, the forward primer was 5′-TGT CAG TGG AAG CCC GGA TAG-3′, and the reverse primer was 3′-AGG TTA TGT GCT TCA GCC ACC AG-5′. The mRNA level of glyceraldehydes-3-phosphate dehydrogenase (GAPDH) was also measured in each sample as an internal control. The forward primer was 5′-ATC ACC ATC TTC CAG GAG CGA-3′, and the reverse primer was 3′-CAG AAG TGG TGG TAC CTC TTC C-5′. Reactions were performed under the following conditions: 10 min at 95 °C for the initial denaturation, 40 cycles of amplification (5 s at 95 °C), and annealing for 31 s at 60 °C, using the ABI Prism 7000 Sequence Detection System (Applied Biosystems). The threshold cycle (Ct) values were determined by ABI Prism 7000 Software (Applied Biosystems) and were normalized by subtracting the Ct GAPDH values. All experiments were performed in triplicate, and the relative amount of mRNA of each sample was calculated using the 2-ΔCt method in individual experiments [Total RNA was extracted from 1×10eriments .Each group of HTFs was lysed in lysis buffer  48 h after the transfection procedure. An equal amount of protein (10 µg) was loaded on 12% sodium dodecyl sulfate-polyacrylamide gel, and electrophoresis was performed for 1 h. The proteins were then electrophoretically transferred to a polyvinylidene diflouride (PVDF) membrane  for probing with mouse monoclonal anti-IKKβ  and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG . Blotting signals were detected by chemiluminescence reagents using an ECL kit  following the manufacturer’s instructions. The β-actin protein amount of each sample was also measured as an internal control.5 cells per well and incubated for 24 h to allow adhesion. Then, 100 nM of IKKΒ-siRNA or 100 nM of scrambled siRNA were transfected into HTFs as described above, and after another 24 h, the cells were stimulated with 20 ng/ml of tumor necrosis factor-α (TNF-α) for 1 h. All the coverslips were taken out 24 h after the TNF-α stimulation and were rinsed three times with PBS. The cells were then fixed by incubation with 4% paraformaldehyde solution at room temperature for 10 min followed by 10 min permeabilization by 0.2% Triton X-100 (Sigma-Aldrich). After blocking the nonspecific binding with goat serum for 30 min, all the samples were incubated with mouse monoclonal anti-p65 of NF-κB  and FITC-conjugated goat anti-mouse IgG  at 37 °C for 1 h, both under light exclusion. The nuclei of the cells were counterstained with 2 µg/ml of DAPI  at room temperature for 10 min under light exclusion. A Zeiss LSM 510 confocal laser scanning device  was used to capture the inflorescence images. Cells not treated with either CS-g-(PEI-b-mPEG)/siRNA complexes or TNF-α were considered the negative control, and cells treated with only 20 ng/ml of TNF-α for 1 h were measured as the positive control.HTFs prepared for the confocal microscopy study were seeded onto preloaded glass coverslips (18 mm×18 mm) in six well plates with a density of 6×10® AQueous One Solution cell proliferation assay. After siRNA transfection, each group of HTFs was trypsinized and seeded in a 96 well plate with an initial density of 5×103 cells per well. Subsequently, cells were cultured in a serum-supplied medium for 72 h, and the absorbance of each sample corresponding to the cell number was measured.The proliferation of HTFs was measured using Cell Titer 96t-test or one-way analysis of variance (ANOVA). Probability (p) of less than 0.05 was considered significant.All data are presented as means±standard deviation (SD). The statistical analyses were conducted using SPSS version 13.0 for windows . Statistical analysis was performed using Student’s q) was calculated by the following equation from the 1H NMR spectrum:CS proton signals appeared at 2.84 ppm (H-2) and 3.3−3.8 ppm  were 1.16×104 and 2.95×104, and the degree of grafted PEI-b-mPEG can be calculated by the following equation according to the data above:From the results of GPC , the molW,CS-CHO is the average molecular weight of repeat unit of CS-CHO.M’g-(PEI-b-mPEG)/siRNA complexes ranged between 200 nm and 250 nm at the determined N/P ratios  and siRNA was assessed by visualizing the mobility of the CS-g-(PEI-b-mPEG)/siRNA complexes using electrophoresis retardation assay. As shown in g-(PEI-b-mPEG) was measured by incubating the CS-g-(PEI-b-mPEG)/siRNA complexes with 10% FBS for 2 h, 4 h, 8 h, 12 h, and 24 h. The remaining intact siRNA was visualized by polyacrylamide gel electrophoresis (PAGE), and the images of the gels are shown in The degree of compaction between CS-g-(PEI-b-mPEG) or 25 kDa PEI/siRNA complexes were added. As shown in g-(PEI-b-mPEG) and 25 kDa PEI increased as the N/P ratio increased. The transfection efficiency of CS-g-(PEI-b-mPEG) reached its peak (more than 60%) at the N/P ratio of 20 in both cell lines. However, it could not be calculated when 25 kDa PEI was used for transfection at the same N/P ratio because most cells had been killed. A tetrazolium-based viability assay, which was based on the bioreduction of the MTS reagent into formazan by living cells, was used to study the cytotoxicity of CS-g-(PEI-b-mPEG) and 25 kDa PEI. The pure CS-g-(PEI-b-mPEG) copolymer exhibited cell viability of more than 70% at concentrations of 1–100 μg/ml, and the number of viable cells was significantly higher than those treated with pure 25 kDa PEI at concentrations of 10 μg/ml and above (g-(PEI-b-mPEG)/siRNA and 25 kDa PEI/siRNA complexes presented less toxicity after being compacted with siRNAs, and the CS-g-(PEI-b-mPEG)/siRNA complexes also performed decreasing cytotoxicity at all the determined N/P ratios compared with that of the 25 kDa PEI/siRNA complexes.Transfection efficiency was evaluated by measuring the percentage of HeLa cells or HTFs containing FITC-conjugated siRNA, using flowmetry 6 h after CS-nd above . Figure IKKβ mRNA (data not shown), so we used IKKΒ-si1 as IKKΒ-siRNA in the subsequent RNAi procedures. Real-time PCR assay revealed that mRNA transcription of IKKβ in the HTFs was suppressed in a dose-dependent manner 24 h after 5–100 nM of IKKΒ-siRNA were transfected  as shown in The RNAi process targeting Glaucoma filtration surgery often fails because of excessive scarring that occurs during the wound healing process. After filtration surgery, fibroblasts are activated by various cytokines and growth factors, eventually resulting in the closure of the drainage channel. Thus, preventing the hyperfunction of fibroblasts is an important strategy for minimizing subconjunctival scarring ,21. CurrIKKβ using a commercial transfection reagent, LipofectamineTM 2000 , as a siRNA-delivering vector. However, this cationic liposome compound cannot be used for in vivo investigation in humans [One goal of our study was to find new physiologic approaches to limiting excessive subconjunctival wound healing that are as effective as antimetabolic agents but have fewer adverse effects. NF-κB is a member of the transcription factor family NF-κB/Rel and was originally described as a necessary element for the transcription of the immunoglobulin κL chain gene in mature B cells . NF-κB hn humans .Therefore, another objective of our study was to design and synthesize new compounds that are non-toxic, non-immunogenic, degradable, and efficient for delivering siRNA into HTFs. Among non-viral nucleotide carriers currently under investigation, PEI is one kind of synthetic polymer with a high cationic charge density and a protonable amino group in every third position. PEI can condense and compact DNA into complexes, and the strong proton capacity of PEI allows it to deliver plasmid DNA and oligonucleotides into mammalian cells, both in vitro and in vivo . However1H NMR spectrum of the copolymer, respectively. CS proton absorption signals  also overlapped the PEI and mPEG proton signals (g-(PEI-b-mPEG) copolymer was successfully obtained. The molecular weight of CS-g-(PEI-b-mPEG) was 2.95×104, which was relatively larger than some reported PEI-alt-PEG copolymers [In light of these results, we speculated that a novel cationic copolymer combined with PEI, PEG, and CS could be synthesized as a vehicle for delivering siRNA into mammalian cells effectively and safely. In our research, we found that after the grafting reaction, characteristic absorption of PEI and mPEG appeared at 2.5−3.3 ppm and 3.5 ppm in the  signals . All of polymers  but smalpolymers .g-(PEI-b-mPEG)/siRNA complexes at five different N/P charge ratios and observed that the complexes represented particles with nanometer size (about 200 nm), which were much less than that of 25 kDa PEI. Furthermore, particle size tended to decrease as the N/P charge ratios of the CS-g-(PEI-b-mPEG)/siRNA complexes increased, indicating that the CS-g-(PEI-b-mPEG) copolymer can condense siRNA into a more compact structure, mainly owing to the net electrostatic repulsive forces between complexes.As indicated in previous reports, the size and shape of nanoparticles play an important role in the delivery process and greatly influence distribution in the body . It has g-(PEI-b-mPEG)/siRNA complexes is also a major factor influencing transfection efficiency. After the compaction between cationic copolymers and siRNA, the negative charge of siRNA is neutralized and the newly assembled nanoparticles may retain a partial positive surface charge to help siRNA pass through the cell membrane and escape the endolysosomes. However, the excessive positive charge of PEI homopolymers may subsequently lead to hyperpermeability of the membrane, resulting in cell death [g-(PEI-b-mPEG) and the negative charge was neutralized. But the zeta potential of the CS-g-(PEI-b-mPEG)/siRNA complexes at each N/P ratio that we measured was still lower than that of the 25 kDa PEI/siRNA complexes. The reason for this result is probably consistent with the reason Petersen et al. [The surface charge of the CS-ll death . The denn et al.  regarding-(PEI-b-mPEG) and siRNA can achieve more efficient delivering capacity. If the complexes are formed efficiently, all siRNAs are bound to the copolymers to form nanoparticles. Hence, the complexes become relatively large and remain immobile in the loading well with no bands of free siRNA apparent. We observed that the migration of siRNA was retarded to different degrees in accordance with the increasing N/P ratio. Complete retardation occurs at an N/P ratio of 10, which means the CS-g-(PEI-b-mPEG)/siRNA complexes are completely formed. The complete complexes of 600 kDa PEI and siRNA cannot be found even at an N/P ratio of 50 (data not shown), which indicates that the condensation ability of CS-g-(PEI-b-mPEG) is better than low molecular weight PEI. It was also detected that the band of siRNA at an N/P ratio of 20 demonstrated much lower fluorescence intensity than that of the band at the N/P ratio of 10. This decrease in fluorescence was also observed by other experiments executed on DNA bands [Condensation ability is one requirement for a siRNA carrier. An optimal binding degree between CS-NA bands ,45. The NA bands .g-(PEI-b-mPEG) we have synthesized has both PEG and CS elements and can provide efficient protection for siRNA against enzymatic degradation.The main hindrance to the use of RNAi as a therapeutic tool for human diseases is that the unprotected dsRNA or siRNA will be rapidly degraded by either nucleases in serum or the endosomal compartment of cells. The enzymatic degradation of siRNA is accompanied by a rapid decline in biological activity and therapeutic efficiency. Therefore, the potential of this technology as a clinical therapy method depends largely on the improvement of siRNA vectors’ protection ability against enzymatic degradation. We found that after incubation in 10% FBS for 24 h, 64% of siRNA was protected from degradation at the N/P ratio of 20 whereas only 0.7% naked siRNA remained intact. We attributed these results to two factors. First, PEG not only has the reported ability to stabilize the structure of nanospheres, but PEG can also protect siRNA from being attacked by nucleases ,47. Secog-(PEI-b-mPEG) and 25 kDa PEI on the cell viability of HTFs. CS-g-(PEI-b-mPEG) showed much less cytotoxicity than 25 kDa PEI, which is consistent with what had been obtained by Kim et al. [g-(PEI-b-mPEG) copolymer did not exhibit significant cytotoxicity at any determined concentration. We hypothesize that the relatively low cytotoxicity of CS-g-(PEI-b-mPEG) could be explained from two aspects. First, the copolymer can be degraded into CS, PEG, and low molecular weight PEI units in cells, all of which can be easily eliminated by excretion pathways, thus making this copolymer relatively less toxic than 25 kDa PEI. Second, PEG reduces toxicity by substituting the amino groups of PEI, which are the main toxic moieties of the copolymer [g-(PEI-b-mPEG)/siRNA and 25 kDa PEI/siRNA complexes showed much lower cytotoxicity, which is due to the neutralization effect of the negative charge of siRNA on the positive charge of the pure polymers.Low toxicity is also a major requirement for an siRNA delivery system. The cytotoxicity of cationic copolymers is mainly caused by the aggregation of nanoparticles on the cell membrane, impairing its normal function. In addition, the excessive positive surface charge of nanoparticles may also interfere with critical intracellular processes of cells . It has m et al.  in testsopolymer . Furtherg-(PEI-b-mPEG)/siRNA complexes was assessed at various N/P ratios in HeLa cells and HTFs. This is mainly because the N/P ratio is directly related to the size, surface charge, compaction degree, and serum-resistant capacity of the CS-g-(PEI-b-mPEG)/siRNA complexes, all of which can affect delivery efficiency. At the N/P ratio of 20, CS-g-(PEI-b-mPEG) outperformed the highest transfection rate in both HeLa cells and HTFs, which can be explained by the appropriate particle size and surface charge as well as the excellent stability of the CS-g-(PEI-b-mPEG)/siRNA complexes. The transfection rate was a little lower than that of 25 kDa PEI at the same N/P ratio partly because of the shielding effect of mPEG on the positive charge of PEI. We also found that both CS-g-(PEI-b-mPEG) and 25 kDa PEI showed a lower transfection rate in HTFs than in HeLa cells, which may be attributed to a cell line dependency of the cationic polymer’s delivery ability [g-(PEI-b-mPEG)/siRNA complexes, and as a result, the CS-g-(PEI-b-mPEG)/siRNA nanoparticles can offer a substantial gene silencing effect with minimal side effects. Moreover, many factors governing the transfection efficiency of cationic copolymers need to be investigated in future studies such as the presence of serum and the pH value of solution [The transfection efficiency of the CS- ability . From ousolution , and ligIKKβ gene was successfully compacted with CS-g-(PEI-b-mPEG) and effectively delivered into HTFs. Subsequently, both the mRNA and protein levels of IKKβ were suppressed, and the activation of NF-κB was inhibited in turn. Finally, IKKΒ-siRNA-mediated blocking of the NF-κB pathway resulted in repression of the proliferation of HTFs, and cells transfected with IKKΒ-siRNA showed growth inhibition up to 42%. All of these findings suggest that blocking the signaling pathway of NF-κB could be an effective way to manipulate the scar formation process by downregulating the proliferation of HTFs. This novel method based on nanotechnology and RNAi could represent a remarkable anti-scarring therapeutic approach for glaucoma filtration surgery. Follow-up studies will focus on the in vivo application of CS-g-(PEI-b-mPEG)/IKKΒ-siRNA complexes.A special siRNA targeting g-(PEI-b-mPEG). We found it to have powerful siRNA binding and protection ability, relatively high transfection efficiency, and low cytotoxicity, making CS-g-(PEI-b-mPEG) a suitable delivery vector for transfecting siRNA into cells. We observed that siRNA targeting IKKβ was successfully transfected into human Tenon’s capsule fibroblasts in vitro, and RNAi processes against the expression of IKKβ can subsequently inhibit the activation of NF-κB and in turn, the proliferation of HTFs. Our results indicate the potential for a safe and effective strategy for preventing scar formation after glaucoma filtration surgery.We have reported on the synthesis and characterization of a novel cationic copolymer, CS-


Background: This study was aimed at evaluating advantages of distal splenorenal shunt (DSRS) with splenopancreatic and gastric disconnection (DSRS-SPGD) over DSRS with splenopancreatic disconnection (DSRS-SPD) and standard DSRS (S-DSRS).Methods: DSRS-SPGD, DSRS-SPD, and S-DSRS were performed on 62, 7, and 55 patients, respectively, from 1970 to 1992. Comparison was performed in the following aspects: (1) long-term results in ratio of rebleeding, survival rate, and quality of life and (2) portal hemodynamics evaluated by preoperative and postoperative angiography. Portal blood flow was assessed by the ratio of the diameter of portal vein (PV) to superior mesenteric vein (SMV), and shunt selectivity was evaluated by selectivity grade.Results: Incidence of rebleeding was significantly lower in patients who underwent DSRS-SPGD than in those who underwent S-DSRS (p< 0.05). Grade 0 and I performance status was better in patients who underwent DSRS-SPGD. Accumulated survival ratio for 5 and 7 years was 78.3% and 70.5% in patients who underwent DSRS-SPGD, 59.7% and 44.1% in patients who underwent S-DSRS, and 75% and 75% in patients who underwent DSRS-SPD. Hemodynamic evaluation showed significantly lower PV/SMV ratio and degree of change in PV/SMV ratio of patients who underwent S-DSRS and DSRS-SPD. Many patients who underwent S-DSRS and DSRS-SPD exhibited loss of shunt selectivity at grades II and III. In contrast, patients who underwent DSRS-SPGD maintained satisfactory PV/SMV ratio and selectivity grade.Conclusions: DSRS-SPGD clearly showed advantages in decrease of rebleeding and improvement of quality of life resulting from maintenance of shunt selectivity and portal blood flow.