We and others have previously documented that the uPAR88-92 sequence, even in the form of synthetic linear peptide (SRSRY), interacts with the formyl peptide receptor type 1 (FPR1), henceforth inducing cell migration of several cell lines, including monocytes. FPR1 is mainly expressed by mammalian phagocytic leukocytes and plays a crucial role in chemotaxis. In this study, we present evidence that the cyclization of the SRSRY sequence generates a new potent and stable inhibitor of monocyte trafficking. In rat basophilic leukaemia RBL-2H3/ETFR cells expressing high levels of constitutively activated FPR1, the cyclic SRSRY peptide ([SRSRY]) blocks FPR1 mediated cell migration by interfering with both internalization and ligand-uptake of FPR1. Similarly to RBL-2H3/ETFR cells, [SRSRY] competes with fMLF for binding to FPR1 and prevents agonist-induced FPR1 internalization in human monocyte THP-1 cells. Unlike scramble [RSSYR], [SRSRY] inhibits fMLF-directed migration of monocytes in a dose-dependent manner, with IC50 value of 0.01 nM. PMA-differentiated THP-1 cell exposure to fMLF gradient causes a marked cytoskeletal re-organization with the formation of F-actin rich pseudopodia that are prevented by the addition of [SRSRY]. Furthermore, [SRSRY] prevents migration of human primary monocytes and trans-endothelial migration of monocytes. Our findings indicate that [SRSRY] is a new FPR1 inhibitor which may suggest the development of new drugs for treating pathological conditions sustained by increased motility of monocytes, such as chronic inflammatory diseases.The receptor for the urokinase-type plasminogen activator (uPAR) is a widely recognized master regulator of cell migration and uPAR We document that both linear SRSRY and [SRSRY] peptides compete with fMLF for binding to FPR1, thus preventing agonist-induced FPR1 internalization in THP-1 cells. However, these peptides exert opposite effect on monocyte motility, the linear SRSRY promotes cell migration, while the peptide [SRSRY] inhibits cell migration in a dose-dependent manner, with IC50 value of 0.01 nM. Finally, we show that [SRSRY] prevents trans-endothelial migration of monocytes and causes a marked inhibition of cytoskeletal re-organization occurring during locomotion.Based on this information, we reasoned that cyclization of the Ser-Arg-Ser-Arg-Tyr peptide could reduce conformational flexibility of its linear form, thus generating a new, more stable peptide that could regulate uPARTable 1was obtained as described in the Material and Methods, using the strategy depicted in the  The stability of the linear peptide Ser-Arg-Ser-Arg-Tyr (SRSRY) and cyclized peptide Ser-Arg-Ser-Arg-Tyr [SRSRY] in human serum was investigated through incubation of the peptide at a 10–2 mol/L in human serum and samples were taken at different time points and analysed by Reversed-phase chromatography (RP-HPLC). Serum-dependent degradation of peptides was monitored for up to 24 hours. The experiments revealed a gradual degradation of SRSRY through serum protease over time, whereas [SRSRY] retained an evident higher stability. As shown in Fig 1, after one hour of exposure to serum, SRSRY and [SRSRY] peptides had a residual concentration lower than 75% and 85%, respectively. After 24 hours, [SRSRY] showed a residual concentration higher than 80% while for compound SRSRY the concentration was lower 50% .Cyclization of peptides listed in Fig 2A). In contrast, FPR1 expressing RBL-2H3/ETFR cells exhibit a specific binding that was reduced to the basal level by unlabelled fMLF as well as SRSRY, as expected. We found that the peptide [SRSRY] inhibits binding of fluorescent agonist to RBL-2H3/ETFR cells to a similar extent as compared to fMLF or SRSRY . To evaluate the effect of [SRSRY] on agonist-dependent FPR1 internalization, binding experiments were performed at 37°C. Upon exposure of RBL-2H3/ETFR cells to FITC-fMLF, FPR1 appeared mainly internalized as indicated by punctuate green fluorescent intra-cytoplasmic spots which were prevented by cell pre-incubation with 100 nM fMLF or 100 nM SRSRY, as expected. An excess of [SRSRY] prevented agonist-dependent FPR1 internalization . Thus, we assessed whether [SRSRY] elicits effects on fMLF-directed cell migration. RBL-2H3/ETFR cell migration toward 10 nM fMLF was monitored in real-time for 4 hours as Cell Index changes due to the adhesion of migrating cells to microelectrodes using the xCELLigence RTCA technology as previously described  did not modify cell growth up to 90 hours, as shown by proliferation curves automatically monitored with the xCELLigence system . These findings indicate that SRSRY is a ligand of FPR1, also when it is in a cyclized form though exerts an opposite effect on cell migration.We have previously documented that SRSRY competes with the bacterial-derived N-formyl-methionyl-leucyl-phenylalanine peptide (fMLF) for binding to FPR type 1 (FPR1) and that both SRSRY and fMLF promote cell migration by triggering FPR1 internalization , 28. To escribed . In the Fig 3A). When binding experiments were performed at 37°C, FPR1 appeared mainly localized within the cytoplasm, adjacent but outside the nucleus of THP-1 cells. Internalization of fluorescent agonist was dramatically reduced in all cell population when THP-1 cells were pre-incubated with an excess of unlabelled fMLF, or [SRSRY] while the control peptide [RSSYR] did not exert such effect . Although we did not determine the binding affinity of [SRSRY] for FPR1, our findings indicate that the peptide [SRSRY] prevents fMLF/FPR1 interaction and inhibits agonist-induced FPR1 internalization in THP-1 cells.Once documented that both SRSRY and [SRSRY] peptides compete with fMLF for binding to FPR1, we investigated the effect of [SRSRY] on the motility of monocytes. Preliminarily, we assessed whether [SRSRY] is a ligand of FPR1 on monocyte cell surface. Similarly to RBL-2H3/ETFR cells, THP-1 cells exposed to 10 nM FITC-fMLF at 4°C, revealed an appreciable binding which was dramatically reduced by an excess of unlabeled fMLF, SRSRY or [SRSRY], but not by the control peptide [RSSYR] . Inhibitory effect of [SRSRY] is dose-dependent, it starts in the high fM range, it seems to level off in the μM range and reaches an overall 50% reduction at 10 pM . Interestingly, a dose-dependent inhibition is exerted by [SRSRY] also on migration of PMA-differentiated THP-1 cells with IC50 value 10 pM . The mechanisms by which monocytes move when subjected to a chemoattractant gradient, involve changes in cytoskeletal organization, which provide both protrusive and contractile forces necessary for cell migration. To gain some insights into the cellular effects exerted by peptide [SRSRY], PMA-differentiated THP-1 cells were grown adherent onto a glass slide, exposed to diluents (CTL), 10 nM fMLF gradient plus/minus 10 nM [SRSRY] or 10 nM [RSSYR] in a DUNN chamber for 6 hours as described [Fig 4C). In order to verify whether the inhibitory effect of [SRSRY] also occurs on primary monocytes and/or macrophages, human monocytes and macrophages were subjected to cell migration assays in Boyden and DUNN chambers, respectively. As shown in Fig 4, 10 nM fMLF (panel D) or 10 nM SRSRY (panel E) elicited a considerable monocyte migration, reaching 644% and 273% of the basal cell migration (CTL), respectively. Unlike [RSSYR], 10 nM [SRSRY] reduced both fMLF- and SRSRY- directed monocyte migration by 50% and 49%, respectively. Next, primary macrophages were subjected to cell migration assay in DUNN chamber as above described. After 6 hours incubation, a total of 100 cells/sample that translocated to the area corresponding to the ring separating the inner and outer chambers were counted. Macrophage exposure to 10 nM fMLF elicited a considerable cell migration, reaching 230% of the basal cell migration (CTL), that was unchanged by 10 nM [RSSYR] . On the contrary, the addition of 10 nM [SRSRY] to the fMLF gradient, reduced migration of primary macrophages to the basal level . These findings indicate that unlike linear SRSRY, the cyclic form of the chemotactic sequence of uPAR inhibits monocyte motility and prevents cytoskeletal re-organization occurring during migration of macrophages.The ability of [SRSRY] peptide to affect THP-1 cell migration was analyzed in Boyden chambers using 10 nM fMLF or 10 nM SRSRY as chemoattractants. Not surprisingly, 10 nM fMLF and SRSRY elicited a considerable cell migration, reaching 211% and 172% of the basal cell migration (CTL), respectively. [SRSRY] at 10 nM concentration, reduced monocyte migration toward 10 nM fMLF or 10 nM SRSRY by 58% and 68%, respectively, whereas [RSSYR] did not exert any effect . To examine the changes in morphology in more detail and quantify monocytes interacting with endothelial monolayer, we performed a subset of experiments using GFP-tagged THP-1, labeling co-cultures for F-actin and recording images by a confocal microscope. In the presence of diluents, or [RSSYR], GFP-tagged THP-1 cells formed F-actin rich lamellipodia and pseudopodia (arrows) which disappeared when GFP-tagged THP-1 were exposed to 10 nM [SRSRY] . Z-stack analysis of confocal images revealed transmigration of THP-1 cells underneath the endothelium (dotted arrows) . In contrast, when the peptide [SRSRY] was added to the co-culture, the majority of THP-1 were seen to rest on top of endothelial cells . Also, a 40% reduction of GFP-THP-1 cell number interacting with endothelial monolayer was achieved by the addition of 10 nM [SRSRY] . These findings indicate that [SRSRY] not only prevents monocyte interaction with endothelium but also reduces their trans-endothelial migration. To further ascertain if [SRSRY] reduces trans-endothelial migration of monocytes, the ability of THP-1 cells to cross an endothelial monolayer was analyzed using the xCELLigence RTCA technology as previously described .Diapedesis of leukocytes plays a key role in the pathogenesis of inflammatory diseases and migration of monocytes from the blood into the sub-endothelial space is one of the earliest events –30. We iescribed . HUVECs Taken together, these data indicate that FPR1-mediated [SRSRY] inhibitory effect involves a marked inhibition of cytoskeletal re-organization occurring during locomotion and trans-endothelial migration of monocytes.Migration of leukocytes across the wall of blood vessels into tissues is one of the key events during inflammation, immune response against infection and tissue remodeling, representing a key process in the pathogenesis of chronic inflammatory diseases. To date, several new targets and inhibitors that mediate leukocyte motility are being developed –32. Howe88Ser-Arg-Ser-Arg-Tyr92 chemotactic sequence even in the form of a synthetic, linear peptide (SRSRY). Herein we provide evidence that the cyclization of the 88Ser-Arg-Ser-Arg-Tyr92 chemotactic sequence of uPAR generates a new peptide [SRSRY] exerting an opposite effect on cell migration, as compared to its linear form. [SRSRY] is a new potent inhibitor of monocyte locomotion with IC50 value of 0.01 nM. Moreover, [SRSRY] displays higher resistance to enzymatic digestion as compared to other uPAR-derived peptide inhibitors  inhibits cell migration by antagonizing FPR1 biologic activity, [SRSRY] may be considered as a potent and stable FPR1 inhibitor which may suggest the generation of new pharmacological treatments for diseases sustained by an excess of monocyte trafficking, such as chronic inflammatory diseases and cancer.Nα-Fmoc-Tyr(tBu)-OH (1.0 equiv) dissolved in 5 mL of DCM to the resin. DIPEA (1.0 equiv) was added to this mixture that was stirred in an automated shaker for 10 min. Then, DIPEA (1.5 equiv) was added again. The mixture was agitated vigorously for 1 h at rt. To endcap any remaining unreacted 2-chlorotritylchloride groups, a mixture of DCM/MeOH/DIPEA (80:15:5) was added and mixed for 1 h. To remove Fmoc from the first amino acid, the resin was suspended in 25% piperidine solution in DMF (1 x 5 min and 1 x 25 min). The following protected amino acids were then added stepwise, Nα-Fmoc-Ser(tBu)-OH and Nα-Fmoc-Arg(Pbf)-OH. Each coupling reaction was accomplished using a 3-fold excess of amino acid with HBTU (3 equiv) and HOBt (3 equiv) in the presence of DIPEA (6 equiv). The N-terminal Fmoc protecting group was removed as described above. The resin was washed with DMF (3x) and DCM (3x) and dried in vacuo. The peptide was released from the resin by a mixture of DCM/AcOH/TFE (80:10:10) for 1 h. The resin was removed by filtration and linear crude peptides were recovered by precipitation in chilled diethyl ether to give pale yellow powders. Head-to-tail cyclization was performed by dissolving the linear peptide in 10 mL of DCM/DMF 1:1 under continuous agitation. Then, DIPEA (2.4 equiv) was added and after 15 minutes HATU (1.2 equiv) and HOAt (1.2 equiv) were added, thus the resulting reaction mixture was stirred for 12 hours to afford the head-to-tail cyclized peptides  of serum were removed at time points varying from 0, 1, 2, 4, 6, and 24 hours and acetonitrile (300 mL) was added to each aliquot before centrifuging . Aliquots (100 mL) of the supernatant were then analyzed by RP-HPLC after passing through Phenomenex Luna 100A C18 5μ, linear gradient from 0–90% acetonitrile in water over 25 minutes [The stability of SRSRY and [SRSRY] was investigated in human serum. The peptides were incubated at 37°C in human serum at a concentration of 10 minutes .2 at 37°C.Rat basophilic leukaemia RBL-2H3, and RBL-2H3/ETFR cells  were gro7 cells were re-suspended in 300 μL complete RPMI 1640 medium containing 20 mM Hepes and incubated with 5 μg pEGFP-N1 vector (Clontech) for 5 minutes on ice before being electroporated at 320 V, 1500 μF. Following electroporation, cells were kept on ice for 5 minutes then grown in 20 mL media for 24 hours. Then, G418-resistant cells expressing the highest levels of GFP were isolated and amplified.THP-1 differentiation into macrophages was induced using 160 nM phorbol-12-myristate acetate (PMA) purchased by Sigma Aldrich for 72 hours. To generate Green Fluorescent Protein (GFP)-tagged THP-1 cells, 2 x 102 to allow monocyte adherence. Plates were then gently washed to remove non-adherent cells, and monocytes were allowed to differentiate for 7 days in RPMI 1640 medium containing 10% autologous human serum.Blood was freshly drawn from healthy volunteers after informed written consent. Human peripheral blood mononuclear cells were harvested following centrifugation of whole blood on a density gradient solution composed of sodium metrizoate and dextran 500  according to the manufacturer’s instructions. The plasma and mononuclear cells were individually collected. The pellet containing mononuclear cells was washed with PBS and transferred to tissue culture plates for 2 hours at 37°C with 5% CO6cells/sample) were pre-incubated with diluents or the indicated unlabeled peptides for 60 minutes at 4°C , extensively rinsed with phosphate buffer saline (PBS), exposed to 10 nM N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein (FITC-fMLF) purchased from Molecular Probes, diluted in PBS, for additional 60 minutes at 4°C and again rinsed with PBS. Quantification of cell-associated fluorescence was assessed by reading cells with a fluorescence plate reader Victor 3 (Perkin Elmer) using 485 nm excitation and 535 nm emission filters.RBL-2H3 cells, RBL-2H3/ETFR cells stable expressing FPR1, or THP-1 cells (1.5x106cells/sample) were incubated with the indicated unlabeled peptides for 30 minutes at 37°C and then exposed to 10 nM FITC-fMLF diluted in PBS for 30 minutes at 37°C as described  or [RSSYR], the last used as a scramble, control peptide. The two compartments were separated by an uncoated 5 μm pore size filter, in the case of THP-1 cells and primary monocytes, or by a collagen-coated (50 μg/mL for 2 hours at 37°C) 8 μm pore size polycarbonate filter (Neuroprobe) for PMA-stimulated THP-1 cells. Incubation, carried out at 37°C in humidified air with 5% CO2 was 90 minutes for THP-1 cells, and 3 hours for PMA-stimulated THP-1 cells. At the end of the assay, cells on the lower filter surface were fixed with ethanol, stained with haematoxylin and 10 random fields/filter were counted at 200x magnification.Migration of RBL-2H3/ETFR cells was analyzed using CIM-16-well plates and the xCELLigence RTCA technology. CIM plates are provided with interdigitated gold microelectrodes on bottom side of a filter membrane which is interposed between a lower and an upper compartment. The lower chamber was filled with 10 nM fMLF diluted in DMEM or growth medium with/without 10 nM [SRSRY]. Cells (3x10escribed . Cell su3/well) were seeded in 16-well plates in growth medium and left to growth for 90 hours in the presence or the absence of 10 μM [SRSRY] or diluents. Microelectrodes placed on the bottom of plates, detect impedance changes which are proportional to the number of adherent cells and are expressed as Cell Index. The impedance value of each well was automatically monitored by the xCELLigence system and expressed as a Cell Index value. Growth medium with/ without [SRSRY] was replaced every 24 hours. The experiments were performed at least twice in quadruplicate.RBL-2H3/ETFR cell proliferation was assessed using E-16-well plates and the xCELLigence technology  as described . BrieflyPMA-stimulated THP-1 cells or primary macrophages were seeded on 20x20 mm coverslips for 24 hours. Before inverting the cover slip on top of a double concentric DUNN chamber, cells on the cover slip covering the outer chamber were carefully scraped away as previously described . A gradi4 cells in 200 μL/well) were seeded onto matrigel and allowed to attach for 3 hours at 37°C, 5% CO2. Coverslips were then flooded with endothelial growth medium and incubated for at least 24 hours before the experiments Then, GFP-THP-1 cells (1x104 cells/well) were exposed to diluents, or the indicated peptides in complete endothelial medium and added to endothelial monolayer at 37°C. After 30 minutes, slides were fixed, stained with rhodamine conjugated phalloidin and finally analyzed using an inverted fluorescence microscope  or a confocal microscope (LSM510), both purchased by Carl Zeiss.Analysis of monocyte diapedesis was performed by seeding GFP-tagged THP-1 cells on an endothelial monolayer according to Ronald et al. . To prov4 cells/well) suspended in growth medium, were seeded in E-16-well plates and allowed to grow for ~24 hours until they form a confluent monolayer, prior to seeding THP-1 cells (1x104 cells/well) in growth medium plus/minus 10 nM [SRSRY]. When HUVECs are challenged with invading cells, there is a drop in electrical resistance within 2–10 hours which is monitored in real-time as the Cell Index changes due to invasion of the endothelial monolayer. The experiments were performed twice in quadruplicate.These assays were performed using E-16-well plates and the xCELLigence RTCA technology (Acea Bioscience) as described . MicroelThe results are expressed as the means ± SD of the number of the indicated determinations. Data were analyzed by one-way analysis of variance and post hoc Bonferonni’s modified t-test for multiple comparisons. p <0.01 was accepted as significant.The research work with human blood samples has been approved by Institutional Ethical Committee of Istituto Nazionale Tumori “Fondazione G. Pascale”-IRCCS, Naples, Italy (protocol CEI 44/13).S1 FigCartoon showing the synthetic strategy employed to synthetize cyclic peptides. Details are included in the Materials and Methods.(PDF)Click here for additional data file.S2 FigR: 7.603  over 25 minutes, flow rate of 1.0 mL/min); molecular formula: C27H46N12O8, calculated mass: 666.26, API2000 ESIMS: m/z 667.36 [M + H]+; [SRSRY]—Purity: 95.41%, tR: 8.127  over 25 minutes, flow rate of 1.0 mL/min); molecular formula: C27H43N11O8, calculated mass: 649.19, API2000 ESIMS: m/z 650.20 [M + H]+; [RSSYR]—Purity: 95.76%, tR: 8.512  over 25 minutes, flow rate of 1.0 mL/min); molecular formula: C27H43N11O8, calculated mass: 649.19 API2000 ESIMS: m/z 650.20 [M + H]+.SRSRY—Purity: 98.83%, t(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.


Arabidopsis over the past 10 years have started to shed some light on the regulatory mechanism of the S response. Key advances in transcriptional regulation , involvement of hormones  and identification of putative sensors  are highlighted here. Although our current view of S nutrient sensing and signaling remains fragmented, it is anticipated that through further studies a sensing and signaling network will be revealed in the near future.Plants have evolved a sophisticated mechanism to sense the extracellular sulfur (S) status so that sulfate transport and S assimilation/metabolism can be coordinated. Genetic, biochemical, and molecular studies in  Consistent with this, several MYB transcription factors, in particular MYB28 and MYB29 which are transcriptionally repressed by S-deficiency, have been shown critical for transcriptional regulation of genes for the biosynthesis of glucosinolate which potentially serves as a critical S storage compound  and NITs (likely involved in auxin synthesis) under S deficiency , indicating a role for auxin transport as well as auxin biosynthesis or response in S signaling  and O-acetylserine (thiol) lyase  and sel1-16 (G208D). These two mutations lie in the predicted transmembrane (TM) helices TM11 and TM5. In contrast to all prior studies in which up-regulation of S response genes in sel1 mutants were interpreted as the result of compromised SO42– uptake and consequently lower accumulation of internal SO42– or its metabolites  could be independent of the compromised SO42– uptake and internal S status of the mutants (42– (10 mM) which did not lead to a difference in internal SO42– concentration and GSH level, sel1-15/16 and a null allele (sel1-18) still had higher gene expression level than their wild-type (WT) backgrounds. Second, treatments with 1 mM Cys or 1 mM GSH in the SO42– deficiency medium  also led to higher gene expression level in sel1-15/16/18. These results strongly suggest that the sel1 seedlings (in particular the expression in roots) grown under sufficient S behave as if they have been treated by certain degrees of S deficiency. In other words, the mutations in SULTR1;2 reduce sensitivity to the S-induced suppression of S response genes. The evidence points toward a novel function for SULTR1;2 in regulating S nutrient response besides its transport function. The possibility that SULTR1;2 acts as an S sensor is discussed in the next section.Most recent genetic and physiological evidence obtained from our groups have shown that besides its high affinity transport function, SULTR1;2 has a novel regulatory function . Using Babolites , we have mutants . First, Dual function transporters, like SULTR1;2 described above, are not unusual. Studies in yeast and animal nutrient transport and sensing have revealed the existence of classic receptors , transceptors  and the majority of common transporters  or TM5 (sel1-16) could abolish both SO42– transport and signaling (as measured by expression of S response genes), but the defect in signaling could be independent of SO42– transport and accumulation  and exert the effect of S sensing in planta? How does SULTR1;2 link to various signaling intermediates acting at the PM, the cytoplasm or the nucleus? Are there additional partners that may form a larger SULTR1;2-based S sensing complex? If such complex cannot account for all S responses, what other sensors are involved? Further, what are the roles of these sensing components in local and systemic S signaling (Exciting findings in the past 10 years have led to the identification of several components from the PM to the cytoplasm and to the nucleus that are involved in S sensing, transport and downstream response. Several outstanding questions remained to be answered. What is the SULTR1;2 topology and does SULTR1;2 have separate sensing and transport domains? Can SULTR1;2 interact with OASTL ignaling ? AlthougThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The human T-lymphotropic virus type 1 (HTLV-1) which is associated with the diseases of adult T-cell leukemia/lymphoma, HTLV-1 associated myelopathy / tropical spastic paraparesis (HAM/TSP) and HTLV-associated uveitis, can cause transfusion-transmitted infections. Although HTLV screening of blood donors was already routinely performed in developed countries, little is know about the HTLV prevalence among blood donors in developing countries which do not perform HTLV screening, such as China.To systematically characterize the prevalence of HTLV infection among bloods in southeast China.A 10-year survey for HTLV prevalence in blood donors was performed in Xiamen, southeast China, during 2004-2013. The HTLV-1/2 of blood donations were screened by enzyme-linked immunosorbent assay, following with confirmation by western blot assay and 9nucleic acid testing. The HTLV-1 prevalences in donors from different cities were calculated. Viral sequences derived from identified HTLV-positive cases were sequenced and analyzed.Among 253,855 blood donors, 43 were confirmed to be seropositive for HTLV-1  and none HTLV-2 infection was found. The HTLV-1 prevalence varied significantly in donors from different cities. Donors from cities in Fujian province  had a significantly higher (p=0.001) HTLV-1 seroprevalence than those who were born in non-Fujian cities . Among nine cities in Fujian province, the highest prevalence was found in blood donors from Ningde  which is a coastal city in the northeast of Fujian. Molecular characterization of viral sequences from 27 HTLV-1 carriers revealed 25 were Transcontinental subtype of genotype A and 2 were Japanese subtype of genotype A. Interestingly, 12 of 25 Transcontinental subtype sequences harbored a characteristic L55P mutation in viral gp46 protein, which was only presented in the Transcontinental subtype sequences from Japan and Taiwan but not in that from other countries.Although China is considered to be a non-endemic region for HTLV, the HTLV-1 prevalence in blood donors is significantly higher in Fujian province, southeast China. A higher prevalence of HTLV-1 in the Fujian may be attributed to endemic foci in the city of Ningde. The human T-lymphotropic virus type 1 (HTLV-1) which is associated with the diseases of adult T-cell leukemia/lymphoma, tropical spastic paraparesis etc., can cause transfusion-transmitted infections, it also can be transmitted by sex or breastfeeding. Globally, approximately 20 million people are estimated to be infected by HTLV-1, and 90% of them remain asymptomatic carriers during their lives. Previous studies had revealed that Japan, Central and Western Africa, the Caribbean islands and Central and South American were the regions with the highest HTLV-1 prevalence in the world. Little is know about the HTLV prevalence in China. We performed a 10-year blood screening survey to systematically characterize the prevalence of HTLV infection among bloods in Fujian province in southeast China since 2004. The HTLV-1 prevalence in blood donors is significantly higher in southeast China, especially in the northern cities of Fujian province, such as Ningde. Moreover, similar molecular characteristics of prevalent HTLV-1 sequences in southeast China, Taiwan and Japan suggested a same origin of these viruses. Human T-lymphotropic virus (HTLV) is a retrovirus which has been known for about 35 years since it was first isolated from a patient with a T-cell malignancy . PreviouGlobally, approximately 20 million people are estimated to be infected by HTLV-1, and 90% of them remain asymptomatic carriers during their lives . PreviouThe study was conducted in accordance with the guidelines of the 1975 Declaration of Helsinki and the principles of good clinical practice. All procedures were approved by the Medical Ethical Committee of Xiamen Blood Services. Written informed consent was obtained from all subjects.From 15 February 2004 and 31 December 2013, all voluntary blood donors at the Xiamen Blood Service were regularly tested for HTLV-1/2 infection.An anti-HTLV-1/2 enzyme-linked immunosorbent assay  was used for initial screening of the infection. The assays were performed on an ELISA STARlet automated system  according to the manufacturers’ instructions. The assay had been validated to be sensitive and specific as previously described . All posTreponemapallidum (the syphilisspirochete), and anti-hepatitis C virus (anti-HCV) antibodies were detected by the use of the Murex ELISA products .Other blood screening markers, including hepatitis B surface antigen (HBsAg), anti-human immunodeficiency virus (HIV), anti-After signing an informed consent, a follow-up samples were collected from all blood donors infected by HTLV. Peripheral blood mononuclear cells (PBMCs) were separated from whole blood and stored at -80°C. Proviral DNA of HTLV-1 in PBMCs were extracted by using QIAamp DNA blood kit , and then were subjected to real-time PCR detection as previously described . Real-tienv gene, collected from a public HTLV-1 molecular epidemiology database (http://htlv1db.bahia.fiocruz.br) [All HTLV-1gp46 sequences obtained in this study and 28 reference sequence in the GenBank were selected for phylogenetic analysis by neighbor-joining with Maximum Composite Likelihood corrected distances in the MEGA4 package, using 1000 bootstrap replicates. The sequences obtained in this study and another 251 HTLV-1 reference sequences derived from different geographical areas and different populations containing viral cruz.br) , were anFor donors with multiple donations during the period, only the screening results of the first samples were calculated. Statistical analyses were performed by the Mantel-Haenszel χ2 test and Fisher’s exact test for categorical variables. Differences were considered significant at a 2-tailed p<0.05. SPSS software version 17.0 was used for the all statistical analyses.Treponemapallidum, 1 was co-infected with HCV, none was found to be co-infected with HBV or HIV. The overall prevalence of HTLV-1 infection was 16.9 per 100,000 .A total of 253,855 donors were tested for HTLV infection, 43 among them were positive for antibody against HTLV-1, none was positive for anti-HTLV-2, as suggested by ELISA and confirmed by Western blot . All 43 The demographic characteristics of the donors were summarized in The HTLV-1 gp46 gene from 27 of the 43 HTLV-1 infected donors were successfully amplified and sequenced. Phylogenetic analysis indicated that 25 sequences  were clustered into Transcontinental subtype of genotype A and 2 remaining sequences (FJ05-FZ and FJ19-ZZ) were clustered into Japanese subtype of genotype A .http://htlv1db.bahia.fiocruz.br). The comparison indicated that the L55P mutation was only observed in Transcontinental subtype strains originated from Japan , Taiwan  and China , but not appeared in strains isolated from Transcontinental subtype strains originated from other Asia countries (0/3), Africa (0/6), Central America (0/29), Europe (0/22), North America (0/12), South America (0/38), but also in other subtypes of genotype A (0/67) or genotype B/C (0/40).The coded amino acid sequences of the isolated gp46 genes were further analyzed . Ten strThis was the first longitudinal and more complete coverage study about HTLV infection of blood donors in China mainland. The key findings include: HLTV-1 infection persist in many cities of Fujian Province, the southeast coast of China. None HTLV-2 was found. The prevalence in blood donors was 16.9 per 100,000 (95% CI: 12.3–22.8), Ningde people up to 171.3(95% CI: 91.3–292.8); molecular analyses demonstrated most of HTLV-1 isolates  in our study belong to Transcontinental subtype of genotype A; 12 of 25 Transcontinental subtype sequences harbored a characteristic L55P mutation in viral gp46 protein, which was only presented in the Transcontinental subtype sequences from Japan and Taiwan but not in that from other countries.The HTLV prevalence varies significantly in different geographic areas. The World Health Organization (WHO) has advised that decisions regarding the screening of blood donations for HTLV be guided by local epidemiological evidence. In most Asia countries except Iran and Japan, the HTLV infection appears rare. However, epidemiological data of most areas of Asia, particularly of the China mainland, were very little due to the lack of large-size and representative studies . In ChinOur study revealed that the HTLV-1 prevalence in donors from Fujian province  was about 7-fold  higher than that in donors from non-Fujian provinces . These data confirmed previous findings regarding Fujian was a HTLV endemic region in China. Furthermore, for the first time, we found the HTLV-1 prevalence significantly varied among donors from nine different cities in Fujian province and it was the highest in donors from Ningde . Statistical analysis demonstrated the HTLV-1 prevalence among donors from Ningde was significantly higher than any other cities of Fujian , and two neighboring cities (Nanping and Sanming) of Ningde also presented relatively higher prevalence . A new sMolecular analyses demonstrated most of HTLV-1 isolates  in our study belong to Transcontinental subtype of genotype A. Interestingly, two cases  carrying Japanese subtype of genotype A were found for the first time in China. Previous study had revealed that the Japanese subtype was a predominant viral strain in some populations of Japan and was also found in neighboring Taiwan . The preIn summary, the HTLV-1 prevalence in blood donors is significantly higher in southeast China, especially in the northern cities of Fujian province, such as Ningde. A surveillance system should be implemented to evaluate residual risk of transfusion-transmitted HTLV-1 infection in different regions of China. Moreover, similar molecular characteristics of prevalent HTLV-1 sequences in southeast China, Taiwan and Japan suggested a same origin of these viruses.S1 Checklist(DOC)Click here for additional data file.S1 Table(XLS)Click here for additional data file.


Structurally similar short peptides often serve as signals in diverse signaling systems. Similar peptides affect diverse physiological pathways in different species or even within the same organism. Assuming that signals provide information, and that this information is tested by the structure of the signal, it is curious that highly similar signaling peptides appear to provide information relevant to very different metabolic processes. Here we suggest a solution to this problem: the synthesis of the propeptide, and its post-translational modifications that are required for its cleavage and the production of the mature peptide, provide information on the phenotypic state of the signaling cell. The mature peptide, due to its chemical properties which render it harmful, serves as a stimulant that forces cells to respond to this information. To support this suggestion, we present cases of signaling peptides in which the sequence and structure of the mature peptide is similar yet provides diverse information. The sequence of the propeptide and its post-translational modifications, which represent the phenotypic state of the signaling cell, determine the quantity and specificity of the information. We also speculate on the evolution of signaling peptides. We hope that this perspective will encourage researchers to reevaluate pathological conditions in which the synthesis of the mature peptide is abnormal. Signaling peptides are amino acid chains with diverse structures that serve as signaling molecules. The lengths of signaling peptides vary greatly from less than ten amino acids (such as oxytocin and vasopressin) to over 100 amino acids (such as the neurotrophic factors). The mature signaling peptide which is secreted is processed from a longer propeptide which contains other domains (prodomains) which are not part of the mature form . 1, serves as both a hormone in mammals and as a mating pheromone in yeast1. It also serves as a paracrine signal in the periphery of the multicellular organism2.Mature peptides of similar structure may function as a neurotransmitter, an endocrine or a paracrine signal within a multicellular organism, and also as a signal between unicellular organisms. For instance, the gonadotropin-releasing hormone (GnRH), which has a significant structural similarity to the yeast mating factor-alpha peptide3, which also function as signals in unicellular organisms4.Thus, though the structure of the mature signal of signaling peptides such as GnRH is conserved, its specific signaling role is not, and their prodomains differ markedly. Moreover, in the same organism, structurally similar signaling peptides may regulate a diverse range of signaling pathways, such as the structurally similar oxytocin and vasopressin6, how may similar peptides provide information regarding such diverse metabolic processes?Assuming that signals elicit a response because they provide specific information that benefits the organism7, glycosylation, glycosulfation or the pairing of S-S bonds8.While the mature peptides of GnRH, oxytocin and vasopressin are short (9-10 amino acids), their propeptides are large proteins (100-160 amino acids). The cleavage of the propeptide to form the comparatively short mature peptide is often dependent on the completion of post-translational modifications, such as sequential enzymatic modificationAs the cleavage of the mature peptide depends on the propeptide completing its various post-translational modifications, and as there is a fixed stochastic relationship between the mature peptide and the propeptide that is determined by the number of repeats of the mature domain within the propeptide, it is reasonable to assume that the ability of the signaling cell to complete the synthesis of the propeptide is the information provided by the mature peptide.We suggest that while the synthesis and modifications of the propeptide are related to the phenotypic state of the signaling cell, the role of the mature peptide is to stimulate cells to be attentive to this information. In this opinion paper we briefly review a number of signaling peptides to support our suggestion and, in addition, speculate why and how a mature peptide is selected to serve as a stimulating molecule.1. Also within the yeast genome, a similar mature peptide  is produced by two different propeptides encoded by the genesMFAL-1 andMFAL-2.MFAL-1 has four tandem repeats of the mature domain, whileMFAL-2 contains two repeats of the mature domain with a slight variation in sequence  ofEscherichia coli15.We know of two cases in which a whole functional protein that has a non-signaling role serves as the propeptide of a mature peptide signal. One of them is the sex pheromone system ofAs far as we are aware, there is no known non-signaling function for the propeptides of the neurotrophic factors. Likewise is the case for oxytocin and vasopressin, and also GnRH. However, cases in which a non-signaling function is known may illustrate how peptide signaling systems evolved.E. faecalis is a bacterium that has a sophisticated mechanism of plasmid transfer governed by signaling peptides of 7-8 amino acids in length. These peptides are produced from specific membranal proteins that perform non-signaling functions in the cell that are unrelated to the plasmid , which provides via a complex transduction mechanism14 reliable information that the signaling cell does not possess the plasmid.plasmid . The matE. coli. The sequence of the pentapeptide is NNWNN, and it is synthesized by the proteolytic cleavage of the enzyme glucose-6-phosphate dehydrogenase17, which includes the sequence NNWDN. The mature peptide requires the modification of the aspartic acid in the propeptide to asparagine.Another example of a signaling peptide whose propeptide serves a non-signaling function in the cell is the extracellular death factor (EDF) pentapeptide that activates the mazEF pathway inE. coli genome only within this enzyme, and is an essential component of the enzyme, its secretion fromE. coli is reliable information that the signaling bacterium does not need the enzyme for its current metabolism.As the propeptide of the pentapeptide is a functional protein, the relationship between its synthesis and degradation links the production of the pentapeptide directly to cell metabolism, and specifically the metabolism of glucose. Since the pentapeptide appears in the18.The main purpose of this article is to propose a solution to the problem we faced when trying to understand how very similar short peptides may provide information that is relevant to receiver cells designed to serve very different roles. Mature peptides are often conserved across the phylogenetic tree, from unicellular organisms to mammals. Hence, it is tempting to attempt to identify what properties of these mature peptides cause them to be adapted for their role. Our perspective is derived from the assumption that signals provide reliable information regarding the behavior of the signaling cellWe suggest that mature peptides were selected as optimal carriers for transferring information due to their ability to stimulate the receiving cell to attend to the information they represent. We speculate that their advantage as stimulating agents is due to their harmful effects which force the receiver to attend to the information .Our suggestion that the mature peptide does not directly represent information regarding the signaling cell's metabolism, but reflects information regarding the synthesis of the prodomain, can be tested. Here we suggest experiments that may test this suggestion:1.Replacing the mature peptide of one propeptide with a similar mature peptide from the same family of peptides (such as oxytocin with vasopressin), may reveal that the mature peptide has the same effect due to its specificity for its receptor; however, we expect that the timing and quantity of the effect will be determined by the protein to which it is attached.2.The harmful effects of the mature peptides may be assessed by increasing their quantities beyond the ability of the receiving cells to counter their noxious effects to which they are usually exposed or by removing any other mechanisms that under normal circumstances counter these harmful effects, such as enzymes that degrade the mature peptide or bind it.It is tempting to speculate how the sequence of mature peptides evolved, even though at present we have succeeded in collecting limited data to support this speculation.E. coli which is a functional element of glucose-6-phosphate dehydrogenase16 and the enterococcus pheromones, which anchor the propeptide to the membrane17.It is reasonable to assume that the first generation of mature peptides were part of large proteins that had non-signaling functions in the cell. During the proteolysis of these large functional proteins, short peptides were secreted. Among the short peptides cleaved and secreted from cells, the peptides that harmed neighboring cells selected, in neighboring cells, for mechanisms that counter the harmful effects of the peptides. The level of the response to the harmful effects is correlated to the level of the secreted peptide and, hence, could be used by the neighboring cells as a source of information regarding the metabolism of the proteins from which they were cleaved in the signaling cells. These large proteins still serve their initial non-signaling function, yet they also serve as propeptides. These are the cases mentioned previously of the EDF pentapeptide of19 and Zahavi & Zahavi5 found this to be the case for many signals used by birds and humans. In addition, Harriset al.20 pointed out that several non-peptide signals such as glutamate and dopamine may cause harm to cells that do not counter their harmful effects in relation to their level of release. They suggested that the toxicity ensures that the response of the receiver cell is correlated to the level of the release from the signaling cell.We suggest that a signal which harms the receiver will force the receiver to respond to a smaller change in its concentration than a signal that provides a positive or neutral effect. ZahaviE. coli by binding to the mazF toxin and interfering with the ability of the mazE antitoxin protein to inhibit the activity of the mazF toxin15.We also suggest that in the case of signaling peptides, the toxicity of the mature peptide ensures that the response of the receiving cell is correlated to the concentration of the secreted peptide. At present we are aware of only one case in which a peptide has a known direct toxicity that is crucial to its function, the EDF pentapeptide. The pentapeptide killsOnce the sequences of mature peptides evolved as carriers of information due to their ability to stimulate cells to attend to information, natural selection could transpose a mature peptide to be a part of other proteins whose synthesis reflected information regarding the phenotypic state of the signaling cell, granted that this information benefitted the organism through its effect on the receiving cells. Small modifications in the mature peptide of the first generation were required to prevent the binding of the new mature peptide to the receptors of the first generation peptides, if the first generation and the second generation mature peptide were to function in the same organism .et al.21 suggested that acetylcholine, which serves as a paracrine signal to coordinate activities between epithelial cells in the airpipe, evolved first as a paracrine signal and was only later adopted by the brain to activate peripheral cells that already respond to acetylcholine. Following this suggestion, we recently proposed a general model for the evolution of non-peptide signals20. It is reasonable to assume that signals which function between cells in the periphery, and are also used by the brain and central glands for their similar effect on peripheral cells, evolved first as paracrine signals in the periphery21 .The third generation of mature peptides is secreted from central glands and from the brain to activate peripheral cells. WesslerIn several cases it is known that the mature peptide by which the brain stimulates peripheral cells is also synthesized in small quantities by the cells that respond to it. Oxytocin and GnRH for example are synthesized in peripheral cells that respond to the same peptides that are secreted in the brain. The present function of the synthesis of the small amounts of mature peptides in peripheral cells is unclear.It is reasonable to assume that the function of the propeptides in the brain is not to provide information relating to the metabolism of the secreting neuron, but to allow the synthesis of the mature peptide in large quantities to regulate and synchronize the activities of various organs to respond to decisions made in the brain. Hence, we expect that the structure of the propeptides may differ between the brain and peripheral cells, as each serves a different need: neurons need to synthesize large quantities of the mature peptide, while in the periphery the propeptide reflects the phenotypic state of the signaling cell.24. Current treatments of these diseases often involve administering synthesized mature peptides (the neurotrophic factors). However, if the inability to process the propeptide correctly (such as the inability to perform the glycosylations) reflects reliably the phenotypic state of the signaling cell, then these treatments provide false information to the neuron. The false information may mask the underlying problem, which may be the deterioration of the signaling cell’s ability to serve its function. If the mature peptide provides retrograde information to the neuron on the ability of the peripheral cell to be activated by the neuron, then supplementing it may mislead the organism that the system is functioning correctly even though it is not. In addition, if, as we suggest, the mature peptide functions due to its harmful effects, then providing an abnormal concentration of it to the receiver cells may cause unnecessary damage to the signaling system. Hence, the attempts to counter the disease should focus on attempting to improve the phenotypic state of the signaling cells, for instance, by providing anti-oxidants25.Many neuropathologies, including amyotrophic lateral sclerosis, Alzheimer's diseases and Parkinson's disease, have been associated with a reduction in the synthesis of neurotrophic factors9. Alignments were calculated using Clustal Omega10 implemented in UniProt9. Jalview v2.8.211 was used for the graphical representation of the alignments.All sequences were taken from the UniProt database This opinion article addresses basic questions about the nature of signals in biological systems. The underlying assumption is the brilliant handicap theory of Amotz Zahavi that signals must have some burden on the signaling cell to be perceived as a reliable signal.The central question that this opinion article is dealing with is how very similar (mature) signaling peptides control different metabolic pathways in different species or even within the same organism.The authors suggest that the message  resides in the propeptides that yield the mature signaling peptides. The amount, location and timing of the very similar  signals provide a reliable and specific information on the capability of the producing cells to synthesize, modify, cleave and secrete the mature signaling peptides.They also suggest that the mature signaling peptides might have toxic effects on the receiving cells that enforce them to respond.The authors also offer an evolutionary prediction of how such signaling peptides might have evolved from propeptides that might have functions which are not necessarily related to the signal. Finally they offer a perspective on the potential impact of their theory on evaluation of pathological treatments, in which the current practice is increasing the amount of the signaling peptides rather than dealing with malfunction of the source of the peptides.As usual, the theories stemming from Zahavi’s and his colleagues (Harris and Barzilai this time) are stimulating, but require validation, as emphasized by the authors themselves. Without validation, the appeal of theories is dependent on how reasonable they are, and if they make (new) sense of disturbing questions. The suggested theories should also be evaluated in comparison with alternative theories. It is not clear if alternative theories exist, and if they do, I would suggest discussing them.The major question raised by the authors is how very similar signaling peptides have different functions?Although very similar, even small signaling peptides are not identical, and it is advised not to exclude the option that the specificity resides within the different amino acids. The examples brought by the authors of cross species effects of similar peptides support the authors’ theory, but can also be explained by the modular nature of biological structures of signaling peptides and receptors. As suggested by the authors, shuffling mature peptides between propeptides may provide an answer.E. coli EDF pentapeptide that has a direct toxicity that is crucial to its function.An important part of the author’s hypothesis is that signals might (should?) be toxic to the receiving cell to enforce a response. The only example is theThe biological logic of toxicity to the receiving cells as a way to enforce response is not clear. Toxicity means that the outcome could be harmful, such as killing or deactivating the receiving cells. Do the authors suggest that in such cases the signaling peptide has also a selection function?The authors also discuss a case in which neurons within the brain produce a signaling peptide that affects peripheral cells and suggest that although processed from propeptides in the brain it does not provide information relating to the phenotype of the secreting neuron.It is not clear why these neurons are excluded from the “handicap principle”? Furthermore, dealing with pathological conditions related to the administration of overdose of the signaling peptides (in Alzheimer disease), the authors suggest that it might mask the “inability to process the propeptide correctly by the signaling cell” which could be the cause of the disease. Wouldn’t that mean that production of signaling peptide from the propeptide in neurons should provide information about the phenotype of the secreting neuron?As hinted above, brain stimulation by various scientific hypotheses is usually positive, and may indicate the excellent “phenotypic state” of the current “signaling” group of authors. Yet, I hope there is some “toxicity” that may enforce few readers to respond and test the hypothesesI have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. A stimulating paper, suggesting a new perspective on the nature of signaling peptides.successful processes undergone by their propeptides, reliably indicates the phenotypic state of the signaling cell.The mere structure of mature signaling peptides does not necessarily convey the information they carry, but rather their sheer existence, which is due toThe information given by signaling peptides is heard by the receiving cell because of the toxic nature of the signaling peptides – their toxicity cannot be ignored by the receiving cell.This is a truly novel idea, which seems to be a solid consequence of natural selection. The authors present a few examples, yet – as they point out – sound experimental evidence is necessary. As a theoretician of evolutionary biology (but by no means an expert in cell biology!) I am not in a position to comment or to suggest such an experimentation. If this idea is valid, the consequences to some medical treatments can be substantial, as indicated in the paper's last section.The main idea can be summarized as follows:I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


The HTLV proviral load was measured using a real-time PCR assay, and T-cell subset counts were determined by flow cytometry. Real-time PCR was used to genotype the rs12979860 SNP. The allelic and genotypic distributions displayed no significant differences among the investigated groups. No significant association between the serum cytokine levels and the presence of the rs12979860 SNP in symptomatic and asymptomatic subjects was observed. A positive correlation (p = 0.0015) between TNF-β and IFN-γ was observed in the asymptomatic group, but a positive correlation was only observed (p = 0.0180) between TNF-α and IL-6 in the HAM/TSP group. The proviral load was significantly higher in HAM/TSP patients than in asymptomatic subjects. The present results do not support a previous report indicating an association between the SNP rs12979860 and HAM/TSP outcome.The present study investigated the association between the rs12979860 polymorphism in the IL-28B gene and HTLV-1 infection as well as the development of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1-infected patients (26 HAM/TSP symptomatic and 53 asymptomatic) and 300 seronegative healthy controls were investigated. Plasma levels of the cytokines TNF- Deltaretrovirus genus [Human T-cell lymphotropic virus 1 (HTLV-1) is a member of the Retroviridae family andus genus –4.Most individuals infected with HTLV-1 remain asymptomatic for life, but 5–10% develop clinical symptoms . HTLV-1-Although recent studies indicate the possible use of genetic biomarkers as predictive factors associated with the outcome of the clinical manifestations –10, the λ  [λ is induced by the same stimulus that induces IFN-α/β [λ inhibits viral replication [Interleukin-28B (IL-28B) is a member of a recently characterized family of cytokines known as interferon , resp.) . IFN-λ i IFN-α/β , 14, andlication , 16.κB, IRF3, and IRF7 which stimulate the expression of the IL-28B gene [During a viral infection, Toll-like receptor-3, Toll-like receptor-4, and Toll-like receptor-7 recognize dsRNA and ssRNA in the cytoplasm or endosomes, activating RIG-1, NF28B gene –21. When28B gene , 22, 23.28B gene  reported28B gene  in a souRegarding these controversial results, the present study investigated whether the rs12979860 polymorphism in the IL-28B gene is associated with HTLV-1 infection and the progression to HAM/TSP in patients from the Amazon region of Brazil, a population ethnically different from the previously studied Spanish and southeastern Brazilian populations.The study group included 79 HTLV-1-infected subjects (26 HAM/TSP symptomatic and 53 asymptomatic) attending the outpatient clinic of the Tropical Medicine Nucleus of the Federal University of Pará and 300 seronegative healthy controls from the Virus Laboratory. To avoid confounding factors associated with ethnic origin, which eventually could cause sample bias and influence the genotypic and allelic frequencies, both groups were composed of subjects residing in Belém and had the same ethnic origin. All individuals were recruited between May 2005 and January 2012 and signed an informed consent form.Blood samples were collected in Vacutainer tubes containing K3-EDTA  as an anticoagulant to obtain plasma and peripheral blood mononuclear cells (PBMCs). The samples were directed to the Virus Laboratory of the Biological Sciences Institute of the Federal University of Pará and stored at −20°C before use. The samples were previously screened for anti-HTLV using an enzyme-linked immunosorbent assay , and infection was confirmed by nested PCR as previously described . The cliα, TNF-β, IL-8, IL-10, IL-6, and IFN-γ from patients were evaluated by indirect enzyme-linked immunosorbent assays .The plasma levels of cytokines TNF-+ T lymphocyte counts in HTLV-1-infected individuals were assessed at the time of entry into the study. Blood samples were processed within 4 hours of collection, and T-cell subset counts were determined by flow cytometry  using the FACScount reagent immunomonitoring kit according to a standard protocol recommended by the manufacturer . The quantification of the HTLV proviral load was performed using a real-time PCR assay as previously described [The HTLV-1 proviral load and CD4escribed .Real-time PCR (qPCR) was used to genotype the single nucleotide polymorphism (SNP) rs12979860 using the TaqMan Gene Expression Assay AHCS19G kit  following the technical procedures recommended by the manufacturer. A custom-designed assay provided by Applied Biosystems was used. The following primer sequences were used in these assays: forward primer, 5′-GCCTGTCGTGTACTGAACCA-3′; reverse primer, 5′-GCGCGGAGTGCAATTCAAC-3′; and the probes (VIC)-5′-TGGTTCGCGCCTTC-3′ and (FAM)-5′-CTGGTTCACGCCTTC-3′ for the C and T alleles, respectively.μL of Master Mix, 3.5 μL of H2O, 0.5 μL of IL-28B assay components (primer set and probe), and 1.0 μL of DNA from each sample. The final volume for each reaction was 10 μL.The PCR reactions were prepared using TaqMan Universal PCR Master Mix components , which consisted of nucleotides, buffer, UNG, AmpliTaq, and a passive reference dye (ROX). The reaction mixture contained 5.0 The Step One Plus Real-Time PCR System  was used to perform the qPCR experiments with the following cycling protocol: one cycle of 60°C for 2 minutes, one cycle of 95°C for 10 minutes, and 50 cycles of 95°C for 15 seconds and 60°C for 20 seconds.The program StepOne v2.2  was employed to interpret the reaction results using the graphical representation of VIC and FAM fluorophore emissions with respect to constitutive ROX emissions.+ and TCD8+ lymphocyte counts were performed using the Mann-Whitney test. Pearson's linear correlation analysis was used to compare the cytokine levels. Hardy-Weinberg equilibrium was calculated using the software BioEstat 5.0 [p value < 0.05 was considered statistically significant.The genotypic and allelic frequencies observed were obtained by direct counting, and comparisons between frequencies were calculated using the Chi-square test. The association analysis between the genotype frequencies and the arithmetic average of the values of proviral load, cytokines, and TCD4stat 5.0 . A p valThe allele and genotype distributions displayed no significant differences between HTLV-infected subjects and healthy controls as well as between HAM/TSP and asymptomatic patients . The CT α was observed in the HAM/TSP patients. In an attempt to correlate each cytokine value with each other  between TNF-β and IFN-γ was observed in the asymptomatic group, whereas TNF-α and IL-6  as well as IL-10 and IL-8  displayed positive correlations in the HAM/TSP group.No significant association  was obsech other , a positp = 0.0002; + and CD8+ T lymphocyte levels and symptomatic and asymptomatic infected subjects or the presence or absence of the SNP rs12979860.The proviral load was significantly increased in HAM/TSP patients compared with asymptomatic subjects (Symptomatic HTLV infection has been described in 5–10% of infected individuals , 30. SevThe results presented herein do not agree with this hypothesis given that we did not observe any differences in the allelic and genotypic frequencies among the symptomatic patients, asymptomatic patients, and control individuals. Furthermore, according to our results, the studied SNP had no influence on the HTLV proviral load, confirming the data previously obtained in a southern Brazilian population ; thus, o+ T lymphocyte response against HTLV-1 to control virus replication and HTLV-infected cell proliferation [α were observed in the HAM/TSP group compared with the controls. Furthermore, a positive correlation between TNF-β and IFN-γ was evident in the asymptomatic group. These results may reflect the systemic antiviral cellular response against CD4+ T cells infected with HTLV-1. Positive correlations between TNF-α and IL-6 as well as IL-10 and IL-8 were observed in the HAM/TSP group, and these results are a potential consequence of the proinflammatory immune responses typically observed in this group of patients. Lal and Rudolph [α levels in culture supernatants from HTLV-1/HTLV-2-infected individuals compared with normal controls, suggesting that these cytokines may regulate lymphocyte proliferation in infected individuals. The in vivo results of Starling et al. [α in HAM/TSP patients compared with asymptomatic subjects.The pathogenesis of HAM/TSP is based on the host's immunological response against infected cells, and cytokines play a central role in the regulation of the CD8feration , 33. Acc Rudolph  reportedg et al.  demonstrKnowledge regarding the immunogenetic and virological profile of HTLV-infected subjects can guide patient clinical counseling, but our results did not reveal any association between the SNP rs12979860 and the TSP/HAM outcome, refuting previous evidence. However, population-scale genetic studies involving larger samples and different ethnic groups are recommended to confirm these results.


FA-CS-UA-NPs entered into lysosome, destructed the permeability of lysosomal membrane, and then got released from lysosomes. Subsequently, FA-CS-UA-NPs localized into mitochondria but not nuclei. The prolonged retention of FA-CS-UA-NPs in mitochondria induced overproduction of ROS and destruction of mitochondrial membrane potential, and resulted in the irreversible apoptosis in cancer cells. In vivo experiments showed that FA-CS-UA-NPs could significantly reduce breast cancer burden in MCF-7 xenograft mouse model. These results suggested that FA-CS-UA-NPs could further be explored as an anti-cancer drug candidate and that our approach might provide a platform to develop novel anti-cancer drug delivery system.Ursolic acid (UA) has proved to have broad-spectrum anti-tumor effects, but its poor water solubility and incompetent targeting property largely limit its clinical application and efficiency. Here, we synthesized a nanoparticle-based drug carrier composed of chitosan, UA and folate (FA-CS-UA-NPs) and demonstrated that FA-CS-UA-NPs could effectively diminish off-target effects and increase local drug concentrations of UA. Using MCF-7 cells as  Cancer remains one of the most devastating diseases threatening public health, causing high mortality worldwide every year. For decades, chemotherapy has served as the preferred treatment. However, conventional chemotherapeutics can’t distinguish cancer cells from normal cells, and inevitably damage healthy cells and tissues with evident toxicity. Therefore, it is of central importance to develop efficacious ant-cancer drugs that selectively target cancer cells with low toxicity.Ursolic acid (UA) is a triterpenoid compound, which exists extensively in food, medicinal herbs, and other plants. Recently, it has been reported that UA can inhibit the growth and development of prostate cancer, liver cancer, and cervical carcinoma1in vivo, leading to maintenances in the body for a long time. Moreover, some inorganic nanoparticles have the intrinsic capability to form large aggregateset al.Nanomaterials have recently been emerging as attractive pharmacological vehicles for drug delivery and cancer therapy. The engineered nanomaterials can gain unusual physiochemical characteristics because of their small sizes, surface structure, solubility and shapes. Importantly, nanomaterials can be designed as nanoscale drug carriers to avoid immune clearance by lymphocyte-macrophage system, and therefore allow drugs to efficiently target cancer cells. Over the past decades, inorganic nanoparticles have been explored as drug carriers for new anti-cancer treatments, as nanoparticles can be synthesized to have regular shapes, size, surface chemical and physical properties for better targeting of cancer cells3et al.et al.While nanoparticles have been used to load UA, some methods for synthesis of UA-loaded nanoparticle exhibit low reproducibilityWe recently made efforts to design novel biodegradable nanoparticles for loading UA and for improving UA solubility/targeting and anti-cancer efficiency. We employed two innovative approaches in the production of potentially useful UA-loaded nanoparticles. First, we used chitosan (CS) for synthesis of UA-loaded nanoparticles. CS is a linear cationic polysaccharide obtained from natural sources of chitin, such as crabs, krill, and shrimps, which has been regarded as a good candidate for the drug delivery system12in vitro and in vivo studies to evaluate novel FA-CS-UA-NPs for the ability to enhance the anti-breast cancer activities and cancer-targeted features or mechanisms. We demonstrated that FA-CS-UA-NPs internalized into cancer cells via folate receptor-mediated pathway and then induced apoptosis in MCF-7 cells through a mitochondria-dependent pathway. Notably, FA-CS-UA-NPs NPs could significantly reduce breast cancer burden in xenograft mouse model. Thus, our approach could provide a platform to design/develop anti-cancer nano-delivery system especially for drugs with poor water solubility.We then conducted The method for synthesis of FA-CS-UA-NPs was straight forward, and undergoing the mild reaction condition. Since the electric charges on nanoparticle surface play important roles in physical stability and biocompatibility of nanoparticle-based suspensions, we sought to examine zeta potential of FA-CS-UA-NPs. As shown in The HPLC results showed that the drug (UA) loading rate was about 50%.Many studies14The images obtained from FITC channel by confocal microscopy showed the red fluorescence of the rhodamine B- encapsulated nanoparticles. As shown in Cellular uptake and endocytic pathways of nanomaterials can critically affect the delivery efficiency and bioavailability of the nano-carrier. There are three main ways of internalizing NPs by cells, i.e. endocytosis, fluid phase endocytosis, and receptor-mediated endocytosisGiven the conjugation of folate on the surface of CS-UA-NPs, we assumed that FA-UA-CS-NPs internalized into cells through folate receptor-mediated endocytosis. To test this hypothesis, MCF-7 cells expressing folate receptors(folate receptor+) and Colo205 cells expressing no or very low-level folate receptors(folate receptor−) were selected as models, respectively. Fluorescence-based flow cytometry was used to quantitatively measure the uptake level of FA-UA-CS-NPs. To facilitate examination of folate receptor-mediated enodytosis, cells were pretreated with 0.1 mM folate for 1 h to make sure that the folate receptors on cell membrane was blocked or ligated.When these two kinds of tumor cells were incubated with non-folate conjugation NPs (CS-UA-NPs) for 30 min at 37 °C, there were no apparent differences in MFI of CS-UA-NPs between MCF-7 and Colo205 cells . In factIt was likely that when folate receptor + MCF-7 cells were pretreated with free folate molecules, the binding sites of folate receptors on cellular membrane were blocked, leading to significant decreases in the folate receptor-mediated endocytosis of FA-CS-UA-NPs . On contThe quantitative analysis also suggested that FA-UA-CS-NPs were internalized distinctly by two types of cancer cells. We then examined the fate of FA-CS-UA-NPs after internalization into cells. To this end, we selected MCF-7 cells as a model to determine the localization of FA-CS-UA-NPs in cells. As shown in It has been reported that the intracellular localization of nanomaterials closely involved lysosomes and mitochondriaThe integrity of lysosome membrane is important indicator to reflect lysosomal physiological functions. It is well accepted that the long-term accumulation and retention of nanoparticles in lysosomes can influence the lysosomal membrane integrity and further impact their functions. Here, the metachromatic fluorescent cationic dye acridine orange (AO) was used as a probe to detect changes in the integrity of lysosomal membrane induced by as-synthesized nanoparticles. AO could emit strong red light when it accumulates in lysosomes at high concentration, and it will emit green light with weak red fluorescence when it’s not concentrated in lysosomes. Nanomaterials such as carbon nanotube, cationic nanoparticles, selenium nanoparticles etc. could destroy the lysosomal membrane and destroy the lysosomal membrane permeabilization (LMP)Next interesting questions would be what would happen and whether FA-CS-UA-NPs could attack and enter into the mitochondria. To address these, Mito Tracker (green color) was used as a mitochondrial fluorescence probe to detect the spatial relationship between FA-CS-UA-NPs and mitochondria in MCF-7 cells. There was only a little overlap of red and green color after 10 min and 30 min incubation. However, appreciable overlay of red and green color could be observed after incubation for 60 min , which wMitochondria, the energy factories of cells, generate most of ATP that used as a source of chemical energy for cell metabolism. Mitochondria are involved in vital processes of cells, such as molecular signal transduction, cellular differentiation, growth and cell death, etc. As shown in MTT assay was performed to detect the cell viability of MCF-7 cells after 48 h treatment with different concentrations of UA and FA-CS-UA-NPs, respectively. As shown in The intracellular ROS level in MCF-7 cells upon engulfing FA-CS-UA-NPs was investigated by MFI of DCFH. DCFH is formed through intracellular esterase hydrolyzation of a cell absorbed inflorescent probe (DCFH-DA). In parallel, the flow cytometry analysis of PI staining was used to determine the changes in cell cycle distribution induced by FA-CS-UA-NPs. th treatment. As shown in The anti-cancer effects of FA-CS-UA-NPs were determined in MCF-7 cancer xenograft mice. Female Balb/C mice were injected with MCF-7 cells, and at 10 days after injection, the mice were randomly divided into three groups. The control group, positive control group and test group were intraperitoneal injection (i.p.)/day with 0.2 ml of physiological saline, and 12.5 mg/kg bw of UA and FA-CS-UA-NPs, respectively. Mice were sacrificed and evaluated for changes in cancer burdens after the 8in vitro and in vivo models. Several studies have assessed high doses of UA for the anti-tumor effects in mouse model. Shanmugam, et al.et al.et al.In the current study, we develop a kind of novel FA-CS-UA-NPs to increase UA solubility, selectively target cancer cells and achieve better therapeutic efficacy against breast cancers in both The enhanced UA efficacy by using FA-CS-UA-NPs might be explained by two main aspects. First, FA-CS-UA-NPs appear to harness UA pharmacological potency by means of an increased solubility of UA and folate-directed on-target effect. As shown by the MTT results , FA-CS-UFA-CS-UA-NPs appear to have the aggregation-resistant properties in suspension as the high absolute value of zeta potential >25 mV) has been reported to produce big repulsive electrostatic force between nanoparticles avoiding aggregation. The zeta potential of FA-CS-UA-NPs is + 39.3 mV, suggesting that they are stable and could be easily internalized by tumor cells. In addition, due to electrostatic interactions, positively charged FA-CS-UA-NPs can be more easily internalized by cancer cells whose membrane is negatively charged24 mV has bThe current study also provides potential intracellular trafficking mechanisms underlying FA-CS-UA-NPs-mediated killing of cancer cells . FA-CS-Uin vivo, with no or very low side effects.In summary, we have synthesized a novel nano-carrier using biodegradable chitosan to encapsulate the anticancer drug UA for enhanced water solubility, with folate loaded on surface to improve the UA’s targeting and anti-cancer activity. We show that the synthesized nano-drugs, FA-CS-UA-NPs, can stably target cancer cells, induce appreciable anti-cancer activities in breast cancer cells with underlying mechanisms, and confer detectable therapeutic effects against breast cancer All chemicals were of analytical grade. UA (98.6%) and chitosan  were purchased from Shanxi Huike Botanical Development Co., Ltd. (China) and Jinan Haidebei Marine Bioengineering Co. Ltd. (China), respectively. Folate, rhodamine B, Ethyl-(3-3-dimethylam -inopropyl) carbondiimide hydrochloride (EDC), N-hydroxy-succinimide (NHS), 3--dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT), N-acetylcysteine (NAC ) were purchased from sigma (USA). Propidium Iodide (PI), Annexin V/PI apoptosis detection kit, 2′,7′-dichlorfluorescein-diacetate(DCFH-DA), 2-(4-Amidinophenyl)-6-indolecarbamidine dihydo-chloride (DAPI), 2-(6-Amino-3-imino-3H-xanthen-9-yl) benzoic acid methyl ester (Rhodamine123), caspase3, caspase8 and caspase9 activity assay kit, Mito-Tracker green, Lyso-Tracker red were purchased from Beyotime institute of biotechnology (China). RPMI-1640 medium, DMEM medium, fetal bovine serum (FBS), trypsin and penicillin-streptomycin were purchased from Gibco BRL (USA). Matrigel was obtained from BD biosciences. Milli-Q water was used in all experimental processes. All experimental protocols were approved by IACUC and IBC in accordance with the guidelines of department of Chemistry, the first affiliated hospital of Jinan University, and UI college of Medicine.32 mg of chitosan was dissolved in 5 ml of 1% (v/v) glacial acetic acid. 10 mg UA, 30 mg EDC and 8 mg NHS were dissolved in 5 ml of THF, after that it was added to the above chitosan solution. Then, the solution was kept on constant magnetic stirring overnight at room temperature. After precipitation with pure water, the solution was centrifuged twice at 11,000 rpm for 20 min to remove excess amounts of THF and unencapsulated UA. Finally, NPs were lyophilized for 48 hours using lyophilizer for storage in powdered form. To determine the NPs uptake by cells, NPs containing a fluorescent dye (rhodamine B) were prepared using the above procedure, except that 250 μg dye was added with the addition of UA to the chitosan solution. The incorporated dye acts as a probe for NPs and offers a sensitive method to determine qualitatively and quantitatively intracellular binding including eventual uptake and retention.To improve the cell-specific targeting of NPs by cancer cells, the NPs were conjugated with folate to target folate receptor bearing tumor cells. Briefly, the solution of NPs was mixed with folic acid (0.5 g/L) for 3 h at 4 °C, and then centrifuged at 3,000 rpm for 15 min to remove the unbinded folate.The size distribution and zeta potential of the nanoparticles was estimated using a Zetasizer Nano ZS . TEM  and AFM  were used to characterize the morphology and dispersed state of the nanoparticles. The micro-graphs were obtained on Hitachi (H-7650) for TEM operated at an accelerating voltage at 80 kV. AFM images were achieved by tapping mode in air. A scanning spectro-photometer  with a 1-cm path length was employed to measure the UV/VIS absorbance of as-prepared products.To measure the drug (UA) loading rate of FA-CS-UA-NPs, 10 mg lyophilized nanoparticles were dissolved in 1 ml of methanol, and then the amount of UA in the solution was determined by High Pressure Liquid Chromatography (HPLC). HPLC detection was performed using a C18 column , whereas the mobile phase, consisting of methanol and 0.1% acetic acid (88:12) (v/v), was maintained at a flow rate of 1.0 mL/minute. The ultraviolet detector wavelength was 215 nm and the injection volume was 20 μL. The drug loading was calculated according to the formula: loading rate % = UA/(CS + UA) × 100%.in vitro release rate of Rh B from the NPs, 10 ml of 1 mg/ml of RhB-loaded NPs were shaken in water bath at 37 °C at different time points. At each time point, 300 μl of samples were taken out and centrifuged. 20 μl of supernatant was collected and measured for Rh B concentration in HPLC. HPLC detection was performed using a C18 column , whereas the mobile phase, consisting of methanol and 0.1% acetic acid (70:30) (v/v), was maintained at a flow rate of 1.0 mL/minute. The ultraviolet detector wavelength was 548 nm of excitation and 578 nm of emission. Release rate was calculated through dividing supernatant RhB by total RhB. The release rates of RhB from NPs were <4% and <10% in 4 and 12 hours, respectively. To detect the 2 atmosphere.MCF-7 breast cancer cells and Colo205 colon adenocarcinoma cells were purchased from American Type Culture Collection . All cell lines were grown in DMEM media supplemented with fetal bovine serum (10%), penicillin (100 units/mL), and streptomycin (100 units/m L) at 37 °C in a humidified incubator with 5% COThe cellular uptake of FA-CS-UA-NPs was qualitatively detected by fluorescence microscopy. Briefly, treated cells cultured on cover glass in 6-well plates until 70% confluence were incubated with different concentrations of rhodamine B-loaded FA-CS-UA-NPs for various periods of time. The cells were then washed three times by PBS and examined under a fluorescence microscope .5 cells/well were plated on 6 well plates and incubated 24 h for attachment. After that, the cells were incubated with designated concentrations of rhodamine B-loaded NPs for different periods. Before analyzed by flow cytometry , the cells were washed triple with PBS and detached by trypsin, washed twice with PBS. The mean fluorescence intensity (MFI) was the indicator of uptake of NPs by cells.Flow cytometry was used to quantify the cellar uptake of FA-CS-UA-NPs in MCF-7 cells. Briefly, 2 × 106 cells/ml) were cultured with 2 μg/ml  UA or FA-CS-UA-NPs for 24 h. Then, the cells were washed three times using cold PBS with the supernatants discarded, and then added to the mobile phase, consisting of methanol and 0.1% acetic acid (88:12) (v/v) for HPLC detection. The flow rate was 1.0 mL/minute. The ultraviolet detector wavelength was 215 nm and the injection volume was 20 μL.MCF-7 cells  was added to the well and incubated for another 4 h. For further confirmation the effects of ROS on cell apoptosis, the ROS inhibitor- N-acetyl-L-cysteine (NAC) (2.5 mM), were added into cells 30 min prior to adding drugs, and then performed MTT assay.Cell viability was determined by measuring the ability of cells to transform MTT to a purple formazan dye. Cells were seeded in 96-well culture plates at 5 × 10MCF-7 cells with or without treatment with FA-CS-UA-NPs for 24 h, were trypsinized, washed twice with PBS, and then, fixed with 70% chilled ethanol overnight. The fixed cells were washed twice with PBS, treated with RNase A, stained with PI (50 μg/mL), and analyzed by flow cytometer.An Annexin V-FITC/PI apoptosis detection kit was used to detect the apoptotic cells according to the manufacturer’s instructions. After treatment with different concentrations of FA-CS-UA-NPs, MCF-7 cells were harvested and washed with cold PBS and suspended in 500 μl Annexin V binding buffer. After incubation with 5 μl FITC labeled-Annexin V and 5 μl PI at room temperature in the dark, the samples were immediately analyzed by flow cytometer at 488 nm.To detect the changes in ΔΨm and ROS levels, the MCF-7 cells were treated with different concentrations of FA-CS-UA-NPs and then harvested to incubate with rhodamin123 and DCFH -DA for 30 min in dark at room temperature, separately. The cells were collected and washed twice with PBS. The resulting fluorescence was measured by flow cytometer both excited by 488 nm.7 cells in 50 μL serum free medium) with 50 BALB/c nude mouse of 4~6 weeks age matrigel were injected into the back of mouse. The mouse were i.p. administrated with physiological saline, ursolic acid (12.5 mg/kgb.w./day), FA-CS-UA-NPs (12.5 mg/kg b.w./day) for nine times. One group of normal healthy mice were used as negative control. There was 5 mice in each group and no mice dead during the whole experimental process.MCF-7 xenograft model was established using BALB/c nude mice of 4~6 weeks age. The animals were obtained from Beijing HFK Bioscience Co., LTD and quality checks were supervised by Institute of Laboratory of Animal Science (CAM&PUMC). MCF-7 cells  were determined by ELISA according to the manual instruction of cytokines ELISA kit from R&D Systems.Statistical analysis was performed using nonparametric t test or ANOVA test, with *P < 0.05 regarded as statistically significant.How to cite this article: Jin, H. et al. Folate-Chitosan Nanoparticles Loaded with Ursolic Acid Confer Anti-Breast Cancer Activities in vitro and in vivo. Sci. Rep.6, 30782; doi: 10.1038/srep30782 (2016).


ATL arises from HTLV-1-mediated transformation of infected T-cells following on average 30+ years post initial infection. Current diagnosis relies upon evaluation of numerous clinical factors and is generally effective for overt Acute and Lymphomatous variants of ATL for which survival is less than 1 year post-diagnosis. As the vast majority of infected individuals >95% will remain disease free and as a significant proportion of those that do develop ATL are “curable/treatable” there is a clear need for early definitive diagnosis of HTLV-1-infected individuals. We hypothesized that immune evasion by HTLV-1 and crosstalk between complement, fibrinolysis and coagulation  provide pathway-specific biomarker events that are concentrated in circulating exosomes. We subjected exosomes isolated from serum of HTLV-1-infected patients to quantitative ultra-high resolution mass spectrometry that identified 784 proteins of which 208 displayed significant expression differences. We further analyzed our data with customized algorithms designed to facilitate a targeted analysis of the potential biomarker candidates inclusive of complex post-translational modifications. This approach allowed for high confidence quantitation of a panel of proteins that effectively discriminate between AS/HAM/ATL. We observed stratification of the disease states across the complement superfamily, over expression of the coagulation proteolytic cascade in HAM/TSP and ATL, and over expression of alpha-1-antitrypsin in ATL. When compared to a parallel analysis of total serum, exosomes reproducibility reflected expected tumor cell phenotype. For example, plasminogen activator inhibitor was over expressed; where as fibrinogen and metalloproteinases were downregulated in ATL exosomes. This is consistent with the function of PAI-1 to inhibit fibrinogen and MMPs. Interestingly, PAI-1 is not normally over expressed in hematopoietic disorders except Acute Lymphocytic Leukemia. We also identified APOBEC3H, which has been reported to bind specifically to HTLV-1 gag, as over expressed in ATL. We also examined the exosomes for evidence of HTLV-1 proteins. We were able to identify Tax and to a lesser extent Hbz each showing a trend of no-expression in AS, detectable expression in HAM/TSP and higher expression in ATL.


Saccharomyces cerevisiae may allow us to understand the mechanisms of yeast stress tolerance in biotechnological processes and it may also helps in searching for effective antifungal drugs, since the CW is a desirable target of action.The cell wall (CW) is one of the most important structures of the yeast cell, accounting for up to 30% of its dry weight. This organelle determines cellular morphology, affords mechanical protection and provides osmotic support. The yeast CW is a dynamic structure susceptible to many modifications, adjusting its composition and thickness to environmental changes. These responses usually involve changes in gene expression, increasing levels of proteins that have protective functions. High hydrostatic pressure (HHP) is a useful model of stress, which causes CW compression . ExploriS. cerevisiae strain BT0510 was subjected to a non-lethal HHP of 50 MPa for 30 min, followed by recovery at atmospheric pressure for up to 15 min. RNA samples were collected to perform a time series microarray expression analysis.In this work HSP12, which protein is present in CW and acts by increasing its flexibility [ROM1. Mtl1p and Wsc3p are related in detecting and signaling CW status to Rho1p; their genes were upregulated by HHP. Related to the same pathway, HHP activates PKH1 and PKH2, paralog genes of which proteins activate components of a signaling cascade required for CWI maintenance. The genes related to β-1,3-glucan, β-1,6-glucan and CW chitin biosynthesis were not strongly affected by HHP. Furthermore, MNN1, MNN9 and MNN10, correlated with protein mannosylation were downregulated by HHP. The products of these genes are subunits of mannose polymerase complexes, what suggest a possible change in the outer layer of the CW. Moreover, HHP induced the coding genes of Pir3p Hsp150p, members of proteins with internal repeats family (PIR), correlated with CW reinforcement by interconnecting two or more β-1,3-glucan molecules providing defense against β-1,3-glucanases, common stress in the wild since these enzymes abound in plant tissues [DSE2, DSE4, EGT2, CTS1, SCW11 and SUN4, related with CW degradation and separation of daughter cell from the mother cell, were downregulated by HHP, suggesting that pressure can affect cell division. Many genes involved in CW biosynthesis and organization had their expression changed after HHP treatment, evidencing the importance of the CW to ensure cell survival against this stress. Knowing the key cell-survival proteins is critical to improve biotechnological processes, and the results presented here may help in development of new drugs or in develop stress tolerant distillery yeast cells.Through bioinformatics, changes in the expression pattern ≥2 fold) of several CW organization and biogenesis genes were identified. HHP induced the expression of  fold of xibility . HHP ind tissues . DSE2, D


HTLV-1 genes are encoded on both strands of the provirus, such as tax in the plus and HBZ in the minus strand. The HBZ gene is constitutively expressed from the negative strand of the integrated provirus, whereas plus-strand expression, required for viral propagation to uninfected cells, is expressed only intermittently in vivo, perhaps to escape from host immune surveillance. However, it remains unknown what regulates this pattern of proviral transcription in vivo. We have found that CTCF binds to the HTLV-1 provirus. CTCF is a DNA-binding protein that plays a fundamental role in controlling higher-order chromatin structure and gene expression in vertebrates. We identified several candidate regions for CTCF binding in the HTLV-1 genome. Chromatin immunoprecipitation assays showed that CTCF bound selectively to the pX region of HTLV-1. Furthermore, electromobility shift assays revealed that CTCF bound directly to the pX DNA sequence. Consistent with the CTCF binding, there was a sharp border of histone modification patterns at the pX region, consistent with CTCF's role as a chromatin insulator. Finally, the CTCF-binding region (1bp) showed enhancer-blocking activity. The CTCF binding and epigenetic border were detectable not only in HTLV-1 cell lines and ATL cell lines but also in fresh PBMCs of ATL patients. These observations suggest that CTCF plays a central role in the regulation of HTLV-1 transcription.#Poster award winnder - 1st place