Virus transmission from various wild and domestic animals contributes to increased risk of emerging infectious diseases in human populations. HTLV-1 is a human retrovirus associated with acute T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), which originated from zoonotic transmission from various African and Asian nonhuman primates (NHPs). Similar to HTLV-1, the simian counterpart, STLV-1, causes chronic infection and leukemia and lymphoma in naturally infected monkeys. However, other clinical syndromes typically seen in human such as a chronic progressive myelopathy have not been observed in NHPs. Little is also known about the development of any neurologic and inflammatory diseases in human populations infected with STLV-1-like viruses following NHP exposure. We identified and analyzed the complete genome of a primate T lymphotropic virus type 1 (PTLV-1) isolated from a patient with typical HAM/TSP who resides in the United States but was born in Liberia. Using a novel droplet digital PCR for the detection of the HTLV-1 tax gene, the proviral load in PBMC was 14.01%; however there was a distinct difference in fluorescence amplitude compared to all other H!M/TSP patient's, suggesting viral heterogeneity. A complete PTLV-1 proviral genome was amplified from DNA extracted from the PBMCs of the HAM/TSP patient using PCR to generate nine overlapping subgenomic fragments. Phylogenetic analysis of PTLV-1 env and LTR regions showed the virus was highly related with PTLV-1 from sooty mangabey monkeys and humans exposed from NHPs in West Africa. These results suggest the patient is likely infected with STLV-1, suggesting for the first time that viral transmission from monkey to human may be associated with a chronic progressive neurologic disease.


Despite phenotypic and functional heterogeneity among TMem, the expression of Fas — a tumor necrosis family receptor (TNFR) superfamily member conventionally known as a death receptor — is held in common among all TMem subsets across multiple species. As Fas has been shown to mediate non-death signaling in other cell types, we set out to elucidate the role of Fas signaling in defined TMem subsets, including T stem cell memory (TSCM), T central memory (TCM), and T effector memory (TEM). We found that augmenting Fas signaling in stimulated TSCM using an oligomerized form of its ligand FasL resulted in augmented cellular differentiation and a loss in IL-2 secretion capacity. Conversely, antibody blockade (anti-FasL) of Fas signaling in TCM retarded cellular differentiation both phenotypically and functionally. To genetically disentangle the pro-apoptotic and differentiation signals from Fas, we made use of a mutant Fas lacking a transmembrane cysteine residue (FasC194V) that is unable to undergo S-palmitoylation and aggregate efficiently in lipid rafts. Using transgenic mice expressing this C194V Fas construct on a Fas-deficient lpr background, we found that FasC194V TMem can still undergo cellular differentiation in the absence of death signaling. In vivo, TMem expanded with anti-FasL showed greater expansion, on-target immunity and withheld differentiation. Additionally, in a relevant syngeneic model of current human T cell immunotherapy, TMem cells expanded with anti-FasL and genetically engineered with an anti-CD19 chimeric antigen receptor (CAR) exhibited enhanced CAR expression, reduced differentiation, and augmented anti-lymphoma activity compared to controls. These studies demonstrate that Fas signaling promotes not only cell death but also TMem effector differentiation, a finding that has implications for the design and execution of T cell-based immunotherapies in patients with cancer or infectious disease.Memory CD8


Ursolic acid (UA), a pentacyclic triterpenoid, is known to have anti-tumor activity in various cancers including human non small cell lung cancer (NSCLC). However, the molecular mechanisms underlying the action of UA remain largely unknown.Cell viability was measured by MTT assays. Apoptosis was analyzed with Annexin V-FITC/PI Apoptosis Detection Kit by Flow cytometry. Western blot analysis was performed to measure the phosphorylation and protein expression of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), DNMT1 [DNA (cytosine-5)-methyltransferase 1], enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) and SP1. Exogenous expression of SP1 and DNMT1 was carried out by transient transfection assays.We showed that UA inhibited the growth and induced apoptosis of NSCLC cells in the dose- and time-dependent fashion. Furthermore, we found that UA induced phosphorylation of SAPK/JNK and suppressed the protein expression of DNMT1 and EZH2. The inhibitor of SAPK/JNK (SP600125) blocked the UA-reduced expression of DNMT1 and EZH2. In addition, UA suppressed the expression of SP1 protein. Conversely, overexpression of SP1 reversed the effect of UA on DNMT1 and EZH2 expression, and feedback attenuated UA-induced phosphorylation of SAPK/JNK. Moreover, exogenous expression of DNMT1 antagonized the effect of UA on SAPK/JNK signaling, EZH2 protein expression, and NSCLC cell growth.Our results show that UA inhibits growth of NSCLC cells through SAPK/JNK-mediated inhibition of SP1; this in turn results in inhibition the expression of DNMT1 and EZH2. Overexpression of DNMT1 diminishes UA-reduced EZH2 protein expression. The negative feedback regulation of SAPK/JNK signaling by SP1 and DNMT1, and the reciprocal interaction of EZH2 and DNMT1 contribute to the overall effects of UA. This study leads to important new insights into the mechanisms by which UA controls growth of NSCLC cells. Lung cancer is the most common malignancy worldwide and a leading cause of cancer-related deaths. Despite recent progress in understanding the tumorgenesis signaling network and in producing new therapeutic strategies, this malignancy especially non-small cell lung cancer (NSCLC) still shows poor prognosis and high incidence of recurrence . MajoritUA, a triterpene compound came from certain traditional medicinal plants , has beeDNA methylation is controlled by DNA methyltransferase (DNMT), an enzyme that catalyzes the transfer of a methyl group to DNA. DNMT1 [DNA (cytosine-5-)-methyltransferase 1] is primarily a DNA methyltransferase that maintains DNA methylation, which is one of the regulatory mechanisms of gene expression . DNMT1 aEnhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2), a polycomb group protein, has been identified as an oncogene in many types of cancers . EZH2 isThe association of UA effect and regulation of either DNMT1 or EZH2 expression have not been reported. In this study, we explored the potential mechanism by which UA inhibited growth of human lung cancer cells. We showed that UA suppressed growth and induced apoptosis of NSCLC cells through stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK)-mediated reduction of SP1 transcription factor; this in turn results in inhibition of DNMT1 and EZH2 expression.Monoclonal antibodies specific for total SAPK/JNK and the phosphor-form (thr183/tyr185) were purchased from Cell Signaling Technology Inc. . The DNMT1 and EZH2 antibodies were obtained from Epitomics . SP600125 (JNK inhibitor) and antibody against SP1 were purchased from Merck Millipore . The 3--2, 5-diphenyltetrazolium bromide (MTT) powder was purchased from Sigma Aldrich . Lipofectamine 3000 reagent was ordered from Life Technologies (AB & Invitrogen) . Ursolic acid was purchased from Chengdu Must Bio-technology Company . The drugs were freshly diluted to the final concentration with culture medium before experiment. Human NSCLC cells  were obtained from the Cell Line Bank at the Laboratory Animal Center of Sun Yat-sen University  and the Chinese Academy of Sciences Cell Bank of Type Culture Collection . The cells were cultured at 37 °C in a humidified atmosphere containing 5 % CO2. The culture medium consisted of RPMI 1640 medium  supplemented with 10 % (v/v) heat-inactivated fetal bovine serum , 100 μg/ml streptomycin and 100 U/mL penicillin. When cells reached 80 % confluence, they were digested with 0.25 % trypsin for passage for the following experiments.4 cells/well). The cells were treated with increasing concentrations of UA for up to 72 h. Afterwards, 10 μL MTT solution (5 g/L) was added to each well and NSCLC cells were incubated at 37 °C for an additional 4 h. After removing the supernatant, 150 μL solvent dimethyl sulfoxide (DMSO) was added to each well and oscillated for 10 min. Absorbance at 490 nm was determined through the use of ELISA reader . Cell viability (%) was calculated as (absorbance of test sample/absorbance of control) × 100 %.The MTT assay was used to determine the cell viability as reported previously . BrieflyCell apoptosis was analyzed with Annexin V-FITC/PI Apoptosis Detection Kit  according to instructions from the manufacturer. Briefly, after treated with UA for 24 h, the apoptotic cells were harvested by Trypsin without EDTA and washed with PBS. Afterwards, the cells were resuspended in 500 μL binding buffer, 5 μL Annexin V-FITC regent and 10 μL PI regents, and incubated for 5 min at room temperature (RT) in the dark, followed by detecting cell apoptosis by Flow cytometry .We measured the activity of caspase-3/7 using the Caspase-Glo 3/7 Assay kit  according to the instruction from the manufacturer. Briefly, NSCLC cells were seeded in 96-well plates and treated with or without UA for 48 h. Afterwards, the cells were lysed and incubated with 100 μL of Apo-ONE Caspase-3/7 reagent (substrate and buffer in the ratio of 1:100). After 1 h incubation in the dark at RT, the fluorescence of each well was measured at 485–520 nm by reading in an Epoch Microplate Reader .The Western blot procedures were performed based on previous study . Briefly7 cells/mL) were transferred into conical tubes and centrifuged at 1200 rpm for 5 min. After centrifuging, the medium were removed and the cells were washed with 1X PBS, and centrifuged again at 160 × g for 5 min. Afterwards, the PBS was aspirated and the tubes were added Bio-Rad Gene Pulser electroporation buffer. After resuspending the cells, the desired control (pCMV6) or expression constructs containing Myc/FLAG-tagged ORFs of human DNMT1and EZH2 were obtained from OriGene Technologies, Inc. , or control (pcDNA3.1) and SP1 overexpression vector  [The procedure was based on the protocol from the provider  and reported previously . BrieflyNC, USA)  at a finP-values <0.05 were considered statistically significant.All experiments were repeated a minimum of three times. All data are expressed as mean ± SD in triplicate measures. Differences between groups were assessed by one-way ANOVA and significance of difference between particular treatment groups was analyzed using Dunnett’s multiple comparison tests . Asterisks showed in the figures indicate significant differences of experimental groups in comparison with the corresponding control condition. We first detected the effect of UA on cell growth in human NSCLC cells H1299 by MTT assay. As show in Fig. Next, we examined the effect of UA on apoptosis in NSCLC cells. We found that H1299 cells treated with increased concentrations of UA for 24 h resulted in induction of apoptosis shown in the lower right (AB4) quadrants of the histograms, which were counted as “early” apoptotic cells Fig.  as detecMultiple growth factors and extracellular stimulus trigger activation of the mitogen-activated protein kinases (MAPK) signaling, such as SAPK/JNK pathway, this in turn lead to wide range of biological responses. The SAPK/JNK signaling pathway controls many facets of cellular function, such as apoptosis and cell growth depending on the cell context. We showed that UA increased the phosphorylation of SAPK/JNK in a time-dependent manner in H1299 and A549 cells  has proved to be an important source for novel drug discovery with promising sources for a more effective and less toxic treatment option for cancer. There has been an increase attention on the study of TCM in the prevention and treatment of cancer including lung cancer , 29. UA,Growing evidence also suggested that complicated signalling networks and multiple genes have been involved in the inhibition of cell growth and induction of apoptosis in NSCLC cells. Consistent with other reports , 35, we Our results demonstrated the involvement of DNMT1 and EZH2 in mediating the effect of UA on NSCLC cell growth inhibition. As crucial tumor promoters and oncogenes, EZH2 and DNMT1 are highly expressed in a wide range of cancer types and overexpression of EZH2 and DNMT1 are correlated with advanced stages of cancers, resulting in poor prognosis , 39. ThuIntriguingly, our results also suggested the role of SAPK/JNK signaling pathway in mediating the effect of UA on inhibition of DNMT1 and EZH2 expression. Activation of SAPK/JNK associated with the regulation of EZH2 and DNMT1 expression has been shown in several other studies –48. One In this study, we revealed the role of SP1 involved in the UA-reduced DNMT1 and EZH2. SP1, a well-known ubiquitous transcription factor, is highly expressed in many types of malignancies and plays an important role in progression and metastasis of cancer through binding to GC-rich sequences, thereby regulating various downstream genes . TargetiInterestingly, our results also demonstrated the negative feedback regulations of SAPK/JNK by SP1 and DNMT1, implying that the complicated regulatory loops were involved in the overall effects of UA. While limited data was available for the feedback links of SP1/DNMT1 to SAPK/JNK signaling pathway, the potential significance of these regulatory mechanisms involved in the overall responses of UA required to be determined.vice versa. Whether the SAPK/JNK /EZH2/DNMT1 signaling cascades also involved in the UA-induced apoptosis needs to be elucidated.We realized that less data were presented involving in the regulation of apoptotic signaling by UA in this study. It is possible that inhibition of proliferation can be in part a consequence of increased cell apoptosis or Collectively, our results show that UA inhibits NSCLC growth through SAPK/JNK-mediated inhibition of SP1; this in turn results in inhibition of EZH2 and DNMT1. Overexpression of DNMT1 diminishes UA-reduced EZH2 protein expression. The negative feedback regulation of SAPK/JNK signaling by SP1 and DNMT1 attenuates, while the reciprocal interaction of EZH2 and DNMT1 contributes to the overall effect of UA Fig. . This st


Human T cell Lymphotropic Virus type 1 (HTLV-1 ) is the etiological agent of a severe form of neoplasia designated Adult T cell Leukemia/Lymphoma (ATLL) It is widely accepted that the viral transactivator Tax-1 is the major viral product involved in the onset but not in the maintenance of neoplastic phenotype as only 30-40% of ATLL cells express Tax-1. It has been recently demonstrated that HBZ (HTLV-1 bZIP factor), a protein encoded by the minus strand of HTLV-1 genome, constantly expressed in infected cells and in ATLL tumor cells, is also involved in the pathogenesis of leukemia. The full role played by HBZ in oncogenesis is still to be explored in detail mainly owing to the unavailability of tools to assess quantitative expression, subcellular location and interaction of HBZ with host factors in ATLL. By the use of the first reported monoclonal antibody against HBZ, 4D4-F3, generated in our laboratory it has been possible to carefully assess for the first time the above parameters in HTLV-1 chronically infected cells and, most importantly, in leukemic cells from patients. Endogenous HBZ is expressed in speckle-like structures localized in the nucleus. The calculated number of endogenous HBZ molecules varies between18.000 to 36.000 molecules per cell, 22-44 fold less than the amount expressed in HBZ transfected cells used by most investigators to assess the expression, function and subcellular localization of the viral protein. HBZ interacts in vivo with p300 and JunD and co-localizes only partially, and depending on the amount of expressed HBZ, not only with p300 and JunD but also with CBP and CREB2.The possibility to study endogenous HBZ in detail may significantly contribute to a better delineation of the role and presence of HBZ during HTLV-1 infection and cellular transformation.


The HTLV-1 bZIP factor (HBZ) gene was discovered in 2002 and has been found to promote T-cell proliferation and cause lymphoma in transgenic mice. Thus HBZ has become a novel hotspot of HTLV-1 research. This review summarizes the current findings on HBZ with a special focus on its potential links to the oncogenesis of ATL. We propose viewing HBZ as a critical contributing factor in ATL development.Human T cell leukemia virus type 1 (HTLV-1) is an oncogenic retrovirus responsible for the development of adult T-cell leukemia (ATL). Although HTLV-1 harbors an oncogene,  Human T-cell leukemia virus type 1 (HTLV-1) is the first human retrovirus to have been identified (in the early 1980s), and it was later demonstrated to be the causative agent of adult T-cell leukemia (ATL), an aggressive cancer of peripheral CD4 T cells . HTLV-1 The HTLV-1 provirus is 9 kb long and has multiple coding regions flanked by two identical 750-bp long terminal repeats (LTRs) in the 5′ and 3′ ends , HAM/TSP and ATL patients . If. If92].


Ciona instestinalis, carrying mutations at conserved residues predicted to be required for chelation of the diiron prosthetic group. The modified protein was stably expressed in mammalian cells or flies, but lacked enzymatic activity and was unable to rescue the phenotypes of flies knocked down for a subunit of cytochrome oxidase. The mutated AOX transgene is thus a potentially useful tool in studies of the physiological effects of AOX expression.The mitochondrial alternative oxidase, AOX, carries out the non proton-motive re-oxidation of ubiquinol by oxygen in lower eukaryotes, plants and some animals. Here we created a modified version of AOX from  The mitochondrial alternative oxidase, AOX, carries out the non proton-motive re-oxidation of ubiquinol by molecular oxygen. Terminal electron transfer by AOX constitutes a parallel system to that provided by OXPHOS complexes III and IV in plants, fungi, protists and many animal phylaIn many organisms this is achieved, at least in part, via the regulated expression of the AOX gene, which is induced by a variety of stresses relevant to OXPHOS dysfunction24Drosophila10Ciona intestinalis, was shown to compensate many of the phenotypes resulting from cytochrome oxidase  deficiency in Drosophila, including the knockdown of structurally essential subunits of the complex10Although the AOX gene has been lost, during the course of evolution, in the lineages leading to the most complex and advanced metazoan groups, including mammals8Trypanosoma brucei, has recently been publishedCiona intestinalis enzyme against this template, predicted amino-acids required for binding the catalytically essential diiron moiety at the active site, and proceeded via alanine-substitution mutagenesis to create an expressible version of the enzyme expected to lack enzymatic activity, despite being predicted to fold to a similar overall structure. In several different contexts , we found that the mutated AOX was stably expressed but devoid of detectable enzymatic activity. Furthermore, expression of the transgene encoding the mutated AOX was unable to rescue engineered COX deficiency in the fly, confirming that this rescue indeed depends on the enzymatic activity of AOX.In order to provide a more applicable test of whether the ability of AOX to rescue COX deficiency depends on its primary enzymatic activity, we sought to engineer the AOX in such a way as to destroy this activity, whilst producing only a minimal effect on the overall structure, stability and expression of the protein. To do this, we took advantage of the fact that AOX is well conserved phylogenetically, that the residues contributing to its active site have been characterized in a number of species, and that the structure of a representative AOX, from the protistan parasite C. intestinalis AOX was generated using the software I-TASSERTrypanosoma brucei AOX (PDB 3VV9:A)C. intestinalis AOX structure could not be modelled with high accuracy, this region was eliminated from the analysis. The dimeric model of C. intestinalis AOX and the positioning of the two diiron centres (one per subunit) were built by overlapping two copies of the model generated by I-TASSER into the crystal structure of the dimeric T. brucei AOX using Pymol (www.pymol.org). Pymol was also used to analyze all structure models and to produce the figures.The sequences of AOX homologues found by BlastP searching were aligned using the MUSCLE algorithm built into the software MEGA6Drosophila expression, the C. intestinalis AOX coding sequence, including its natural stop codon, was recloned from the pMT/V5-His B vector (Invitrogen), in which it had been previously propagated, into the EcoRI site of pUASTattBEcoRI site of pAc5.1/V5-His B  to create pAC/AOXkanR plasmid containing the CAG promotertet operator, loxP sites, insulator elements and portions of the porcine Ggta1 gene), to create the expression constructs pCAG-AOX and pCAG-mutAOX. The nucleotide sequences of all clones were confirmed by Sanger sequencing using the Big Dye Terminator v3.1 kit (Life Technologies) and an ABI3130xl Genetic Analyzer, according to the manufacturer’s specifications.For w1118, the RNAi line for CG9603 (Vienna Drosophila RNAi Center line 106661), the ubiquitous da-GAL4 driver (Bloomington line 8641) and the driver line bearing elavC155-GAL4 on chromosome X and UAS-Dcr2 on chromosome 2 (Bloomington line 25750), were obtained from stock centres. ΦC31 recombinase-mediated-site-directed transgenesis was used to generate transgenic fly lines , using recipient lines with the following integration sites: attP18 (chromosome X), attP40 (chromosome 2) and attP2 (chromosome 3), according to Pfeiffer et al.F24 and UAS-AOXF6 were described previouslyExcept where stated, flies were maintained and grown on standard medium at 25°C, using a 12 h light/dark cycle, as previously106 cells were transfected with 24 μg of the pCAG-AOX or pCAG-mutAOX plasmids or, as control, empty vector , using 60 μl Lipofectamine® 2000 (Invitrogen) under manufacturer’s recommended conditions. Drosophila S2 cells were grown and transfected with pAc5.1/V5-His B or derivatives as previouslyHEK293T cells were cultured as previouslyRpL32 RNA as an internal normalization standard were as previously describedDrosophila adults and Western blots were conducted essentially as by Fernandez-Ayala et al.TM EX Running and Transfer Buffers (Lonza), and membranes were treated in PBS-Tween® instead of TBS. Primary antibodies used were customized rabbit anti-AOXst Centrury Biochemicals, 1:10,000), rabbit anti-α-actininin C-20-R  and mouse anti-ATP5A . Secondary antibodies were Peroxidase Goat Anti-rabbit IgG and Horse Anti-mouse IgG . Post-nuclear extracts (PN) from HEK293T cells were prepared according to Cannino et al.RNA extraction and QRTPCR to measure AOX transcript levels using 6 human cells was measured 48 h after transfection, following permeabilization with 80 μg/ml digitonin, in a Clark-type electrode (Hansatech Oxytherm system) using respiratory buffer An-propyl gallate. Respirometry on S2 cells was as described previouslyDrosophila males. Briefly, 25 males were gently homogenized in 0.8 ml ice-cold isolation buffer  and muslin-filtered. Respirometry was performed on 150 μl aliquots of this homogenate, mixed with 500 μl assay buffer , substrates (15 mM glycerol-3-phosphate and 5 mM ADP) and inhibitors as for permeabilized mammalian cells.Oxygen consumption of 5 × 10Drosophila crosses was measured as previouslyTime to eclosion following The work reported here did not use human subjects or any materials derived from human subjects, other than the freely available cell-line HEK293T.Ciona intestinalis AOX amino-acid sequence with the corresponding protein from other taxa, including Trypanosoma brucei, revealed conservation of residues implicated in the organization of the diiron centre of the enzyme, as previously reported by Shiba et al.Ciona AOX with E200, E239, E290, E344, H242 and H347  was first verified, following transient transfection into cultured human cells. Based on Western blotting , the mutg QRTPCR , C and Wg QRTPCR , E.In both females  and maleda-GAL4 driver, the AOX and mutAOX transgenes produced only very small changes in developmental timing, most of them non-significant compared with the corresponding vector-only line  was due to the enzymatic activity of AOX or some other property conferred by the AOX protein, when expressed in da-GAL4 driver to induce simultaneous transgene expression and Cox7a knockdown. Wild-type AOX rescued the lethality, as previously , exceeds a threshold value required to maintain metabolic homeostasis and complete development. In contrast, the lower expression level of the targeted integrants gave a clearly weaker rescue of locomotor dysfunction, when COX was knocked down only in neurons, roughly in proportion to the decreased expression level.Drosophila indeed resides in supercomplexesIt may also be noted that the amount of antimycin-resistance conferred upon respiration in homogenates from the targeted integrants was still approximately 14%, compared with approximately 20% for the P element-mediated integrants, even though they are expressed at a much higher level. The level of respiratory antimycin-resistance in the fly may vary between tissues, and this 20% maximum may reflect only the properties of the predominant class of mitochondria. Most of the respiratory capacity in adult flies is vested in the flight muscles, where mitochondria make up almost one-third of the total tissue massCiona AOX modifies the phenotypes of model organisms, potentially contributing the eventual development of AOX-based therapies.In conclusion, mutAOX offers a useful tool for future studies of the mechanism(s) whereby expression of How to cite this article: Andjelković, A. et al. Diiron centre mutations in Ciona intestinalis alternative oxidase abolish enzymatic activity and prevent rescue of cytochrome oxidase deficiency in flies. Sci. Rep.5, 18295; doi: 10.1038/srep18295 (2015).


GJA5 gene, which encodes Cx40, resulting in a truncated Cx40 (Q49X) was identified in a large Chinese family with lone (idiopathic) atrial fibrillation (AF). This mutation co-segregated with seven AF probands in an autosomal-dominant way over generations. To test the hypothesis that this Cx40 mutant affects the distribution and function of atrial gap junctions, we studied the Q49X mutant in gap-junction-deficient HeLa and N2A cells. The Q49X mutant, unlike wild-type Cx40, was typically localized in the cytoplasm and failed to form gap-junction plaques at cell-cell interfaces. When the Q49X mutant was co-expressed with Cx40 or Cx43, the mutant substantially reduced the gap-junction plaque formation of Cx40 and Cx43. Electrophysiological studies revealed no electrical coupling of cell pairs expressing the mutant alone and a significant decrease in the coupling conductance when the mutant was co-expressed with Cx40 or Cx43. Further colocalization experiments with the organelle residential proteins indicate that Q49X was retained in the endoplasmic reticulum. These findings provide evidence that the Q49X mutant is capable of impairing gap-junction distribution and function of key atrial connexins, which might play a role in the predisposition to and onset of AF.Connexin40 (Cx40)-containing gap-junction channels are expressed in the atrial myocardium and provide a low-resistance passage for rapid impulse propagation. A germline mutation in the  Atrial fibrillation (AF) is the most common cardiac arrhythmia and predisposes affected individuals to an increase in morbidity and mortality . The majCx40 gene-knockout mice suggested that Cx40 is responsible for the decreased conduction velocity in the atrial myocardium and an increased susceptibility to inducible arrhythmias (Cx40 knockout mouse (GJA5 gene (encoding Cx40) mutations as well as other genetic variants in the regulatory regions of GJA5 were found to associate with AF  . In the hythmias . Howeverut mouse , questio with AF . This cl with AF . A decre with AF . The onl with AF . The Cx4 with AF .Clinical issueGJA5 gene, which encodes an atrial gap-junction protein, connexin40 (Cx40). The mutation, which co-segregated with the seven AF probands in an autosomal-dominant manner, results in a truncated form of Cx40. The functional basis for the association of truncated Cx40 with AF remains unknown.Atrial fibrillation (AF) is the most common form of cardiac arrhythmia (irregular heartbeat), affecting millions of individuals worldwide. AF increases the risks of morbidity and mortality and is a major cause of embolic stroke, because of a greater potential for blood clots to form in the heart. Genetic factors are known to play an important role in AF, and researchers have examined familial cases of AF to identify genetic mutations associated with the disease. Recently, analysis of a Chinese family with seven AF individuals led to the identification of a putative causative mutation in the ResultsIn this study, the authors sought to test the hypothesis that the AF-linked Cx40 mutant (Q49X) affects the distribution and function of atrial gap junctions. Using gap-junction-deficient HeLa and N2A cell lines, they demonstrate that the Q49X mutant is retained primarily within the endoplasmic reticulum (ER), rather than being localized to cell-cell junctions. In line with this, cells expressing the mutant protein failed to form gap-junction plaques at cell-cell interfaces. When the Q49X mutant was co-expressed with wild-type Cx40 or another major atrial gap-junction protein, Cx43, gap-junction-plaque formation was substantially reduced and the wild-type proteins were also sequestered in the ER, indicating a dominant-negative effect. Finally, electrophysiological analysis revealed a lack of electrical coupling of cell pairs expressing the mutant alone and a significant decrease in the coupling conductance when the mutant was co-expressed with Cx40 or Cx43.Implications and future directionsThese findings provide evidence that the Q49X mutant is unable to form functional gap junctions because it is retained intracellularly, in the ER. Furthermore, the Q49X mutant impairs gap-junction formation and coupling conductance driven by the two major atrial connexins. Thus, the truncated Cx40 mutant is likely to slow down heart impulse propagation in the atria and promote atrial arrhythmias. This study provides a molecular basis for the association of truncated Cx40 with AF. Understanding the mechanisms by which AF-linked Cx40 mutants impair gap-junction coupling could help in the development of effective treatments for this prevalent cardiac arrhythmia.In the present study, we characterized the Q49X AF-linked mutant, which consists of only the N-terminus, first transmembrane domain and a small part of the first extracellular loop of Cx40 . Our resn=330 cell pairs, n=300 cell pairs, j) at 20 mV transjunctional voltage gradient  of cell pairs expressing Cx40-YFP was similar to that of untagged Cx40 (data not shown), further suggesting that the YFP tag did not interfere with channel function.YFP-tagged Cx40 and the Q49X mutant were expressed in HeLa cells to assess their ability to form gap-junction plaques at the cell-cell interfaces. In Cx40-expressing cells, there were 71% YFP-positive cell pairs exhibiting clear gap-junction plaque-like structures at cell-cell interfaces  and, to a much lesser extent, to the same intracellular compartments  cells. Anti-Cx43 antibody labeling showed an abundant expression of Cx43 in the intracellular compartments and at the cell-cell interfaces in non-transfected NRK cells . ExpressHere, we characterized the first and only nonsense AF-linked germline Cx40 mutant. This mutation introduced a premature stop codon at amino acid position 49, predicting to generate a truncation mutant of Cx40, Q49X. This mutation is the most frequently found Cx40 mutant to date and affected individuals are prone to the onset of AF early in their adult life see . Our finGJA5 gene (encoding Cx40) associated with lone AF   . Howevergle exon  without gle exon . Althouggle exon , at presgle exon . HoweverCx40 gene exhibit a decrease in the action-potential conduction velocity  or only Cx43 gap junctions (NRK cells) are expressed. These mammalian model cells are excellent in delineating individual actions of a mutant and its interactions with a particular connexin. Future studies are required to examine whether the mutant also has similar actions in the native human or animal atrial myocytes.In summary, mutations in the Human wild-type Cx40-YFP, Cx40-RFP, Cx43-RFP and Cx40-IRES-GFP expression vectors were generated as described earlier . Mutant Yellow fluorescent protein (YFP)-tagged wild-type and mutant connexins were localized following expression in HeLa or N2A cells. These cell lines are gap-junction-deficient, easily transfected with connexin cDNA constructs and capable of assembling the expressed connexin into morphological and functional gap junctions. Briefly, cells were grown to 70–80% confluence in glass-bottom dishes  at 37°C in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). X-tremeGENE HP DNA transfection reagent  was used to transfect cDNAs encoding mutant or wild-type connexins. To mimic the patient context, in which both wild type and the mutant are co-expressed in the same cells, cDNAs encoding the mutant and Cx40 (or Cx43) were co-transfected at 1:1 ratio. Live HeLa cells were visualized with a confocal microscope  or a CCD camera  mounted on a fluorescent microscope (Olympus BX51) using a 40× water-immersion lens.For subcellular localization of mutant connexin proteins, HeLa cells were rinsed in PBS  24 hours after transfection. The cells were fixed for 10 minutes in 1:1 acetone/methanol at −20°C and blocked for 1 hour with 5% BSA in PBS. Antibodies against the resident proteins of the ER  or Golgi apparatus  were incubated for 1 hour at room temperature. Cells were washed twice and subsequently labeled for 30 minutes with Alexa-Fluor-594-conjugated secondary antibody. Following further washing, the coverslips were mounted on glass slides in preparation for confocal microscopy. For live-cell studies, cells were co-transfected with YFP-tagged mutant (or Cx40) and a DsRed-tagged ER marker protein . Double-transfected cells were observed with a confocal microscope 24 hours later. Pearson’s correlation coefficient was measured with Image Pro-Plus software  to estimate the colocalization of the mutant and the organelle markers. Pearson’s correlation coefficient=1 represents perfect colocalization and zero represents random localizations.NRK cells  were cultured in DMEM supplemented with 10% FBS. NRK cells were transfected with cDNA vector Cx40 Q49X-IRES-GFP. 24 hours after transfection, cycloheximide (30 μg/ml) was added in some dishes for 9 hours in 37°C incubator to block protein synthesis. Both cycloheximide- treated and untreated cells were fixed with 4% paraformaldehyde for 10 minutes at room temperature. The cells were permeabilized for 10 minutes with 0.2% Triton X-100 in PBS. After two washes, the cells were blocked with 3% BSA and then labeled overnight at 4°C with rabbit anti-Cx43 antibody . The secondary antibody was conjugated with Alexa Fluor 594 (Invitrogen). Counterstaining with Hoechst 33342 (10 μg/ml) for 2 minutes was used to show cell nuclei. Fluorescent images were obtained from a CCD camera  mounted on a fluorescent microscope (Olympus BX51) using a 40× water-immersion lens.j), while the junctional currents (Ij) were measured in the other cell. Macroscopic gap junctional conductance (Gj) was calculated as follows: Gj=Ij/Vj. Data were obtained from multiple independent transfections. To reduce possible cytoplasmic bridge contamination on the Gj measurement, on occasion we included the gap-junction-impermeable high-molecular-weight dextran Texas red dye  in the pipette solution in one cell of the pair. Only cell pairs that did not show transfer of the dextran were selected for Gj analysis. In some experiments, we used the gap-junction blockers carbenoxolone (200–400 μM) or flufenamic acid (50 μM) to ensure that the measured Gj was due to gap-junction channels.Electrophysiological recordings were carried out in the connexin-deficient N2A cells engineered to express wild-type or mutant connexins. To test the effect of the Q49X-YFP mutant on the function of Cx40-RFP (or Cx43-RFP), we co-transfected these two constructs at 1:1 ratio. The dual whole-cell patch-clamp technique was performed to assess the gap-junctional conductance between cell pairs co-expressing mutant and wild-type connexins . Brieflyt-test was used to test statistical significance if data were from two groups. Statistical probability of P<0.05 was considered significant (***P<0.001 shown in figures).All data are expressed as mean ± s.e.m. The statistical comparison of multiple groups of data was performed using one-way ANOVA with Student-Newman-Keuls test with SPSS software. An unpaired Student’s


In the opening plenary lecture, Prof McCormack discusses the trials and tribulations of HIV prevention.


Since September 2010, in Japan serological screening for the detection of human T-cell leukemia virus type 1 (HTLV-1) antibodies can be performed for all women during pregnancy with the Japanese public funds for strategies for prevention of HTLV-1 vertical transmission, because Japan, especially Kyushu area, has been reported to be one of the areas of highest prevalence of HTLV-1 in the world , we examRecently, the migration of Japanese people from Kyushu area to the metropolitan areas has been thought to contribute to a significant decrease of HTLV-1 carriers in Kyushu area and an increase in Kanto (including Tokyo) area in Japan . To confIn 2013, the total rates of positive HTLV-1 screening tests and positive WB test in positive screening tests were 0.35% and 50.8%, respectively. Considering the response rate and the rate of implementation of WB test, the number of HTLV-1 carrier in Japanese pregnant women in 2013 was estimated to be 1,780 (0.18%). Although the migration of Japanese people from Kyushu area to the metropolitan areas has been supposed to contribute to a significant decrease of HTLV-1 carriers in Kyushu area, the estimated number of HTLV-1 carrier of pregnant women seemed to be increased in Kyushu area. In addition, the estimated rate of HTLV-1 carrier in pregnant women in Kyushu area was still significantly higher than that in the other areas (P < 0.01 by the Chi-square test). Therefore, there are still remaining problems concerning the locality for strategies for prevention of HTLV-1 vertical transmission in Japan.


Platelets play an essential role in the body's ability to respond to vascular injury (hemostasis). To form a hemostatic plug and prevent bleeding from the damaged blood vessel, these cells need to be equipped with a powerful signaling machinery that allows them to respond to trace amounts of agonists produced at injury sites, and to rapidly transition from an anti-adhesive/patrolling state to an adhesive state (integrin inside-out activation) required for plug formation. However, this highly sensitive signaling machinery can also pose a significant risk, as premature platelet activation can lead to thrombocytopenia (impaired platelet homeostasis) and/or thrombosis. Thus, negative feedback within the signaling machinery is crucial to balancing sensitivity and speed.Circulating platelets are also important in various aspects of cancer . They suD~80nM) [in vitro and in vivo. In addition to sensitivity, CalDAG-GEFI is also critical for the near-immediate activation of RAP1, triggered by rapid calcium mobilization from stores in the endoplasmic reticulum. Both RAP1 activation and integrin inside-out activation are delayed in platelets from mice and humans lacking functional CalDAG-GEFI [We and others identified the small GTPase RAP1 as a critical regulator of platelet adhesiveness . RAP1 seD~80nM) , 4. ThesDAG-GEFI , 5, 6.IP4bp, R-RAS-GAP) as a critical negative regulator of RAP1 signaling in platelets [Rasa3 mutant mice, strongly suggesting that RASA3 is critical to restrain the highly sensitive CalDAG-GEFI/RAP1 signaling pathway. Thus, one can imagine RASA3 as a “hand brake”, which is pulled in circulating platelets to maintain the cells in a resting state. At sites of vascular injury, however, this brake would impair RAP1 activation and firm platelet adhesion and thus needs to be released to allow for hemostatic plug formation. Our studies strongly suggest that RASA3 inactivation is dependent on signaling by phosphoinositide 3 kinase (PI3K), downstream of the platelet receptor for ADP, P2Y12. More specifically, we observed that Rasa3 mutant platelets did not require feedback signaling via ADP and P2Y12, and that RAP1-dependent integrin activation in these cells was insensitive to inhibitors of P2Y12 or PI3K. Consistent with this data, platelet adhesion at sites of injury in Rasa3 mutant mice was insensitive to inhibitors of P2Y12.As outlined above, a highly sensitive signaling machinery needs to be tightly controlled to prevent premature platelet activation. In recent work, we identified the RAP-GAP, RASA3 (GAP1latelets . RASA3 iTogether, these studies suggest that the antagonistic balance between CalDAG-GEFI and RASA3 signaling is critical for the fine-tuning of platelet adhesiveness, both in the circulation and at sites of vascular injury. Genetic and environmental factors that shift the balance towards RAP1 activation may lead to thrombocytopenia and thrombosis, while those that lead to impaired RAP1 activation may cause bleeding in patients (Figure


TEK tyrosine kinase is primarily expressed on endothelial cells and is most commonly referred to as TIE2. TIE2 is a receptor tyrosine kinase modulated by its ligands, angiopoietins, to regulate the development and remodeling of vascular system. It is also one of the critical pathways associated with tumor angiogenesis and familial venous malformations. Apart from the vascular system, TIE2 signaling is also associated with postnatal hematopoiesis. Despite the involvement of TIE2-angiopoietin system in several diseases, the downstream molecular events of TIE2-angiopoietin signaling are not reported in any pathway repository. Therefore, carrying out a detailed review of published literature, we have documented molecular signaling events mediated by TIE2 in response to angiopoietins and developed a network map of TIE2 signaling. The pathway information is freely available to the scientific community through NetPath, a manually curated resource of signaling pathways. We hope that this pathway resource will provide an in-depth view of TIE2-angiopoietin signaling and will lead to identification of potential therapeutic targets for TIE2-angiopoietin associated disorders. Angiopoietin-TIE2 is one of the major signaling systems that regulates development and remodeling of vascular system , 2. TIE2 via Tyr-1101 and activates PI3K-AKT pathway which inhibits Smac release from mitochondria and increases the expression of survivin leading to survival and chemotaxis of endothelial cells , a resource of signaling pathways. These formats include Proteomics Standards Initiative for Molecular Interaction (PSI-MI version 2.1), Biological Pathway Exchange (BioPAX level 3), and Systems Biology Markup Language (SBML level 2.1).Our analysis resulted in the cataloging of 140 unique molecules that are reported in TIE2 signaling. These molecules were part of 43 PPIs and 102 catalytic events, 23 activation/inhibition events, and 11 protein translocation events. We have also documented 124 and 65 genes that were reported to be upregulated and downregulated, respectively, by TIE2 signaling in response to angiopoietin(s) in human cells. The curated data for TIE2 signaling pathway is freely available to the scientific community for visualization and download in different community standard data exchange formats through NetPath [http://www.netpath.org/netslim/TIE2_pathway.html), a resource that provides a smaller version of the pathway by filtering data based on predefined confidence threshold criteria [For effective visualization, we have graphically represented the reactions and various cellular processes that those reactions mediate in the context of specific studies on TIE2 signaling . PathViscriteria . At NetS vice versa, in the subsequent versions in NetPath. We encourage scientific community to help us maintain this resource up-to-date and error-free through http://www.netpath.org/comments.This open-access pathway data enables better analysis of high-throughput experimental data and hypothesis-driven approaches to study the dynamics of TIE2 signaling for therapeutic interventions. Information on TIE2 pathway in NetPath will be periodically updated to reflect novel findings relevant to TIE2 signaling. We intend to provide information pertaining to cross-talks of other ligand/receptor systems such as VEGF, TNF-alpha, and integrins with TIE2 and


Caenorhabditis elegans for mutants defective in dense-core vesicle function. We previously reported the identification of two conserved proteins that interact with the small GTPase RAB-2 to control normal dense-core vesicle cargo-sorting. Here we identify several additional conserved factors important for dense-core vesicle cargo sorting: the WD40 domain protein EIPR-1 and the endosome-associated recycling protein (EARP) complex. By assaying behavior and the trafficking of dense-core vesicle cargos, we show that mutants that lack EIPR-1 or EARP have defects in dense-core vesicle cargo-sorting similar to those of mutants in the RAB-2 pathway. Genetic epistasis data indicate that RAB-2, EIPR-1 and EARP function in a common pathway. In addition, using a proteomic approach in rat insulinoma cells, we show that EIPR-1 physically interacts with the EARP complex. Our data suggest that EIPR-1 is a new interactor of the EARP complex and that dense-core vesicle cargo sorting depends on the EARP-dependent trafficking of cargo through an endosomal sorting compartment.The dense-core vesicle is a secretory organelle that mediates the regulated release of peptide hormones, growth factors, and biogenic amines. Dense-core vesicles originate from the trans-Golgi of neurons and neuroendocrine cells, but it is unclear how this specialized organelle is formed and acquires its specific cargos. To identify proteins that act in dense-core vesicle biogenesis, we performed a forward genetic screen in  C. elegans to identify proteins that are important for early steps in the generation of dense-core vesicles, such as packaging the correct molecular cargos in the vesicles. We identify several factors that are conserved between worms and humans and point to a new role for a protein complex that had previously been shown to be important for controlling trafficking in other cellular compartments. The identification of this complex suggests new cellular trafficking events that may be important for the generation of dense-core vesicles.Animal cells package and store many important signaling molecules in specialized compartments called dense-core vesicles. Molecules stored in dense-core vesicles include peptide hormones like insulin and small molecule neurotransmitters like dopamine. Defects in the release of these compounds can lead to a wide range of metabolic and mental disorders in humans, including diabetes, depression, and drug addiction. However, it is not well understood how dense-core vesicles are formed in cells and package the appropriate molecules. Here we use a genetic screen in the microscopic worm  The dense-core vesicle is a specialized secretory organelle found in neurons and endocrine cells. In endocrine cells, these vesicles are often called secretory granules; for simplicity, we will here use the term ‘dense-core vesicles’ to refer to both the neuronal and endocrine carriers. Dense-core vesicles package several classes of cargo, including peptide hormones, growth factors, and biogenic amines. In response to elevated calcium, dense-core vesicles release their cargos to modulate a variety of biological processes ranging from blood glucose homeostasis to development, function and plasticity of the nervous system. Despite the importance of the signaling molecules released by dense-core vesicles, surprisingly little is known about how dense-core vesicles are generated and acquire their proper cargos –4.2+ milieu of the trans-Golgi . Lysates were passed 10 times through a 20G needle and incubated for 30 min at 4°C. Lysates were pre-cleared by a 20,000 g centrifugation at 4°C for 15 minutes and the supernatant was incubated with 30 μg of anti-GFP nanobody bound to magnetic beads, for two hours at 4°C. The beads were washed three times with 250 μL wash buffer . Samples were eluted in two steps with (1) 0.2 M glycine pH 2.5 and (2) 1 M NH4OH. Eluents were neutralized with 0.33 M Tris-HCl pH 7.5 and stored at -80°C.For immunoprecipitation, 4x10Samples were processed using modified FASP (filter aided sample preparation) to remove mass spec incompatible substances . SamplesA total of 0.15 μg of peptides was chromatographically separated onto a Thermo LTQ-Velos Pro mass spectrometer coupled with a Waters nanoACQUITY liquid chromatography system. A 75 μm fused silica column (Polymicro Technologies) was loaded with 30 cm C12 Jupiter (Phenomenex), 4 μm reverse-phase beads, and a 100 μm fused silica Kasil frit trap (PQ Corporation) loaded with 4 cm of C12 Jupiter. Peptides were separated by a 110 minute gradient consisting of buffer A  and buffer B . Two analytical replicates were run for each sample with each set of replicates randomized. Quality control samples were run every sixth sample and at the beginning of the runs to assess column chromatography stability.Rattus norvegicus FASTA database containing contaminants. False discovery rates were determined via a decoy database using Percolator [Data were searched with SEQUEST  against rcolator  at a q-vrcolator . Lists oFor co-immunoprecipitation, 832/13 cells were cultured and lysed as described above (Mass spectrometry). The lysates were incubated with 20 μg anti-GFP nanobody bound to magnetic beads, the beads were washed three times with lysis buffer and resuspended in Laemmli loading buffer. Samples were resolved on 10% SDS-polyacrylamide gels and blotted onto nitrocellulose or PVDF membranes. To detect co-precipitated proteins, we added the following primary antibodies: mouse monoclonal anti-GFP , monoclonal anti-mCherry , monoclonal anti-VPS50 , rabbit polyclonal anti-SNAP29 , or monoclonal anti-beta-tubulin as a loading control . The secondary antibody was an Alexa Fluor 680-conjugated goat anti-mouse antibody  or Alexa Fluor 680-conjugated goat anti-rabbit antibody . A LI-COR processor was used to develop images.Worms were mounted on 2% agarose pads and anesthetized with 50 mM sodium azide. Images were obtained using a Nikon 80i wide-field compound microscope except for the images shown in 5 832/13 cells per well were seeded onto sterilized cover slips placed in 12 well cell culture plates. The next day, cells were rinsed twice with ice-cold PBS and fixed with 4% paraformaldehyde in PBS for 20 minutes at room temperature. The cells were rinsed twice with PBS and permeabilized with 0.5% Triton X-100 in PBS for 5 minutes at room temperature. The cells were again washed twice with PBS and then placed in 5% milk in PBS for 1 hour at room temperature, followed by immunofluorescence staining with rabbit polyclonal anti-CCDC132  and mouse monoclonal anti-GFP  in 0.5% milk in PBS at room temperature for 1 hour. The cells were then washed with PBS three times for 5 minutes each, and incubated with Rhodamine anti-rabbit secondary antibody  and Alexa Fluor 488 anti-mouse secondary antibody  at room temperature for 1 hour. The cells were washed with PBS three times for 5 min each and examined by fluorescence microscopy (Nikon 80i wide-field compound microscope). The anti-CCDC132 antibody recognizes VPS50.2x10P values were determined using GraphPad Prism 5.0d (GraphPad Software). Data sets with multiple comparisons were analyzed by a one-way ANOVA followed by a Bonferroni posthoc test to examine selected comparisons or by Dunnett’s test if all comparisons were to the wild type control. Pairwise data comparisons were analyzed by two-tailed unpaired t tests.S1 Figegl-30(tg26) has hyperactive locomotion and deep body bends. The egl-30(tg26) locomotion phenotype is suppressed by the eipr-1(ox316), vps-50(ok2627), vps-52(ox345), and vps-53(ox339) mutations. (B) eipr-1 acts in neurons to control egg laying. The graph shows the number of eggs laid by 5 animals in a 2.5 hour period. eipr-1 mutants show egg-laying defects and this phenotype is rescued by panneuronal expression of either the worm gene or its mouse ortholog . Error bars = SEM; n = 10 plates of 5 worms each. (C) eipr-1 acts in the same pathway as rab-2 to control egg-laying. Double mutants of eipr-1(tm4790) with rab-2(nu415) or rund-1(tm3622) do not have stronger egg-laying defects than the single mutants. Though rund-1(tm3622) mutants have a visible Egl (egg-laying defective) phenotype indicating that they retain more eggs, they did not have a significantly reduced egg-laying rate as measured by this assay. Error bars = SEM; n = 5 plates of 5 worms each. ns, not significant, P>0.05.(A) The activated Gq mutant (TIF)Click here for additional data file.S2 FigC. elegans EIPR-1  and its orthologs from Arabidopsis thaliana , Drosophila melanogaster , and Homo sapiens . Identical residues are shaded in black and similar residues are shaded in gray. The WD40 repeats  are marked with single black bars. Using SMART, worm EIPR-1 has four predicted WD40 repeats, Arabidopsis has six, fly has three, and human has five. WD40 repeats are difficult to identify by primary sequence and are often missed by prediction programs. The position of the ox316 stop mutation is marked with an asterisk. Alignment was made with MUSCLE (http://www.ebi.ac.uk/Tools/msa/muscle/) using default parameters and exhibited with Boxshade 3.21 (http://www.ch.embnet.org/software/BOX_form.html).Alignment of (TIF)Click here for additional data file.S3 FigceIs61) fluorescence in dorsal cord motor neuron axons of the wild type and eipr-1(tm4790) mutant strains. Scale bar: 10 μm. eipr-1(tm4790) mutants have decreased fluorescence in the dorsal cord, indicative of a FLP-3::Venus sorting or trafficking defect. (B) Representative images of INS-22::Venus (nuIs195) fluorescence in dorsal cord motor neuron axons. Scale bar: 10 μm. (C) Representative images of IDA-1::GFP (ceIs72) fluorescence in the dorsal nerve cord. Scale bar: 10 μm. (D) Levels of the synaptic vesicle cargo synaptobrevin, SNB-1::GFP (nuIs152), in the dorsal nerve cord. eipr-1(tm4790) mutants do not have a defect . Error bars = SEM; n = 21–26. (E) eipr-1 and vps-50 mutants do not have enlarged lysosomes. Representative images of LMP-1::GFP (pwIs50) fluorescence in coelomocytes of wild-type (WT), eipr-1(tm4790) and vps-50(ok2627) mutant strains. Scale bar: 10 μm.(A) Representative images of FLP-3::Venus ((TIF)Click here for additional data file.S4 Figeipr-1 is located. The image on the left shows expression in the hypodermis (skin) while the image on the right shows expression in the pharynx. (B) Representative images of neurons expressing eipr-1::GFP under panneuronal  and head cholinergic  promoters. Scale bars: 5 μm. (C) Representative images of neurons coexpressing rab-3p::eipr-1::GFP and GTP-bound RAB-2 (RAB-2QL). Scale bars: 5 μm. (D) Representative images of 832/13 cells expressing GFP::RAB2A and costained for endogenous VPS50. Scale bar: 10 μm. VPS50 partially colocalizes with RAB2A. (E) Representative images of 832/13 cells expressing CCCP1::GFP and costained for endogenous VPS50. Scale bar: 10 μm. VPS50 largely colocalizes with CCCP1. (F) Representative images of wild-type (WT) and eipr-1(tm4790) mutant neurons expressing VPS-52::tagRFP. Scale bars: 1 μm. VPS-52 localizes normally in eipr-1 mutants.(A) Representative images of animals expressing GFP under the promoter region upstream of the first gene (W09C5.1) of the operon where (TIF)Click here for additional data file.S5 Fig(A) List of top hits from two independent experiments performing mass spectrometry of a pulldown of rEIPR1::GFP in 832/13 cells. On the left side of the table, we show the list of hits found in a mass spec experiment after subtracting hits found in a GFP control pulldown. On the right side of the table, we show the list of hits found in an independent mass spec experiment after subtracting hits found in either the pulldown of CCCP1::GFP or RUNDC1::GFP. In both cases, EIPR1::GFP pulldowns were performed in parallel to controls. # seq = number of unique peptides from each protein. All proteins with two or more unique peptides are shown. (B) EGFP-tagged rat EIPR1 or EGFP was expressed in 832/13 cells. Immunoprecipitation of EIPR1::EGFP pulled down more SNAP29 than untagged EGFP. IN: input; IP: immunoprecipitation.(TIF)Click here for additional data file.S6 Figvps-50 acts in the same genetic pathway as eipr-1 to control FLP-3::Venus trafficking. Left: representative images. Right: quantification. vps-50(ok2627) and eipr-1(tm4790) have reduced FLP-3::Venus (ceIs61) fluorescence, but an eipr-1(tm4790); vps-50(ok2627) double mutant does not have a stronger phenotype than either single mutant indicating that eipr-1 and vps-50 act in the same genetic pathway. A vps-54(ok1473) mutant does not have decreased axonal levels of FLP-3::Venus. ***, P<0.001 compared to wild type. Error bars = SEM; n = 9–14. (B) eipr-1 and GARP/EARP mutants have reduced secretion of NLP-21::Venus. NLP-21::Venus (nuIs183) fluorescence levels in the coelomocytes of the indicated strains. Like rab-2(nu415) mutants, eipr-1(tm4790), vps-50(ok2627), and vps-52(ox345) mutants show reductions in the accumulation of Venus fluorescence in coelomocytes that is approximately proportional to the decrease in axonal fluorescence seen in these mutants, suggesting that these mutants are not defective in dense-core vesicle release. Double mutants between eipr-1(tm4790) and rab-2(nu415) or vps-50(ok2627) do not have stronger phenotypes than the single mutants, suggesting that these genes all act in the same dense-core vesicle cargo sorting pathway. **, P<0.01; ***, P<0.001 compared to WT. Error bars = SEM; n = 14–33. (C) vps-51 mutants have defects in trafficking FLP-3::Venus. FLP-3::Venus (ceIs61) fluorescence levels in the dorsal nerve cord of wild type and vps-51(tm4275) mutant strains. Error bars = SEM; n = 8. (D) GARP mutants vps-54(ok1463) and vps-54(ok1473) do not have a reduced locomotion rate. Error bars = SEM; n = 10. (E) eipr-1(tm4790) and vps-50(ok2627) mutants do not have defects in trafficking synaptic vesicle cargos. SNB-1::mCherry (ceIs61) fluorescence levels were measured in the dorsal nerve cord. ns, not significant, P>0.05 compared to wild type. Error bars = SEM; n = 11–14.(A) (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


AGE/RAGE signaling has been shown to increase oxidative stress to promote diabetes-mediated vascular calcification through activation of Nox-1 and decreased expression of SOD-1. AGE/RAGE signaling in diabetes-mediated vascular calcification was also attributed to increased oxidative stress resulting in the phenotypic switch of VSMCs to osteoblast-like cells in AGEs-induced calcification. Researchers found that pharmacological agents and certain antioxidants decreased the level of calcium deposition in AGEs-induced diabetes-mediated vascular calcification. By understanding the role the AGE/RAGE signaling cascade plays diabetes-mediated vascular calcification will allow for pharmacological intervention to decrease the severity of this diabetic complication.AGE/RAGE signaling has been a well-studied cascade in many different disease states, particularly diabetes. Due to the complex nature of the receptor and multiple intersecting pathways, the AGE/RAGE signaling mechanism is still not well understood. The purpose of this review is to highlight key areas of AGE/RAGE mediated vascular calcification as a complication of diabetes. AGE/RAGE signaling heavily influences both cellular and systemic responses to increase bone matrix proteins through PKC, p38 MAPK, fetuin-A, TGF- Diabetes mellitus is highly prevalent in the United States with approximately 29 million people living with diabetes or 9.3% of the population [Diabetes mellitus is a family of diseases characterized by elevated blood glucose levels or hyperglycemia resulting from the body's inability to produce and/or use the insulin hormone. Type I diabetes mellitus is associated with pancreatic pulation . It is rpulation . IncreasType II diabetes has been heavily linked to vascular calcification through several different mechanisms, some of which include oxidative stress, hyperglycemia, hyperkalemia, and hypercalcemia with oxidative stress being the main focus of this review –5. Vascu In vitro experiments, using bovine vascular smooth muscle cells (BVSMCs) grown in euglycemic  and hyperglycemic conditions, revealed that CBFA1, ALP, and osteocalcin levels were significantly higher in cells grown in a high glucose media. In addition, calcium deposition was also significantly higher in high glucose than in normal glucose media, and this trend was also observed when both types of growth media conditions were supplemented with calcification media. Calcification media contain elevated levels of inorganic phosphate to promote calcification through utilization of the cells that need to maintain homeostasis. To determine the signaling mechanisms responsible for the increased bone matrix protein expression, BVSMCs were exposed to high glucose levels and protein kinase C (PKC) activity was pharmacologically inhibited in both normal and high glucose treated cells. PKC was selected as the signaling pathway focus due to its predetermined role in cellular responses to diabetes and hyperglycemia [In a series of studies performed by Chen et al., arteries harvested from diabetic and nondiabetic patients were analyzed to determine the amount of calcium, OPN, ALP, type I collagen, and BSP. With the exception of BSP, all investigated bone matrix proteins were significantly increased as a result of diabetes . In vitrglycemia , 23. As glycemia , 24. Supglycemia –27. Takeζ to trigger the downstream activation of a signaling cascade that works through p38 mitogen activated protein kinase (MAPK), transforming growth factor-β (TGF-β), and nuclear factor κB (NFκB) [α (SM22α) [ζ signaling, increased expression of ALP, and decreased expression of VSMC gene markers.In addition to increased bone matrix protein expression in VSMCs during diabetic and calcification treatments, studies have also shown that advanced glycation end products (AGEs) and their receptors (RAGEs) play a role in vascular calcification . Type IIB (NFκB) , 35. Sug (SM22α) . This do (SM22α) . This is (SM22α) . These s in vitro calcification model increasing the levels of AGEs significantly increased the amount of calcium deposition after 7 and 14 days when compared to BSA treated and control samples [In studies performed by Tanikawa et al., using an HVSMC samples . Additio samples . VSMC ex samples . In a si samples . Therefo samples .While these findings demonstrate the importance of the AGE-RAGE pathway in diabetes-mediated vascular calcification, Ren et al. demonstrated that AGEs also significantly increased intracellular calcium levels in rat VSMCs , 39, 40.α2-Heremans-Schmid glycoprotein (Ahsg or fetuin-A), a systemically circulating glycoprotein, has been implicated in insulin resistance in type II diabetic patients [Serum protein patients . Patientpatients , 44. Colpatients . VasculaConversely, fetuin-A can also elicit an innate immune response elicited in part by toll-like receptors (TLRs). This mechanism can be activated by free fatty acids (FFAs) to induce a proinflammatory response . Pal et −/−) determined that the glycoprotein is an inhibitor of calcification [−/− animals to perform an in vitro basic calcium phosphate (BCP) precipitation assay. Fetuin-A decreased the amount of BCP precipitate within the serum, indicating that fetuin-A can inhibit the formation of BCP deposition [ in vitro resulted in BCP structure changing from a rigid to a fragile appearance [3 nanoparticles [Fetuin-A (Ahsg) has a high affinity for hydroxyapatite crystals, which are located in sites of vascular calcification, such as bone and teeth , 50, 51.fication . X-ray iposition . Within pearance , 55. Thiarticles . in vitro and in vivo HVSMCs model system. Reynolds et al. demonstrated that fetuin-A was localized in the matrix vesicles of calcified HVSMCs in the medial layer of the artery [The relationship between fetuin-A, BCP, and calcified VSMCs was determined usinge artery . These ce artery , 59. Mice artery . In simie artery , 61. Takβ, NF-κB, and Nox-1 [ in vivo system can be reduced after diabetes-mediated vascular calcification has occurred [ in vivo model of vascular calcification [The AGE/RAGE signaling cascade has been demonstrated to be akin to a feed-forward loop whereby outcomes such as increased fibrosis, increased RAGE expression, and increase oxidative stressors are produced , 63. Oxind Nox-1 . In a stnd Nox-1 . Brodeuroccurred . Pyridoxfication . Collectβ. In a study by Li et al. when VSMCs were treated with AGEs, members of the AGE/RAGE signaling cascade  were found to be phosphorylated upon RAGE activation [β signaling resulted in the phosphorylation of its family of mediators, Smads, which serve as transcriptional modulators [β dependent. Western blot analysis revealed that when RAGE expression was downregulated, Smad 2 phosphorylation was also inhibited indicating the AGE/RAGE cascade in Smad activation and TGF-β signaling. Since the accumulation of AGEs is within the extracellular matrix (ECM), it is important to note that an increase in TGF-β has been implicated in fibrosis within disease [β through mediation by RAGE [Another ROS signaling cascade activated by AGEs is transforming growth factor- (TGF-) tivation . In addidulators . These c disease . Fibrosi by RAGE , 66, 68.κB through RAGE signaling in VSMCs. Studies have demonstrated that VSMCs will maintain a compliant, contractile phenotype within the artery; however, increases in NFκB signaling will interfere with this phenotype resulting in increased rigidity and stiffness commonly associated with cardiovascular diabetic complications [κB activity. Western blot analysis revealed that ERK1/2 activation was significantly increased with AGE-HSA treatment, and AKT activation was slightly increased. Both of these pathways activate NFκB, which would allow for the conclusion that RAGE signaling increases NFκB activity [κB transcription activity can lead to an increase in mRNA expression of type I collagen a1 and a2 in murine VSMCs treated with AGEs as shown in Peng et al. [κB in VSMCs, which can lead to remodeling of the type I collage in the ECM or to a change in cell morphology. Also, when they are treated with AGE-HSA, the mRNA levels of smooth muscle-myosin heavy chair (SM-MHC) and SM-22α were decreased, and additionally protein expression of SM-α-actin, SM-22α, and myocardin (MyoC) was also decreased. Overall, the researchers demonstrated that RAGE signaling interferes with the expression of smooth muscle phenotype markers in A7r5 cells. The loss of smooth muscle phenotype markers offered an explanation for the changes in smooth muscle mechanical cell properties as AGE/RAGE signaling increased. There was also an increased granularity within the A7r5 cells demonstrating a visual change in cell morphology due to increased RAGE signaling. While the overall actin density was unchanged with AGE-HAS treated cells, Young's modulus, a measure of elasticity, revealed that basal cell rigidity was significantly increased indicating a stiffer, less elastic cell type. Protein expression levels of phosphorylated myosin light chain (MLC) were also measured to determine changes in contractile function and actin-myosin-mediated motor activity. These results revealed that no changes in contractile function occurred when A7r5 cells were treated with AGE-HSA. Taken together, increased AGE/RAGE signaling alters the mechanical properties of VSMCs resulting in a stiffer, less compliant cell type.AGEs have also been shown to increase the activity of NFications . Simard activity . An incrg et al. . Collectβ mediated fibrosis, NFκB, and ERK1/2 pathways and decreased expression of SOD-1. Researchers found that pharmacological agents and certain antioxidants decreased the level of calcium deposition in AGEs-induced diabetes-mediated vascular calcification. Overall, the role of AGE/RAGE signaling in diabetes-mediated vascular calcification was attributed to oxidative stress and the phenotypic switch of VSMCs in AGEs-induced calcification conditions as shown in AGE/RAGE signaling is a complex and intricate cascade and has been studied in many different disease states. Particularly, diabetes-mediated vascular calcification exhibits several factors that allows for AGE/RAGE signaling to heavily influence both cellular and systemic responses. Vascular calcification has been demonstrated to increase bone matrix proteins through PKC signaling in hyperglycemic and calcification conditions. AGEs-induced vascular calcification caused downregulation of VSMCs markers and an upregulation of bone matrix proteins, thus, suggesting that the VSMCs undergo a phenotypic switch to an osteoblast-like cell. RAGE signaling can also mediate VSMC calcification through a number of mitogenic pathways. Of those, the p38 MAPK pathway was demonstrated to be an essential component for AGE/RAGE mediated VSMC differentiation. Fetuin A was also shown to play a more controversial role in vascular calcification. Fetuin A acts as a mediator for both procalcification by artificially selecting for AGEs as a RAGE ligand as well as anticalcification in certain models of CDK. Fetuin-A represents an exciting area for more work to be done to understand its role in vascular calcification as a diabetic complication. AGE/RAGE signaling has been implicated in oxidative stress associated with diabetes-mediated vascular calcification through activation of Nox-1, TGF-


Brazil has one of the highest adolescent fertility rates in the world. Income inequality has been frequently linked to overall adolescent health, but studies that analyzed its association with adolescent fertility have been performed only in developed countries. Brazil, in the past decade, has presented a rare combination of increasing per capita income and decreasing income inequality, which could influence future desirable pathways for other countries.We analyzed every live birth from 2000 and from 2010 in each of the 5,565 municipalities of Brazil, a total of 6,049,864 births, which included 1,247,145 (20.6%) births from women aged 15 to 19. Income inequality was assessed by the Gini Coefficient and adolescent fertility by the ratio between the number of live births from women aged 15 to 19 and the number of women aged 15 to 19, calculated for each municipality. We first applied multilevel models separately for 2000 and 2010 to test the cross-sectional association between income inequality and adolescent fertility. We then fitted longitudinal first-differences multilevel models to control for time-invariant effects. We also performed a sensitivity analysis to include only municipality with satisfactory birth record coverage.Our results indicate a consistent and positive association between income inequality and adolescent fertility. After controlling for per capita income, college access, youth homicide rate and adult fertility, higher income inequality was significantly associated with higher adolescent fertility for both 2000 and 2010. The longitudinal multilevel models found similar results. The sensitivity analysis indicated that the results for the association between income inequality and adolescent fertility were robust. Adult fertility was also significantly associated with adolescent fertility in the cross-sectional and longitudinal models.Income inequality is expected to be a leading concern for most countries in the near future. Our results suggest that changes in income inequality are positively and consistently associated with changes in adolescent fertility. Brazil has one of the highest adolescent fertility rates in the world. According to a report by the World Health Organization (WHO) , it is oVarious theories have been put forward to account for adolescent pregnancies. Most focus on individual factors such as the absence of sex education and the availability of contraceptive measures ,3. More th century [Income inequality is a growing source of concern for most developed countries, as the disparity between rich and poor individuals has been consistently growing since the last decades of the 20 century . It is f century ,8.A recent review of the determinants of adolescent health, part of a special series on adolescents by The Lancet , includeDespite growing evidence that income inequality affects overall adolescent health, there is a limited number of studies that have analyzed the specific relationship between income inequality and adolescent fertility, all of which used data from developed countries. An ecological study based in 39 US states found an association between income inequality and adolescent fertility, but the association was substantially explained by the correlation between income inequality and poverty . AnotherBrazil has historically presented one of the highest levels of income inequality in the world . During We analyzed every live birth that occurred in the year 2000 and in 2010  in each of the 5,565 municipalities of Brazil, a total of 6,049,864 births-which included 1,247,145 (20.6%) births from women aged 15 to 19 [All variables were calculated separately for each municipality. Municipalities in Brazil are the smallest regions with administrative power, encompassing both urban and rural areas, and dividing the entirety of the country - i.e. every area of Brazil is part of a municipality . In geneAlto Feliz) to 0.87 (Campos de Júlio) in the year 2000, and from 0.28 (São José do Hortêncio) to 0.80 (São Gabriel da Cachoeira) in 2010.Income inequality was assessed by the Gini Coefficient with data from the United Nations Development Program . The GinWe also included variables that could confound the relationship between income inequality and adolescent health. First, we controlled for the per capita income of the municipality, to take account of the correlation between absolute income and income inequality . Second,We first tested the association between adolescent fertility and income inequality separately by year (2000 and 2010) by fitting a multilevel regression model. The first level of the model referred to the aggregated results for each municipality. As adolescent pregnancies could be affected by regional factors, we included each of the 27 states of Brazil as the second level of the model to test for a broader cultural influence on adolescent fertility. The first model included only the municipality-level income inequality as the independent variable. The second model added the control variables, i.e. per capita income, college access, adult fertility and youth homicide rate. The third model included state-level income inequality. The same sequence was repeated for 2010. We added a seventh model to test if the association between income inequality and adolescent fertility significantly changed from 2000 to 2010, by including the results from both years  and adding an interaction term for the year 2010 for all the covariates. For each model, we calculated the deviance of the multilevel model (-2*log-likelihood), a badness-of-fit statistics, where lower values indicate that the model has a better fit to the data.As adolescent fertility may be affect by other cultural and geographic factors not included in the models, we fitted longitudinal first-difference multilevel models to control for time-invariant effects . We firsFor every model, each municipality was weighted by its total population. For models that included 2000 and 2010, municipalities were weighted by the average population for the two years. Descriptive statistics were assessed with Stata 12. Multilevel models were estimated by using MLwiN V.2.29 software.Table Table Table We then introduced a sensitivity analysis to check for the robustness of the results, by including only municipalities with satisfactory birth record coverage Table . The lonOur results indicate a consistent and positive association between income inequality and adolescent fertility. We first tested this association cross-sectionally and then fitted a first-differences longitudinal model to control for time-constant effects, with similar results. For the final first-differences model, a longitudinal increase of 0.10 in the Gini Coefficient was associated with a longitudinal increase of 0.41 live births for every 100 adolescent mothers. Adult fertility (from women aged 20 to 49 years old) was also positively associated with adolescent fertility for every model. Results for youth homicide rate were mixed. Finally, per capita income does not seem to be associated with adolescent fertility.A number of authors have suggested that when there is a perception of future economic advancement, either due to a more equal or mobile society, adolescents have an incentive to delay motherhood and invest in human capital, decreasing adolescent pregnancies ,5. AdoleAdolescent pregnancies have been associated with a number of adverse future socioeconomic outcomes for the mother, such as a lower probability of receiving high school diploma, lower income as a young adult and a higher probability of receiving cash assistance . AdolescPrevious studies have mostly analyzed individual risk factors for adolescent fertility. Recent studies in Brazil have suggested that individual risk factors may have lost at least some of its influences. In a recent Brazilian study of teenage girls from the municipality of São Paulo, most of them answered positively when asked if one should always wear a condom when having sexual relationships, a proportion that did not change whether the teenager was from a public (96.6%) or private school (96.7%) . AnotherOur results suggest the existence of an effect of broader, contextual factors on adolescent fertility. Besides income inequality, adult fertility was also independently and positively associated with adolescent fertility. This suggests that adolescent fertility and adult fertility are likely to move in the same direction, indicating the presence of a local peer effect on motherhood, as found by Vundule et al. .Youth homicide rate was cross-sectionally associated with adolescent fertility, an association also previously reported by Pickett et al.  on a samOur results also indicate that the association of income inequality on adolescent fertility happens primarily on the municipality-level. State-level income inequality was not significant in any of the models. As most of the previous studies that analyzed the effects of inequality in the US focused on state-level differences, our results suggest that by not including municipalities the association between inequality and health outcomes could have been underestimated.The present study adds to a growing literature on how social determinants affect the health of populations. It is well established that some social characteristics such as poverty and education are associated with a large number of health outcomes, but the effect of income inequality has only recently entered the public debate in developed countries, especially after the analyses of Wilkinson & Pickett  that shoOur analyses focused solely on the effect of income inequality on live births. Another possible effect of income inequality would be on the abortion rate of the municipalities. In the case of Brazil, that analysis is still not possible, as the great majority of abortions are illegally induced and thus not reported by municipalities or states . The posThe study has a few other limitations. First, Brazil is a large and heterogeneous country, where the quality of the data differs significantly between the regions. We tried to account for that by adding an analysis that included only municipalities with satisfactory live birth coverage, but other biases may remain. Second, some of the determinants of adolescent fertility may be clustered within smaller areas than municipalities, such as neighborhoods, for which data is not available. Third, the study relied on secondary data that can be subject to measurement errors, especially in very small and poor municipality where per capita income is unclear due to informal work and where homicides are underreported. Fourth, the direct generalization of our finds to other countries should be handled with caution given the particular socioeconomic pathway that Brazil went through during the period .In developing countries, complications from pregnancy and childbirth are the leading cause of death for women aged 15 to 19 . It is e


The 23 HTLV-1+ bitten individuals reported a gorilla (17), chimpanzee (3) or monkey (3) bite. Interestingly, 13/23 were coinfected by a simian foamyvirus, for which cross-species transmission from NHP to humans through bites is demonstrated. Moreover, familial studies excluded the other established routes of virus acquisition among some positive bitten individuals. Lastly, a phylogenetic analysis showed a HTLV-1 subtype F in a bitten subject closely related to the STLV-1 strain from a Cercocebus agilis with whom he was in contact, thus strengthening these new findings.HTLV-1 infection is endemic in Central Africa, as well as the closely related STLV-1 found in several non-human primates (NHPs). Like other retroviruses, acquisition through interspecies transmission is strongly suggested but needs to be investigated. We analyzed 269 selected individuals  for whom a direct contact (mainly a severe bite) with a NHP occurred. This happened mostly during hunting activities and involved bleeding and body fluids exchange with at least saliva/blood contact. The same number of persons who live in the same villages/settlements but did not report any bite by NHPs was matched according to sex, age and ethnicity. Both groups include either Pygmies or Bantus living in the rain forest of South Cameroon. Plasma were tested for HTLV serology by WB, and proviral DNA was searched in buffy-coat DNA by 3 HTLV generic and one HTLV-1 specific PCR. HTLV-1 prevalence was of 8.5% (23/269) among bitten individuals


Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL) and chronic inflammatory diseases. HTLV-1 bZIP factor (HBZ) is transcribed as an antisense transcript of the HTLV-1 provirus. Among the HTLV-1-encoded viral genes, HBZ is the only gene that is constitutively expressed in all ATL cases. Recent studies have demonstrated that HBZ plays an essential role in oncogenesis by regulating viral transcription and modulating multiple host factors, as well as cellular signaling pathways, that contribute to the development and continued growth of cancer. In this article, I summarize the current knowledge of the oncogenic function of HBZ in cell proliferation, apoptosis, T-cell differentiation, immune escape, and HTLV-1 pathogenesis. Fouor (HBZ)  6,7]. I,7. Igag,ormation ,9,10. Hoormation ,11. In 2ormation . Thus, Tormation ,11. In cormation ,16,17. Tormation ,18,19. Tin vivo . Fu. Fuin viammation . Yamamotry cells . Cell adet al. reported that HBZ is not required for efficient infectivity or HTLV-1-mediated immortalization of primary human T lymphocytes in vitro [in vivo [in vivo, and further studies are necessary to clarify their distinct pathobiologies.HTLV-1 and HTLV-2 are closely related human retroviruses that have been studied extensively ,86. HTLVin vitro . However[in vivo . Unlike [in vivo . In addi[in vivo . Thus, tIt has been 35 years since the discovery of HTLV-1. Numerous studies have focused on elucidating the molecular mechanisms by which HTLV-1-encoded viral proteins induce viral replication, cellular transformation, and oncogenesis. Tax is thought to have an important role in the leukemogenesis of HTLV-1 because of its pleiotropic functions. However, Tax expression is frequently lost during the development of ATL suggesting that Tax may not be essential for this process. By contrast, HBZ is the only viral gene that is constitutively expressed in ATL cases and thus is a plausible player to critically control the ATL leukemogenesis. In addition, APOBEC3G frequently introduces mutations to HTLV-1 genes before proviral integration but HBZ seems to be spared from this alteration. As discussed in this review, current findings support the view that HBZ is indispensable for leukemogenesis by HTLV-1. Further studies are needed to elucidate the precise molecular mechanisms by which HBZ induces oncogenesis so that novel therapies targeting HBZ could be developed.


This study proposes to investigate the vertical and sexual HTLV-1 transmission rate in a coorte followed up in São Paulo, Brazil. The latter 173 HTLV-1-infected patients were selected until july 2014 . Data were recorded using RedCap (Research Electronic Data Capture), transported to SPSS – Statistical Package for Social Sciences (v 20) and subjected to statistical analysis. To compare results between groups, one-way analysis of variance (one-way ANOVA test) was used. The vertical transmission rate found among 73 individuals born to HTLV-1-positive mothers was 6.8%. HTLV-1 concordant couples were compared to HTLV-1 discordant couples. Those coinfected with HIV were excluded from the sample. Among 91 individuals with stable partnership and without HIV, 49% did not know about the current partner serology: 39% of wives and 54.5% of husbands have not been tested. The horizontal transmission rate of HTLV-1 among those tested couples was 55.3%. Among men 68.2% of its wives were seropositive for HTLV-1, while among women 44.0% of its husbands were seropositive. HTLV-1 concordant couples  were also compared to HTLV-1 discordant couples , with respect to HTLV-1 proviral load. Those without HTLV-1 proviral load were excluded. In turn, the control group was subdivided into two subgroups: discordant couples with male index partner (n=5) and discordant couples with female index partner (n=9). Because of the impossibility of defining whether sexual transmission had occurred from man to woman or woman to man, for group 1 (concordant couples) we chose to use the average of the proviral loads of both partners in this group. Serodiscordant couples showed higher mean proviral loads ) compared with serodiscordant couples (282 ± 293 cópias/104 PBMC) (p=0.045), probably associated with increased genital shedding of this virus and resulting increased risk of sexual transmission.


HTLV-1 is linked to adult T cell leukemia (ATL) and several chronic inflammatory diseases, such as HTLV-1 associated myelopathy (HAM). The minus strand of HTLV-1 provirus encodes HTLV-1 bZIP factor (HBZ), which is expressed in all ATL cells and a key player in the proliferation of infected cells. One of the hallmarks of cancer cells is a property to escape from apoptosis. The aim of this study is to clarify the effects of HBZ on apoptosis in HTLV-1-infected cells. There are two major pathways for apoptosis: the extrinsic and intrinsic apoptotic pathways, which are mediated by Fas and Bim respectively. We analyzed the transcriptional profile of HBZ expressing cells, and found that the transcription of pro-apoptotic genes, Bim and Fas ligand (FasL), were both suppressed by HBZ. HBZ physically interacts with FoxO3a, a critical transcription factor of Bim and FasL genes, and attenuates the DNA binding ability of FoxO3a. In addition, HBZ induces the mislocalization of inactive phosphorylated FoxO3a in the nucleus, whereas this form exists in the cytoplasm in HTLV-1-negative cells. Besides of FoxO3a perturbation by HBZ, we have identified the epigenetic aberrations, DNA methylation and histone modification, in the promoter region of the Bim gene in ATL cells. The suppression of pro-apoptotic genes, Bim and FasL, by HBZ results in the reduction of activation-induced apoptosis. These results suggest that the attenuating FoxO3a function by HBZ may play an important role in increasing the number of infected cells and might be associated with HTLV-1-induced inflammation and leukemogenesis.


RNA interference (RNAi) is a natural cellular mechanism to silence gene expression and is predominantly mediated by microRNAs (miRNAs) that target messenger RNA. Viruses can manipulate the cellular processes necessary for their replication by targeting the host RNAi machinery. This study explores the effect of human T-cell leukemia virus type 1 (HTLV-1) transactivating protein Tax on the RNAi pathway in the context of a chromosomally integrated viral long terminal repeat (LTR) using a CD4(+) T-cell line, Jurkat. Transcription factor profiling of the HTLV-1 LTR stably integrated T-cell clone transfected with Tax demonstrates increased activation of substrates and factors associated with chromatin remodeling complexes. Using a miRNA microarray and bioinformatics experimental approach, Tax was also shown to downregulate the expression of miRNAs associated with the translational regulation of factors required for chromatin remodeling. These observations were validated with selected miRNAs and an HTLV-1 infected T cells line, MT-2. miR-149 and miR-873 were found to be capable of directly targeting p300 and p/CAF, chromatin remodeling factors known to play critical role in HTLV-1 pathogenesis. Overall, these results are first in line establishing HTLV-1/Tax-miRNA-chromatin concept and open new avenues toward understanding retroviral latency and/or replication in a given cell type.


Engineering T cells with chimeric antigen receptors (CARs) is proving to be an effective method for directing T cells to attack tumors in an MHC-independent manner. Current generation CARs aim to recapitulate T cell signaling by incorporating modular functional components of the TCR and costimulatory molecules. Development of next generation CARs has relied upon trial and error evaluation of signaling domains. We sought to develop an alternate method to re-direct the T cell receptor which does not rely upon the incorporation of signaling domains into the chimeric receptor. To this end, we developed a tri-functional molecule which is membrane-anchored and redirects the TCR in the presence of tumor antigen. We also included components of the CD4 co-receptor to provide requisite Lck signaling upon ligation of the tumor antigen. Our prototype receptor was directed against the HER-2 proto-oncogene. We have determined that engineering peripheral blood T cells with this novel receptor (termed a Tri-TAC) engenders tumor-antigen specific activation of numerous T cell functions, including cytokine production, degranulation and cytolysis - equivalent to, if not greater than, a 2nd generation CAR bearing the CD28 and CD3zeta signaling domains. Future iterations of the engineered T cells will include chimeric costimulatory receptors to enhance T cell functionality and reduce off target toxicity. This research was supported by the Canadian Institutes of Health Research and the Terry Fox Foundation.


To assess the hypothesis that HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) runs in families, we compared the frequency of HAM/TSP among HTLV-1-positive relatives of HAM/TSP patients with the frequency of HAM/TSP among HTLV-1-positive relatives of asymptomatic HTLV-1 carriers. We reviewed available information at the Instituto de Medicina Tropical Alexander von Humboldt in Lima (period 1990-2012). Index cases with HAM/TSP were defined as unrelated, HTLV-1-positive patients with a clinical diagnosis of HAM/TSP for whom HAM/TSP was the motive for HTLV-1 testing and who brought ≥1 relative (blood relatives and/or partners) for HTLV-1 testing. Asymptomatic index cases were defined as unrelated, asymptomatic HTLV-1 carriers who were tested for HTLV-1 as candidate blood donors and brought ≥1 relative for screening. In the family studies of 334 index cases with HAM/TSP, 1124 relatives were tested, 318/1124 (28%) were HTLV-1 positive, and 30/318 (9%) had HAM/TSP. In the family studies of 230 asymptomatic index cases, 544 relatives were tested, 204/544 (38%) were HTLV-1 positive, and 15/204 (7%) had HAM/TSP. We classified the relatives in groups based on sex and age. HAM/TSP frequency increased with age and was higher in women than in men. We found no significant differences in HAM/TSP frequency between relatives of HAM/TSP index cases and relatives of asymptomatic index cases. In total, there were 21 families with 2 HAM/TSP cases, 4 families with 3 HAM/TSP cases, and 1 family with 5 HAM/TSP cases. This analysis approach suggests that HAM/TSP usually affects isolated people, but that in some, particular families, HAM/TSP clusters can occur.


Human T cell leukemia virus type 1 (HTLV-1) gene expression is controlled by the key regulatory proteins Tax and Rex. The concerted action of these proteins results in a two-phase kinetics of viral expression that depends on a time delay between their action. However, it is difficult to explain this delay, as Tax and Rex are produced from the same mRNA. In the present study we investigated whether HTLV-1 may produce novel mRNA species capable of expressing Rex and Tax independently.tax/rex mRNAs followed by the monocistronic mRNAs coding for Rex isoforms.Results revealed the expression of three alternatively spliced transcripts coding for novel Rex isoforms in infected cell lines and in primary samples from infected patients. One mRNA coded for a Tax isoform and a Rex isoform, and two mRNAs coded for Rex isoforms but not Tax. Functional assays showed that these Rex isoforms exhibit activity comparable to canonic Rex. An analysis of the temporal expression of these transcripts upon ex vivo culture of cells from infected patients and cell lines transfected with a molecular clone of HTLV-1 revealed early expression of the dicistronic The production of monocistronic HTLV-1 mRNAs encoding Rex isoforms with comparable activity to canonical Rex, but with distinct timing, would support a prolonged duration of Rex function with gradual loss of Tax, and is consistent with the two-phase expression kinetics. A thorough understanding of these regulatory circuits will shed light on the basis of viral latency and provide groundwork to develop strategies for eradicating persistent infections. However, we provided experimental evidence that Tax protein is more rapidly degraded than Rex, a property that might contribute, at least partially, to a temporal separation between maximal Tax and Rex function [Human T cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia-lymphoma (ATLL) and tropical spastic paraparesis/HTLV-1-associated myelopathy (TSP/HAM)  sites in the vicinity of the canonical exon 3 SA at nt 6950. We focused our attention on potential sites defining 5′ exon boundaries located at positions 6875, 6878 and 6962, and performed RT-PCR with primers  are adjacent to the nuclear localization signal and upstream the multimerization domain of Rex [Figure n of Rex .Figure 21-2-3a transcript was detected at levels similar to that of 1-2-3, while the 1-2-C and 1-2-Ca transcripts were less abundant. In ACH-transfected HLtat and PBMC samples, all three novel transcripts were less abundant than the canonical 1-2-3 tax/rex mRNA.We next employed qRT-PCR to measure the levels of expression of these transcripts in the HTLV-1-infected cell line C91PL Figure a, in thea, Rexb and Rexc were constructed and transfected into HLtat cells. Immunofluorescence with anti-Rex antibodies followed by laser scanner microscopy (IF-LSM) showed that Rex and Rexa accumulated in the nucleus , while Rexb and Rexc were mainly localized to the cytoplasm  from a Rex knock-out HTLV-1 molecular clone  or the leucine-rich nuclear export signal/activation domain (NES/AD). However, the presence of 25 or 24 extra amino-acids in Rexb and Rexc respectively, immediately downstream of the NLS and 55 amino-acids upstream of the NES/AD  is predicted to quickly shut down viral transcription due to loss of the rex mRNA , 8. It irex mRNA . To testct mRNAs , 10, 21.These findings provide insight into the mechanisms controlling HTLV-1 expression and suggest that the production of alternatively spliced monocistronic mRNAs coding for Rex isoforms contributes to the two-phase kinetics of HTLV-1 expression. The resulting pattern of viral gene expression might be important to temporally restrain the expression of highly immunogenic viral epitopes , thus favouring escape from the immune response and establishment of long term persistence in the host, a key feature of HTLV-1 infection. A better understanding of how these regulatory circuits are established and maintained will shed light on the basis of viral latency and may provide groundwork for the development of new therapies for eradicating persistent infections.