The innate immune system is essential for controlling viral infections, but several viruses have evolved strategies to escape innate immunity. RIG-I is a cytoplasmic viral RNA sensor that triggers the signal to induce type I interferon production in response to viral infection. RIG-I activation is regulated by the K63-linked polyubiquitin chain mediated by Riplet and TRIM25 ubiquitin ligases. TRIM25 is required for RIG-I oligomerization and interaction with the IPS-1 adaptor molecule. A knockout study revealed that Riplet was essential for RIG-I activation. However the molecular mechanism underlying RIG-I activation by Riplet remains unclear, and the functional differences between Riplet and TRIM25 are also unknown. A genetic study and a pull-down assay indicated that Riplet was dispensable for RIG-I RNA binding activity but required for TRIM25 to activate RIG-I. Mutational analysis demonstrated that Lys-788 within the RIG-I repressor domain was critical for Riplet-mediated K63-linked polyubiquitination and that Riplet was required for the release of RIG-I autorepression of its N-terminal CARDs, which leads to the association of RIG-I with TRIM25 ubiquitin ligase and TBK1 protein kinase. Our data indicate that Riplet is a prerequisite for TRIM25 to activate RIG-I signaling. We investigated the biological importance of this mechanism in human cells and found that hepatitis C virus (HCV) abrogated this mechanism. Interestingly, HCV NS3-4A proteases targeted the Riplet protein and abrogated endogenous RIG-I polyubiquitination and association with TRIM25 and TBK1, emphasizing the biological importance of this mechanism in human antiviral innate immunity. In conclusion, our results establish that Riplet-mediated K63-linked polyubiquitination released RIG-I RD autorepression, which allowed the access of positive factors to the RIG-I protein. The cytoplasmic viral RNA sensor RIG-I recognizes various types of pathogenic viruses and evokes innate immune responses, whereas several viruses have evolved strategies to escape the host innate immune responses. RIG-I triggers a signal to induce type I interferon and inflammatory cytokines. RIG-I activation is regulated by K63-linked polyubiquitin chains mediated by the ubiquitin ligases TRIM25 and Riplet; however, the functional difference between the two ubiquitin ligases remains unclear, and the molecular mechanism underlying Riplet-mediated RIG-I activation is unknown. We revealed sequential roles of the two ubiquitin ligases during RIG-I activation and found that Riplet-mediated polyubiquitination of the RIG-I repressor domain released RIG-I autorepression of its N-terminal CARDs responsible for triggering the signal, which resulted in an association with TRIM25 ubiquitin ligase and TBK1 protein kinase. Interestingly, we found that this mechanism was targeted by hepatitis C virus, which is a major cause of hepatocellular carcinoma. This result emphasizes the vital role of Riplet-mediated release of RIG-I RD autorepression in antiviral responses. Our results establish that Riplet releases RIG-I RD autorepression and demonstrated the biological significance of this mechanism in human innate immune responses. The innate immune system is essential for controlling virus infections, and several viruses have evolved strategies to evade host innate immune responses. Cytoplasmic viral RNA is recognized by RIG-I-like receptors, including RIG-I and MDA5 Several ubiquitin ligases are involved in regulating the RIG-I-dependent pathway, and RIG-I itself is regulated by ubiquitin chains Another E3 ubiquitin ligase, Riplet, binds RIG-I RD and mediates the K63-linked polyubiquitination of RIG-I RD in vitro and in vivoHepatitis C virus (HCV) is a major cause of hepatocellular carcinoma (HCC) worldwide. HCV RNA is primarily recognized by RIG-I In mouse embryonic fibroblasts (MEFs), TRIM25 is essential for type I IFN production in response to SeV infection We performed reporter gene assays with p125luc (IFN-β reporter) using either RIG-I CARDs fragment or full-length RIG-I expression vectors. It is known that ectopic expression of RIG-I CARDs fragment activates the signaling even in the absence of stimulation with RIG-I ligand We then investigated whether the two ubiquitin ligases associate with RIG-I after stimulation. First, we performed immunoprecipitation assay and found that endogenous Riplet and TRIM25 bound to endogenous RIG-I after stimulation with HCV dsRNA . Second,ΔCTD) abrogated Riplet effect on RIG-I signaling  activity. Thus, Riplet disruption by NS3-4A might be a cause of liver disease induced by HCV infection.All animal studies were carried out in strict accordance with Guidelines for Animal Experimentation of the Japanese Associations for Laboratory Animal Science. The protocols were approved by the Animal Care and Use Committee of Hokkaido University, Japan (Permit Number: 08-0245 and 09-0215).HEK293, Vero, and HepG2 cells were cultured in Dulbecco's modified Eagle's medium low glucose medium (D-MEM) with 10% heat-inactivated fetal calf serum (FCS) (Invitrogen). HeLa cells were cultured in minimum Eagle's medium with 2 mM L-glutamine and 10% heat-inactivated FCS. HEK293FT cells were maintained in D-MEM high glucose medium containing 10% of heat-inactivated FCS (Invitrogen). Human hepatocyte cell line with HCV 1b full-length replicons (O cells) and O curred cells (Oc cells) were kindly gifted from Kato N VSV Indiana strain and SeV HVJ strain were amplified using Vero cells. To determine the virus titer, we performed plaque assay using Vero cells. HCV JFH1 was amplified using HuH7.5 cells.Generation of IPS-1 KO and Riplet KO mice were described previously ΔRD (RIG-I-dRD), FLAG-tagged RIG-I RD, C-terminal HA-tagged TRIM25, C-terminal HA-tagged Riplet, and Riplet-ΔRING (Riplet-DN) plasmids were described previously TTC CCG TGT GGC TGG CCG CGG CCG CCC TCG GCT GCA TCA TCT GCC, and Riplet-3A-R: GGC AGA TGA TGC AGC CGA GGG CGG CCG CGG CCA GCC ACA CGG GAA. RIG-I K172R and RIG-I K788R expression vectors was constructed by PCR-mediated mutagenesis using primers, RIG-I K172R-F, RIG-I K172R-R, RIG-I K788R-F and RIG-I-K788R-R, and pEF-BOS/FLAG-RIG-I plasmid as a template. The primer sequences are RIG-I K172R-F: GGA AAA CTG GCC CAA AAC TTT GAG ACT TGC TTT GGA GAA AG, RIG-I K172R-R: CTT TCT CCA AAG CAA GTC TCA AAG TTT TGG GCC AGT TTT CC, RIG-I-K788R-F: TGC ATA TAC AGA CTC ATG AAA GAT TCA TCA GAG ATA GTC AAG AA, and RIG-I-K788-R: CTT GAC TAT CTC TGA TGA ATC TTT CAT GAG TCT GTA TAT GCA G. RIG-I 5KR expression vectors were constructed by PCR-mediated mutagenesis using primers, RIG-I 849 851 RR-F, RIG-I 849 851 RR-R, RIG-I 888R-F, RIG-I 888R-R, RIG-I 907 909 RR-F, RIG-I 907 909 RR-R, and pEF-BOS/FLAG-RIG-I plasmid as a template. The primer sequences are RIG-I 849 851 RR-F: AGT AGA CCA CAT CCC AGG CCA AGG CAG TTT TCA AGT TTT G, RIG-I 849 851 RR-R: CAA AAC TTG AAA ACT GCC TTG GCC TGG GAT GTG GTC TAC T, RIG-I 888R-F: GAC ATT TGA GAT TCC AGT TAT AAG AAT TGA AAG TTT TGT GGT GGA GG, RIG-I 888R: CCT CCA CCA CAA AAC TTT CAA TTC TTA TAA CTG GAA TCT CAA ATG TC, RIG-I 907 909RR-F: GTT CAG ACA CTG TAC TCG AGG TGG AGG GAC TTT CAT TTT GAG AAG, RIG-I 907 909RR-R: CTT CTC AAA ATG AAA GTC CCT CCA CCT CGA GTA CAG TGT CTG AAC. HCV cDNA fragment encoding NS3-4A of JFH1 strain was cloned into pCDNA3.1 (-) vector. The mutation on catalytic site of NS3-4A S139A was constructed by PCR-mediated mutagenesis using primers, NS3-4A S139A-F and NS3-4A S139A-R, and pCDNA3.1 (-)/NS3-4A plasmid as a template. The primer sequences are NS3-4A S139A-F: TTC GAC CTT GAA GGG GTC CGC GGG GGG ACC GGT GCT TTG C and NS3-4A S139A-R: AAG CAC CGG TCC CCC CGC GGA CCC CTT CAA GGT CGA AAG G.Expression vectors encoding for N-terminal FLAG-tagged RIG-I, N-terminal FLAG-tagged RIG-I CARDs (dRIG-I), FLAG-tagged RIG-I Total RNA was extracted with TRIZOL (Invitrogen), after which the samples were treated with DNaseI to remove DNA contamination. Reverse transcription was performed using High Capacity cDNA Reverse Transcription Kit (ABI). Quantitative PCR analysis was performed using Step One software ve2.0. (ABI) with SYBER Green Master Mix (ABI). HCV ss and dsRNA was in vitro synthesized with SP6 and/or T7 RNA polymerase using 3′ UTR of HCV cDNA as template as described previously Cells were plated onto microscope cover glasses (matsunami) in a 24-well plate. The cells were fixed for 30 min using 3% formaldehyde in PBS and permeabilized with 0.2% Triton X-100 for 15 min. Fixed cells were blocked with 1% bovine serum albumin in PBS for 10 min and labeled with the indicated primary Abs for 60 min at room temperature. Alexa-conjugated secondary Abs were incubated for 30 min at room temperature to visualize staining of the primary Ab staining. Samples were mounted on glass slides using Prolong Gold (Invitrogen). Cells were visualized at a magnification of ×63 with an LSM510 META microscope (Zeiss). Data collected with confocal microscopy were analyzed with ZEISS LSM Image Examiner software. NS3, RIG-I, TBK1, IPS-1, and p-TBK1 were stained with anti-NS3 goat pAb (abcam), anti-RIG-I mouse mAb , anti-NAK (TBK1) rabbit mAb , anti-MAVS (IPS-1) rabbit pAb (Bethyl Laboratories Inc), and anti-p-TBK1 rabbit mAb ,Renilla luciferase. The total amounts of plasmids were normalized using an empty vector. Cells were lysed in a lysis buffer (Promega), and luciferase and Renilla luciferase activities were determined using a dual luciferase assay kit (Promega). Relative luciferase activities were calculated by normalizing the luciferase activity by control. HCV dsRNA (3′ UTR polyU/UC region) was synthesized using T7 and SP6 RNA polymerase as described previously HEK293 cells were transiently transfected in 24-well plates using FuGene HD (Promega) or lipofectamine 2000 (Invitrogen) with expression vectors, reporter plasmids (IFN-β: p125luc), and an internal control plasmid coding AAA CUG AAA GGG AGA AGU GAA AGU G; and (antisense strand) UCAC UUU CAC UUC UCC CUU UCA GUU . Biotin was conjugated at the U residue at the 3′-end of the antisense strand (underlined). Biotinylated dsRNA was phosphorylated by T4 polynucleotide kinase (TAKARA). dsRNA was incubated for one hour at 25°C with 10 µg of protein from the cytoplasmic fraction of cells that were transfected with Flag-tagged RIG-I, Riplet, and/or HA-tagged ubiquitin expressing vectors. This mixture was added into 400 µl of lysis buffer  containing 25 µl of streptavidine Sepharose beads, rocked at 4°C for two hours, harvested by centrifugation, washed three times with lysis buffer, and resuspended in SDS sample buffer.RNA used for the assay was purchased from JBioS. The RNA sequences are as follows: (sense strand) 7) were infected with or without VSV at MOI = 10 for eight hours, after which cell extracts were prepared with lysis buffer . Immunoprecipitation used an anti-RIG-I Rabbit monoclonal antibody . To detect endogenous K63-linked polyubiquitin chain that is ligated to RIG-I, 6×107 of mouse splenocyte were infected with SeV at MOI = 0.2 for 24 hours. Immunoprecipitation was performed with anti-RIG-I mAb (D14G6). Anti-K63-linkage specific polyubiquitin (D7A11) Rabbit mAb  was used for western blotting. HEK293FT cells were transfected with or without 0.8 µg of HCV dsRNA in a 6-well plate. HCV dsRNA (HCV 3′ UTR polyU/UC region) was synthesized using T7 and SP6 RNA polymerase as previously described 3VO4, 20 mM iodoacetamide, and 2 mM phenylmethylsulfonyl fluoride) to dissociate unanchored polyubiquitin chain Splenocytes (1×10GGA ACA UCU UGU AGA CAU Utt and (anti-sense) AAU GUC UAC AAG AUG UUC CCac. siRNA was transfected into cells using RNAiMax Reagent (Invitrogen) according to the manufacture's instructions.siRNAs for human Riplet  and negative control were purchased from Ambion. siRNA sequences for Riplet are: (sense) FLAG-tagged Riplet was expressed in HEK293FT cells, and cell lysate was prepared with the lysis buffer described above. The protein was immunoprecipitated with anti-FLAG antibody and protein G sepharose beads, and washed with Buffer B . The samples were suspended in 50 µl of Buffer B, and incubated with 400 ng of recombinant NS3-4A (rNS3-4A) protein at 37°C for one hour, and then subjected to SDS-PAGE analysis. The NS3-4A protein was purchased from AnaSpec Inc (CA). N-terminal GST-fused Riplet (1–210 aa) (rRiplet) was purchased from Abnova. 500 ng of rRiplet was incubated with or without 500 ng of rNS3-4A in 10 µl of reaction buffer (20 mM Tris-HCl (7.5), 4% Glycerol, 5 mM DTT, 150 mM NaCl, 0.1% of Triton-X100, 0.9% polyvinyl alcohol) at 37°C for 30 min.The accession numbers are Riplet (BAG84604), TRIM25 (NP_005073), TBK1 (NP_037386), IKK-ε (AAF45307), IPS-1 (BAE79738), RIG-I (NP_055129), and G3BP (CAG38772).Figure S1K63-linked polyubiquitination of RIG-I RD. HA-tagged ubiquitin and FLAG-tagged RIG-I RD expression vectors were transfected into HEK293FT cells. 24 hours after transfection, the cells were infected with VSV at MOI = 1 for six hours. Then, cell lysate was prepared. Immunoprecipitation was carried out using anti-FLAG antibody. The samples were subjected to SDS-PAGE, and the proteins were detected by western blotting using anti-HA, FLAG, and K63-linked polyubiquitin specific antibodies.(TIF)Click here for additional data file.Figure S2Intracellular localization of RIG-I, NEMO, and p-TBK1 proteins. (A) HeLa cells were transfected with HCV dsRNA using lipofectamine 2000 reagent. The cells were fixed six hours after transfection. The microscopic analysis was performed using anti-RIG-I mAb (Alme-1) and anti-NEMO pAb. (B) HeLa cells were transfected with HCV dsRNA using lipofectamine 2000 reagent (Invitrogen). The cells were fixed at indicated hour. The microscopic analysis was performed using anti-RIG-I mAb (Alme-1). (C) HepG2 cells were transfected with HCV dsRNA using lipofectamine 200 reagent. The cells were fixed six hours after the transfection. The microscopic analysis was performed using anti-RIG-I (Alme-1) mAb and anti-p-TBK1 mAb.(TIF)Click here for additional data file.Figure S3NS3-4A of HCV cleaves IPS-1 and Riplet but not IKK-ε. (A) HA-tagged Riplet was transfected into HEK293 cells together with NS3-4A. 24 hours after transfection, cell lysate was prepared and subjected to SDS-PAGE. The proteins were detected by western blotting and CBB staining.  HA-tagged IKK-ε (B) or IPS-1 (C) expression vectors were transfected into HEK293FT cells with or without NS3-4A of HCV expression vector. 24 hours after the transfection, the cell lysate was prepared, and analyzed by SDS-PAGE. The proteins were detected by western blotting using anti-HA or anti-β actin antibodies. (D) HA-tagged IPS-1 or HA-tagged Riplet expression vector was transfected into HEK293FT cells with or without NS3-4A expression vectors. 24 hours after transfection, cell lysate was prepared and subjected to SDS-PAGE. The proteins were detected by western blotting using anti-HA antibody.  N-terminal FLAG-tagged Riplet (E) or C-terminal HA-tagged Riplet (F) expression vector was transfected into HEK293FT cells with NS3-4A or NS3-4A*. 24 hours after the transfection, cell lysates were analyzed by SDS-PAGE. (G) HA-tagged wild-type Riplet or mutant Riplet-C21A expression vector were transfected into HEK293FT cells with NS3-4A or NS3-4A*. 24 hours after the transfection, the cell lysate was prepared, and analyzed by SDS-PAGE. The proteins were detected by western blotting using anti-HA or anti-β actin antibodies.  RIG-I, Riplet, Riplet-3A (H), and/or Riplet C21A (I) mutant expression vectors were transfected into HEK293 cells together with p125luc reporter and Renilla luciferase. 24 hours after transfection, luciferase activity was measured.(TIF)Click here for additional data file.Figure S4siRNA for Riplet or control was transfected into HeLa cells in 24-well plate using RNAi MAX (Invitrogen) according to manufacture's protocol. 48 hours after transfection, the cells were transfected with 100 ng of HCV dsRNA. Six hours after transfection, the cells were fixed and stained with anti-RIG-I mAb (Alme-1) and anti-mouse Alexa-488 Ab.(TIF)Click here for additional data file.


Objective(s): Although HTLV-I infection is endemic in different geographical parts of the world including Northeast of Iran, there have been no documents of HTLV-II infection in this region. It is reported that one possible reason for seroindeterminate state in HTLV western blot is HTLV-II virus. This study aimed to investigate the presence of HTLV-II among blood donors with seroindeterminate western blot results.Materials and Methods: Three ml whole blood obtained from 50 blood donors referring to Mashhad Blood Transfusion Organization who had reactive Elisa for HTLV-I and seroindeterminate HTLV western blot state. A conventional PCR was applied to detect HTLV-I provirus using specific primers while a nested PCR was designed with specific external and internal primers for the detection of HTLV-II.Results: The average age of participants, 39 males and 11 females, was 37.12± 14.36 years. The average OD of the Elisa assay was 1.767± 1.195. The most common indeterminate patterns were Rgp46-II alone , Rgp46-I alone , and Rgp46-I with GD21 .After introducing the DNA to the PCR tests, results revealed 10 (20%) HTLV-I PCR positive samples while no HTLV-II positive sample was detected by nested PCR. There were no significant age, blood group, Optical Density of the Elisa assay, and western blot indeterminate pattern differences between HTLV-I PCR positive and negative samples.Conclusion: No HTLV-II positive sample was detected in this study which confirms the absence of HTLV-II infection in this region. However, high frequency of HTLV-I PCR positive samples among the seroindeterminate cases implies on the important role of molecular techniques for further confirmation of the infection. Human T-Lymphotropic Virus (HTLV) types I and II were the first discovered human retroviruses. HTLV-I was first reported in 1980 while HTLV-II was first reported in 1982 . HTLV-I HTLV-I is endemic in a number of geographical regions including Japan, Africa, Caribbean islands, South America, and Northeast Iran while the epidemiology of HTLV-II is not well identified -15.Within the last decade newly developed serologic assays made us capable to accurately differentiate between antibodies to HTLV-II and HTLV-I. These new techniques include synthetic Enzyme Immunoassays (EIA) , 17 and Although such techniques greatly improve the identification and differentiation of HTLV-II from HTLV-I a frequent problem in HTLV-I/II diagnosis which is the high prevalence of indeterminate serological results by WB still leads to difficulties in the interpretation .The current diagnosis of HTLV-I and HTLV-II infection is mainly based on Enzyme-linked immunosorbent assays (ELISA) followed by the confirmation of ELISA reactive samples with WB analysis . To distMashhad, located in Northeast of Iran, is an endemic region for HTLV-I infection. It has been recently reported that 2.12% of the general population and 0.77% of blood donors were infected with HTLV-I . Howeverbetter, it would be quite beneficial to determine the proportion of HTLV-I/II infection among HTLV-seroindeterminate individuals. The current study aimed to investigate the presence of HTLV-II and HTLV-I infections in Mashhad among WB indeterminate samples from blood donors by nested PCR.To better understand the molecular epidemiology of HTLV infection in this endemic area In this descriptive cross-sectional study blood samples of 50 subjects with WB indeterminate result for HTLV collected from Khorasan Blood Transfusion Organization (BTO), who had donated blood during 2009 and 2010 were selected.Serum samples of all blood donors in Khorasan BTO were routinely tested for HTLV-I/II antibodies using ELISA assay  according to the manufacturer’s instructions and reactive samples were reevaluated in duplicate with the same kit. Repeatedly reactive samples were further evaluated for confirmation of the infection with a WB assay  following the manufacturer’s criteria. According to World Health Organization (WHO) criteria for HTLV seropositivity in WB assay, a sample would be regarded as positive when either gp46 or gp462/68 and one of p19, p24, or p53 were present. The HTLV European Research Network proposed the stringiest criteria for WB seropositivity that defines a WB assay as HTLV-I positive only if bands for the gag proteins p19 and p24 and the env proteins gd21 and MTA-1 were present while it scored as HTLV-II positive if p24, gd21, and K-55 bands were identified and was HTLV positive but nontyped if p24, p19, and gd21 were present . In the To perform the molecular analysis, 3 ml whole blood was collected in sterile Ethylenediaminetetraacetic acid (EDTA) tubes and genomic DNA was extracted from peripheral blood of WB indeterminate individuals using a commercial DNA extraction kit  according to the manufacturer’s instructions.Extracted DNA stored at -20 until PCR analysis. In order to confirm HTLV-I infection, Long Terminal Repeat (LTR) and TAX genes were targeted and amplified using designed specific primers .2, 2 µl , dNTPs 0.5 µl , 0.5µl of each primers (10mM) , 0.5 unit of Taq DNA polymerase enzyme  and 50-100 ng DNA. The PCR machine  was programmed to 94˚C 5min, 35 cycles of 94˚C 45 sec, 60˚C 50 sec, 72˚C 50 sec and a final extension of 72˚C 7min.PCR condition for LTR and TAX genes were 10X buffer 2.5 µl, MgclAmplified products were introduced on 2% gel agarose, stained with ethidium bromide and observed under ultraviolet (UV) luminometer. To determine HTLV-II infection the regions of LTR and TAX genes were selected and amplified using designed specific primers .2, 0.2 mM of each of the Deoxynucleoside triphosphates, and 10 pmol of each outer primer. The PCR cycling conditions consisted of 35 cycles at 95°C for 45 sec, 58°C for 60 sec, and 728C for 180 sec and a final extension of 72˚C for 7min.LTR fragment was amplified by a nested PCR. Briefly a first-round PCR reaction was carried out on 50-100 ng of extracted DNA in a 25 µl reaction mixture containing 0.5 U of Taq DNA polymerase  2.5 µl 10x buffer, 2 mM MgClFor the second round 3µl of the first PCR product was used. The PCR mixture was the same as first round PCR but with inner primer set. The PCR machine  was programmed to 94˚C for 5 min, 35 cycles of 94˚C for 30 sec, 60˚C for 30 sec, 72˚C for 60 sec and a final extension of 72˚C for 7min.A 159 base pair fragment of TAX gene was amplified by the outer primer . PCR macAll amplified products were run on 2% gel agarose, stained with ethidium bromide and observed under UV luminometer.The current study is approved by the research deputyship of Iranian Academic Centre for Education, Culture, and Research (ACECR) Mashhad branch regarding ethical and methodological issues. A written consent was obtained from each individual after describing the aims of the study and assuring the participants about the results to be remained anonymous.P-values <0.05 were considered as statistically significant.Demographic data as well as WB and PCR results were registered and analyzed using SPSS V.18.0. Chi-square and Fisher’s exact tests as well as T-test were employed to perform categorical and numerical comparisons respectively. Related tables were produced to discuss the results. + and AB- with 35.4% (n=17) and 0% respectively  were male and 11 (22%) were female. The mean age of participants was 37.12±14.36 years. The most and the least common blood groups were Oectively .All samples had HTLV-I/II positive reactivity in ELISA. The mean value of Optical Density (OD) in ELISA for the samples was 1.767±1.195.The most common bands in WB were gd21 (47.7%), rgp46-I (38.6%), rgp46-II (31.8%), gp21 (9.1%), p24 (6.8%), p53 (2.3%), and p26 (2.3%) respectively.WB indeterminate patterns were identified for all samples. The most common WB indeterminate patterns were rgp46-II alone (27.3%), rgp46-I alone (15.9%), rgp46-I and gd21 (15.9%), gd21 alone (11.4%), rgp46-I, p24, and gd21 (4.5%), rgp46-II and gd21 (4.5%), p24 and gd21(2.3%), rgp46-I, gd21, and gp21 (2.3%), and p53 alone (2.3%) respectively .P>0.05).Nested PCR revealed no HTLV-II proviral DNA in samples. However 10 samples (20%) were positive for HTLV-I. From the 10 HTLV-I positive individuals 8 were male (80%) and 2 were female. Also statistical analysis showed no significant difference between HTLV-I PCR positive and negative individuals regarding age, blood group, ELISA OD, and WB indeterminate pattern  or HTLV-I related endogenous sequence (HRES-I) or a novel virus, antibody responses to a malaria parasite with epitope homology to HTLV-II like Plasmodium Falciparum, a defective HTLV-I or HTLV-II, and low copy numbers of prototype HTLV-I in the affected patient having indeterminate state .To better understand the status of HTLV-I and HTLV-II infections in WB indeterminate samples we investigate 50 indeterminate samples from Mashhad blood donors.et al in Argentina, Berini et al in Argentina, and Santos et al in Brazil with 9.2%, 13.8%, 13.2%, and 22% frequencies respectively (The current study results demonstrate a high rate of HTLV-I infection among WB indeterminate samples (20%) using PCR technique that may be due to low copy number of prototype HTLV-I. Similar results reported by previous studies indicated the ability to amplify HTLV-I tax region from PBMCs of some HTLV WB indeterminate individuals , 34. Morectively , 28, 35.Similar to other studies performed on WB indeterminate samples, the current study observed no significant difference regarding age, gender, and blood group of HTLV-I PCR positive and negative individuals , 36.Nested PCR results showed no evidence of HTLV-II infection among indeterminate WB individuals in the present study which was predictable due to no previous reports of HTLV-II in this region , 15. Simet al but in contrast to the findings of Rouet et al and Costa and Segurado (The most common indeterminate patterns in the current study were single bands of rgp46-II, rgp46-I, and gd21 which were similar to the findings of Mangano Segurado , 27, 28.The results of the current study revealed a high rate of HTLV-I infection among WB indeterminate samples as well as no HTLV-II infection. Therefore it can be concluded that HTLV-II is not a matter of concern in this region while special attention to indeterminate WB individuals regarding HTLV-I infection is necessary. It is recommended to determine the status of HTLV-I infection in WB indeterminate blood donors in Northeast of Iran using molecular techniques such as PCR to control the infection.


Nef disturbs the organization of immunological synapses (IS) between infected CD4+ T lymphocytes and antigen-presenting B lymphocytes. These effects include the potent retargeting of the T cell receptor (TCR) proximal kinase Lck from the plasma membrane to intracellular membranes. Consequently, Nef interferes with TCR proximal signaling. Paradoxically, Nef enhances distal TCR signaling in infected CD4+T lymphocytes, an effect thought to be involved in its role in AIDS pathogenesis.The Nef protein of human immunodeficiency virus type-1 (HIV-1) facilitates viral replication and disease progression Using live-cell imaging, quantitative confocal microscopy and cell fractionation of Nef-expressing cells and HIV-1-infected primary human T lymphocytes, we analyzed by which mechanism Nef affects the subcellular localization of Lck and addressed the functional consequences of this retargeting for HIV-1 replication.We found that Nef induces intracellular compartmentalization of TCR signaling to adjust TCR responses to antigenic stimulation. Nef prevents anterograde transport of newly synthesized pools of the TCR signaling master switch Lck to reroute kinase-active Lck away from the plasma membrane (PM) to the trans-Golgi network (TGN). Via this mechanism, Nef prevents recruitment of active Lck to the IS upon TCR engagement and limits signal initiation at the PM. Instead, Nef triggers Lck-dependent activation of TGN-associated Ras-Erk signaling to promote production of the T lymphocyte survival factor IL-2 and to enhance virus spread. Overexpression of the Lck PM transporter Unc119 restores Nef-induced subversions of Lck trafficking and TCR signaling. Nef thus hijacks Lck sorting to selectively activate TGN-associated arms of compartmentalized TCR signaling.These findings reveal a mechanism by which Nef simultaneously restricts proximal and enhances select aspects of distal TCR signaling, respectively. By tailoring T lymphocyte responses to antigenic stimulation, Nef optimizes the environment for HIV-1 replication.


Giardia duodenalis is highly endemic in East Africa but its effects on child health, particularly of submicroscopic infections, i.e., those below the threshold of microscopy, and of genetic subgroups (assemblages), are not well understood. We aimed at addressing these questions and at examining epidemiological characteristics of G. duodenalis in southern highland Rwanda.G. duodenalis assemblages were genotyped. Cluster effects of villages were taken into account in statistical analysis. The prevalence of G. duodenalis as detected by microscopy was 19.8% but 60.1% including PCR results. Prevalence differed with residence, increased with age, and was reduced by breastfeeding. In 492 community children without, with submicroscopic and with microscopic infection, underweight (weight-for-age z-score <−2 standard deviations) was observed in 19.7%, 22.1%, and 33.1%, respectively, and clinically assessed severe malnutrition in 4.5%, 9.5%, and 16.7%. Multivariate analysis identified microscopically detectable G. duodenalis infection as an independent predictor of underweight and clinically assessed severe malnutrition. Submicroscopic infection showed respective trends. Overall, G. duodenalis was not associated with gastrointestinal symptoms but assemblages A parasites  were increased among children with vomiting and abdominal pain.In 583 children <5 years of age from communities and health facilities, intestinal parasites were assessed by triplicate light microscopy and by PCR assays, and G. duodenalis in high-endemicity areas may be greatly underestimated by light microscopy, particularly when only single stool samples are analysed. Children with submicroscopic infections show limited overt manifestation, but constitute unrecognized reservoirs of transmission. The predominance of assemblage B in Rwanda may be involved in the seemingly unimposing manifestation of G. duodenalis infection. However, the association with impaired child growth points to its actual relevance. Longitudinal studies considering abundant submicroscopic infections are needed to clarify the actual contribution of G. duodenalis to morbidity in areas of high endemicity.The prevalence of  Giardia duodenalis is a protozoan parasite causing gastroenteritis. Although the parasite occurs worldwide, its regional prevalence varies considerably. Using PCR as a highly sensitive molecular diagnostic tool, we detected G. duodenalis in 60% of 583 children younger than five years in southern Rwanda. It was by far the most frequent intestinal parasite detected in this population. Importantly, two out of three infections would have been undetected if only the commonly used light microscopy had been applied. Genotyping revealed the presence of two distinct types of parasites, and only the infrequent subtype showed a weak association with gastrointestinal symptoms. However, G. duodenalis infection was associated with underweight and clinically assessed severe malnutrition. The data call for the establishment of more sensitive than light microscopy, yet simple diagnostic tools to identify infected children as well as for the consideration of abundant submicroscopic infections in evaluating the significance of G. duodenalis in high endemicity areas. Giardia duodenalis  is among the most common intestinal protozoa and is the most frequent parasitic agent of gastroenteritis worldwide. The regional prevalences of infection differ enormously and may be >30% in children in the African and the Eastern Mediterranean region G. duodenalis is known for causing gastrointestinal symptoms, such as acute or chronic diarrhoea, bloating, and stomach cramps, asymptomatic infections may occur, particularly in highly endemic areas, and there is also evidence for protection against acute diarrhoea in infected individuals G. duodenalis have been revealed, i.e., assemblages A1, A2, and B–H with A1, A2, and B being considered pathogenic in humans G. duodenalis is traditionally based on the detection of the parasites by light microscopy in direct stool smears or following concentration techniques, e.g., by formalin-ethyl acetate centrifugation. Multiple rather than single sample testing is recommended to improve sensitivity but this is often difficult to implement. Immunoassays are of superior sensitivity but also more expensive G. duodenalis infections, i.e., infections detected by PCR but not by light microscopy, in highly endemic areas.Diagnosis of G. duodenalis surpassed only by the Indian subcontinent G. duodenalis infection among children in communities and health facilities applying both microscopy and PCR for diagnosis. Also, we aimed at examining the influence of G. duodenalis infection, particularly of submicroscopic infection and different assemblages, on the children's clinical status.East Africa is considered to be among the most endemic regions for All children's parents were thoroughly informed on the purpose and procedures of the study, and recruitment was preceded by HIV pre-counseling and obtaining informed written consent from the children's parents. The study was reviewed and approved by the National Ethics Committee, Republic of Rwanda.Centre Hospitalier Universitaire de Butare). Due to Rwanda's mandatory health insurance system, treatment of common diseases is basically free of charge including utilization of district and provincial hospitals provided there is adherence to a strict referral system The study was conducted from January to March 2010, i.e. during a delayed rainy season, in Butare and the neighbouring rural Huye subdistrict. Butare  is the capital of Huye district, southern province of Rwanda. Located on the country's central plateau , the city is surrounded by densely populated farmland hills. Governmental health services in the area are provided by several primary health centres, Kabutare district hospital and the University Teaching Hospital of Butare . In parallel and during 16 and 11 days, respectively, 103 and 101 pediatric health facility attendants aged five years or less and presenting with a health problem at the primary Sovu health center and at the referral Kabutare district hospital, respectively, i.e. the health facilities serving this population, were consecutively recruited. Leading primary diagnoses in the health facility attendants were respiratory tract infection, gastro-intestinal tract affection, and malaria. Clinical details are reported elsewhere The study was designed as a cross-sectional survey to assess the prevalences of malaria, HIV, and intestinal pathogens in children under five years of age in the CHUB catchment area, i.e., at the levels of community, health center, and district hospital. The present report focuses on vs. none. Using standardized forms, children were examined by a physician and a medical history was obtained. For the latter, parents were specifically asked about the presence of gastrointestinal and other symptoms during the preceding two weeks, e.g., vomiting, diarrhoea, and abdominal pain. For this study, we used two categories to describe impaired child growth and/or malnutrition, i.e. underweight and clinically assessed severe malnutrition. Underweight was defined as a weight-for-age z (WAZ)-score of <−2 standard deviations (SD) following calculation of WAZ-scores using WHO Anthro Brief questionnaires were filled in on socio-economic aspects of the children's families including household assets. Age, sex, weight, and fever (axillary temperature ≥37.5°C) were documented. Breastfeeding was documented as any In the communities, stool collection containers were handed to the parents who were instructed to bring a fresh sample of the selected child at the scheduled day of examination. In the health facilities, fresh samples were collected. During presentation of the children, intestinal pathogens were screened for by direct wet mount microscopic stool examination. A second microscopic examination was performed later on the examination day following the ether-based concentration technique Necator americanus, Ancylostoma duodenalis, or Ascaris lumbricoides were detected by real-time PCR assays G. duodenalis, Entamoeba histolytica (or E. dispar), and Cryptosporidium parvumG. duodenalis samples as well as negative controls. Cycle threshold (Ct) values of >36 were considered to reflect limited reproducibility due to low copy numbers, and all respective assays were repeated. For G. duodenalis, the real-time PCR was repeated for 62 samples with an initial Ct-value of 36.01–45.1 , of which 47 were reproducibly positive  while 15 were negative. No sample had to be excluded because of evidence of fecal inhibitory factors .For molecular assays, aliquots of stool were stored at −70°C and transported on dry ice to Berlin. Of the 622 microscopically assessed samples, 583 were available for PCR analysis. DNA was extracted by commercial kits . For each sample, phocine herpesvirus 1 (PhHV-1), kindly provided by Dr. Martin Schutten, Department of Virology, Erasmus MC, Rotterdam, The Netherlands, was added to the isolation lysis buffer to serve as an internal control for the extraction process G. duodenalis genotyping, stool DNA samples were analysed by a previously described method with slight modifications Giardia triosephosphate isomerase (TPI) gene was amplified by nested PCR using primer TPI-f1, AAATYATGCCTGCTCGTCG and TPI-r1, CAAACCTTYTCCGCAAACC for the first PCR reaction and primer TPI-f2, CCCTTCATCGGNGGTAACTT and TPI-r2 GTGGCCACCACNCCCGTGCC for the second PCR reaction. The PCR products were purified  and unidirectional sequencing reactions were performed with the ABI Big dye 3.1 Terminator kit using TPI-f2 primer and send for in-house sequencing service at the Robert Koch-Institute. Sequences were analyzed with Geneious Pro software tool . To determine Giardia genotypes, a sequence fragment from base 610 to 1052 of a reference sequence (Genbank accession number L02120) was compared to previously defined TPI references tpi locus was not attempted because of the known heterozygosity at this locus For G. duodenalis infection and of clinical symptoms was performed by simple and multiple logistic regression analysis, and odds ratios were reported. Confirmatory analyses ) were adjusted for intracluster correlations (cluster = villages) by use of Generalized Estimating Equations  and SAS for Windows (release 9.2) (SAS Institute Inc.). Descriptive analyses include absolute frequencies and percentages or means, medians, and ranges. Evaluation of determinants of Socio-demographic and selected clinical characteristics of the 583 children are shown in G. duodenalis was the most prevalent parasite identified, detected by microscopy in 19.8% (114/583) and by PCR in 59.7% (366/583)  and that of PCR was 99.5% .Gastrointestinal parasites were assessed in all children . G. duod366/583) . All butP = 0.02).Of 208 successfully typed isolates, 85.9% (179/208) were assemblage B and 12.7% (27/208) were assemblage A2, in addition to one assemblage A1 and one mixed assemblage A+B. Infections with assemblage B isolates were more frequently submicroscopic  than infections caused by assemblage A . Overall, 18.0%, 33.3%, 25.9%, 13.2%, and 9.6% of the children harbored 0, 1, 2, 3 or ≥4, respectively, of the parasites listed in G. duodenalis mono infections (22.1%), followed by co-infestations with G. duodenalis and A. lumbricoides (9.8%) or with E. coli (5.7%), and triple infestations with G. duodenalis, A. lumbricoides, and E. coli (3.9%). Assemblages A and B did not associate with any other intestinal parasite species (data not shown).ely rare . The preG. duodenalis were analyzed including available socio-economic data. G. duodenalis was more common in community children (65.2%) than in health facility attendants  (P = 0.002). Among community children, prevalences in the villages ranged from 26.7% to 87.0% (P<0.0001) but no geographic focus was discernible  in infants to 81.1% (79/90) in children ≥4 years of age (P<0.0001). This was not observed in health facility attendants (P = 0.10). Also, the proportion of microscopic among all G. duodenalis infections in community children tended to increase with age. While only 7.4% (1/15) of infections in infants were microscopically detectable, this was the case in 30.8% (21/68), 30.3% (26/88), 43.3% (36/82), and 29.3% (21/73), in the age groups 1<2, 2<3, 3<4, and 4<5 years, respectively (P = 0.06).Factors potentially associated with the presence of = 0.001) , and varcernible . MoreoveG. duodenalis (P = 0.04) while a high number of siblings showed the opposite effect. In multivariate analysis, G. duodenalis remained positively associated with increasing age many siblings, and negatively with health facility attendance and breastfeeding . Only 11 children (1.8%) had reportedly taken drugs with anti-giardial effects within the preceding two weeks . In these, G. duodenalis was found in 45.5% (5/11) as compared to 60.5% (361/581) of respectively untreated children (P = 0.27).Several factors expected to be associated with G. duodenalis were of assemblage A in community children  and health facility attendants , and in households with  and without cattle possession . The age  of infected children with assemblage A or assemblage B parasites did not differ (38.0 (22–59) vs. 37.0 (8–60), P = 0.24). A notable exception was residence: in the community children's villages, the proportion of assemblage A among detected G. duodenalis ranged between 0% and 50% (P = 0.02) but again no focus was discernible.For assemblages B and A, no differences with respect to the above mentioned factors were discernible. In particular, similar proportions of G. duodenalis infection on community children because of their large sample size and because of the diverse additional morbidity of health facility children G. duodenalis . Abdominal distension tended to be increased in infected children .We focused the analysis of the clinical significance of odenalis . DefininG. duodenalis infected children more frequently exhibited clinically assessed severe malnutrition  than uninfected children. This was paralleled by slightly decreased WAZ scores (P = 0.14) and an increased proportion with underweight (P = 0.14) among infected children (Underweight (WAZ score <−2 SD) was observed in 23.7% (117/490) of community children. Of note, children .P<0.0001] and of underweight  as well as reduced WAZ-scores (P = 0.01), as compared to uninfected children. Children with submicroscopic infections had an intermediate prevalence of clinically assessed malnutrition  but WAZ scores were not significantly reduced (P = 0.47).Looking separately at submicroscopic and microscopic infections, children with microscopic infections exhibited significantly increased odds of clinically assessed severe malnutrition , adjusted for lacking paternal education , and number of siblings . All other covariates  were not significant. In this model, the adjusted odds of clinically assessed severe malnutrition in microscopic G. duodenalis infection was 3.34  while submicroscopic infection showed a smaller effect . An identical approach identified microscopic G. duodenalis infection to be an independent predictor of underweight  but not submicroscopic infection  or G. duodenalis infection per se , adjusted for maternal education, and absent household possessions.In multivariate analysis, G. duodenalis infection per se were pronounced for assemblage B but less obvious for the small group of children infected with assemblage A parasites. The latter, however, were more frequently affected by abdominal pain (P = 0.02) and vomiting (P = 0.03) than uninfected children. This was not the case for children with assemblage B isolates. Compared to them, children with assemblage A parasites more frequently reported abdominal pain (P = 0.004) and vomiting (P = 0.04).Lastly, we examined the clinical relevance of assemblages A and B . The incG. duodenalis infects two of three predominantly asymptomatic children, is underestimated by conventional microscopy of single stool samples, and contributes to underweight and clinically assessed malnutrition. This suggests an underrated but considerable burden of disease due to G. duodenalis.In rural communities in southern highland Rwanda, G. duodenalis of 60% in Rwandan children – who mainly were randomly selected from communities – considerably exceeds figures reported from other parts of the world G. duodenalis has recently been shown in Danish patients G. duodenalis infections. Repeated microscopic examinations (which are difficult to implement) or the use of immunoassays could have reduced this discrepancy. In addition, chronically and/or repeatedly infected individuals in highly endemic settings might shed low cyst numbers. Thus, microscopy-based prevalence figures from high-endemicity areas may be underestimations, and analyses comparing individuals categorized by microscopy into Giardia infected and non-infected may be confounded by a significant number of false negatives. While some false negatives may persist even when applying PCR assays, that risk cannot be determined accurately in the absence of a well defined gold standard.The prevalence of G. duodenalis among community children, we herein describe associations of symptoms with the presence of the parasite. G. duodenalis infection is known to vary widely in clinical manifestation including acute, chronic, and asymptomatic courses a priori define clinical giardiasis in our study. Nevertheless, no evidence for causation of gastrointestinal symptoms was seen, with the potential exception of abdominal distension. The association with malnutrition, however, was impressive: approximately every third and every sixth child with microscopic infection had underweight and clinically assessed severe malnutrition, respectively, although causation cannot be clarified in a cross-sectional study. Association does not mean causality, and reduced sample sizes in subgroup analyses increases the chance of type 1 statistical errors. Selection bias during recruitment at home, e.g., due to preferential presentation by the parents of children with (a recent history of) sickness cannot completely be excluded. However, recruitment teams were instructed to select children from households randomly. Also, confounding cannot completely be excluded but our results remained stable in multivariate analyses adjusting for socio-economic parameters and for the number of other intestinal pathogens serving as a proxy parameter for polyparasitism (data not shown). Notably in this regard, A. lumbricoides infections found in one third of the children had no clear-cut effect on malnutrition (data not shown), similar to findings from Brazil and Iran G. duodenalis of clinically assessed severe malnutrition as compared to underweight may also indicate a greater selectivity of the former. Unfortunately, we were unable to analyze the influence of G. duodenalis on stunting and wasting, i.e., categories based on children's height, because this parameter was inconsistently measured in the field study, had implausible values, and respective Z-scored were consequently omitted. However, our interpretation of malnutrition as a consequence of giardiasis is in line with other studies As to the clinical significance of G. duodenalis infection had a prevalence of clinically assessed severe malnutrition, which was intermediate between children without and with microscopic infection. In multivariate analysis, submicroscopic infections still tended to be associated with clinically assessed severe malnutrition but showed no link with underweight. Longitudinal observations and immunological studies may help to understand the relevance of this type of infection and the underlying mechanisms.Children with submicroscopic G. duodenalis assemblage B parasites clearly predominated (86%), which is in accordance with most larger studies .es e.g.,  this is In our study, children infected with assemblage A isolates showed increased proportions of abdominal pain and vomiting while assemblage B infections associated with underweight and clinically assessed severe malnutrition. Much of the available data on the role of assemblages in clinical disease is inconsistent G. duodenalis in the present study. One reason involved may be a limited selectivity of the questionnaires used in this generally poor agricultural and rural population. Also, we were unable to identify hot spots, let alone their characteristics, potentially underlying the pronounced spatial differences in infection prevalence. In multivariate analysis, only the increases of G. duodenalis with children's age and with a high number of siblings as well as the reduction with breastfeeding remained statistically significant. Increasing prevalence with increasing number of siblings supports the role of transmission within households. Protection from G. duodenalis infection by breast milk has previously been reported G. duodenalis in the study area it seems plausible that protective antibodies Giardia spp. in murine studies Socio-economic factors had no great influence on the presence of G. duodenalis infection to be potentially rewarding, e.g. by preventive chemotherapy. Yet, the influence of repeated treatment of G. duodenalis infection on anthropometric development is unclear G. duodenalis infection in highly endemic areas has been associated with protection from acute diarrhea G. duodenalis infection. To date, there is insufficient data to balance the potential effects of anti-Giardia treatment against each other. Lastly, food supplementation in areas highly endemic for G. duodenalis also appears to be complex. G. duodenalis associated protection from diarrhea has recently been reported to be lost in Tanzanian children with multi-nutrient supplementation G. duodenalis among the interacting factors contributing to malnutrition and diarrhea in high-endemicity regions and to estimate the potential impact of control measures.The association with impaired child growth as observed in the present and other studies G. duodenalis assemblage B without causing diarrhoea but associated with underweight and clinically assessed severe malnutrition in Rwandan children. The underestimation of G. duodenalis by light microscopy suggests that prevalences and consequences have previously been underrated and asks for the implementation of more sensitive diagnostic, yet simple diagnostic tools. The clarification of the clinical significance of G. duodenalis in high endemicity areas needs to take account of an abundance of submicroscopic infections.In conclusion, our data provide evidence of a very high prevalence of Table S1Intestinal parasites in community children and children attending health facilities in southern Rwanda.(DOC)Click here for additional data file.Checklist S1STROBE checklist.(DOC)Click here for additional data file.


Cells reliably sense environmental changes despite internal and external fluctuations, but the mechanisms underlying robustness remain unclear. We analyzed how fluctuations in signaling protein concentrations give rise to cell-to-cell variability in protein kinase signaling using analytical theory and numerical simulations. We characterized the dose-response behavior of signaling cascades by calculating the stimulus level at which a pathway responds (‘pathway sensitivity’) and the maximal activation level upon strong stimulation. Minimal kinase cascades with gradual dose-response behavior show strong variability, because the pathway sensitivity and the maximal activation level cannot be simultaneously invariant. Negative feedback regulation resolves this trade-off and coordinately reduces fluctuations in the pathway sensitivity and maximal activation. Feedbacks acting at different levels in the cascade control different aspects of the dose-response curve, thereby synergistically reducing the variability. We also investigated more complex, ultrasensitive signaling cascades capable of switch-like decision making, and found that these can be inherently robust to protein concentration fluctuations. We describe how the cell-to-cell variability of ultrasensitive signaling systems can be actively regulated, e.g., by altering the expression of phosphatase(s) or by feedback/feedforward loops. Our calculations reveal that slow transcriptional negative feedback loops allow for variability suppression while maintaining switch-like decision making. Taken together, we describe design principles of signaling cascades that promote robustness. Our results may explain why certain signaling cascades like the yeast pheromone pathway show switch-like decision making with little cell-to-cell variability. Cells sense their surroundings and respond to soluble factors in the extracellular space. Extracellular factors frequently induce heterogeneous responses, thereby restricting the biological outcome to a fraction of the cell population. However, the question arises how such cell-to-cell variability can be controlled, because some cellular systems show a very homogenous response at a defined level of an extracellular stimulus. We derived an analytical framework to systematically characterize the cell-to-cell variability of intracellular signaling pathways which transduce external signals. We analyzed how heterogeneity arises from fluctuations in the total concentrations of signaling proteins because this is the main source of variability in eukaryotic systems. We find that signaling pathways can be highly variable or inherently invariant, depending on the kinetic parameters and the structural features of the cascade. Our results indicate that the cell-to-cell variability can be reduced by negative feedback in the cascade or by signaling crosstalk between parallel pathways. We precisely define the role of negative feedback loops in variability suppression, and show that different aspects of the dose-response curve can be controlled, depending on the feedback kinetics and site of action in the cascade. This work constitutes a first step towards a systematic understanding of cell-to-cell variability in signal transduction. External stimuli typically induce cellular responses by binding to cell surface receptors. Intracellular signaling networks transduce the signal, ultimately triggering gene expression responses in the nucleus. The basic building blocks of eukaryotic signaling networks are protein kinase cascades : The sigSignaling cascades can transduce information in different ways The dose-response curve of a signaling pathway relates the signaling activity to the amount of extracellular stimulus applied. The dose-response curve of signaling pathways is typically sigmoidal in shape and can be quantitatively described by the so-called Hill equation  manner see . The shmCell-to-cell variability is introduced into the models by assuming fluctuations in initial signaling protein expression levels. In contrast to previous studies on variability Our results show that generic five-step protein kinase cascades exhibit much stronger cell-to-cell variability than the yeast pheromone pathway, unless certain robustness requirements are fulfilled.Multi-step signaling cascades show a gradual dose-response behavior if the response of each individual cascade level is gradual as well The following ordinary differential equation describes the temporal evolution of the active kinase Each phosphorylation step is described as a reaction between the phosphorylated form  kinase  . The corThe steady state activity of each cascade level describing the activity upon long-term stimulation can be calculated by assuming that the kinase concentrations do not change over time  the variability cannot be reduced significantly by lowering the expression noise of certain signaling proteins; instead, a simultaneous noise reduction of all species would be required: The cell-to-cell variability can be quantified using the inter-quartile ratio (IQ ratio) which expresses the difference of cells with high and low signaling activities by dividing the third and first quartiles of the distribution (see Methods). Assuming realistic protein concentration fluctuations in Eq. 6, the IQ ratio only drops from 4.1 to 3.8 if the noise of 1 out of 9 signaling protein concentrations is eliminated, implying that the cell-to-cell variability remains essentially unchanged. (ii) the variability does not depend on the choice of the kinetic parameter values The variability principles derived from the analytical model (Eq. 1–7) were confirmed by explicit cell-to-cell variability simulations. To this end, each of the nine protein concentrations in the cascade was sampled from a log-normal distribution with a coefficient of variation that matches the experimentally observed variability of eukaryotic protein expression We investigated how the variabilities of the maximal pathway activation This protein concentration insensitivity can be explained as follows: strong negative feedback shifts the species The effects of strong negative feedback on the maximal pathway activation variability are less pronounced. Assuming log-normally distributed gene expression noise, the variance of The The We confirmed our findings concerning negative feedback regulation for more realistic feedback cascades with limited feedback strength Signaling cascades are often equipped with multiple negative feedback loops, some acting close to the receptor level, while others modulate the terminal cascade levels The steady state condition of the upstream feedback The steady state of We analyzed the downstream feedback model with limited feedback strength iability . Taken tiability .Our models predict that upstream negative feedback in the pheromone pathway may contribute to the invariant shmooing threshold, while downstream negative feedbacks may primarily ensure that all cells exhibit a similar maximal activation upon strong stimulation. One limitation of the negative feedback models is their shallow dose-response behavior which is inconsistent with the reported ultrasensitivity of the pheromone pathway The term ultrasensitivity describes signaling cascades with steep, sigmoidal dose-response curves that allow for all-or-none decision making. Ultrasensitive behavior has been reported for the yeast pheromone pathway, although the steepness of the dose-response curve differs between literature reports Two strategies exist to establish a very steep overall dose-response curve in a signaling cascade: Firstly, the all-or-none behavior may be primarily established at a single level, while the rest of the cascade shows gradual behavior (in isolation). Localized switching at the terminal cascade level has been reported for the yeast mating pathway Multi-step signaling cascades are capable of strong ultrasensitivity amplification, implying that a combination of multiple weak switches establishes a very steep overall dose-response curve. To simplify the mathematical analysis, we initially analyze a two-step signaling cascade with ultrasensitivity at each levelWe analyzed the overall dose-response curve relating the signaling output The activation resistances Numerical simulations were performed for a five-step signaling cascade, where each level was modeled using a Hill equation with n = 2 (similar to Eq. 14). The five-step signaling cascade exhibits very strong ultrasensitivity if low phosphatase activities are assumed for all cascade levels ; . The sde steps .The inherent invariance of the ultrasensitive system can be understood intuitively by considering an extreme case scenario, where each cascade level is a very steep switch : The cell-to-cell variability of a gradual cascade with realistic protein concentration fluctuations, quantified as the inter-quartile ratio (see Methods), only drops from 4.1 to 3.5 if a perfect correlation is introduced for a single kinase-phosphatase pair. This suggests that correlations in upstream signaling protein concentrations specifically promote the robustness of ultrasensitive systems.A way to increase the variability of the ultrasensitive cascade relative to seen in  , panel 2iability , panel 3Switch-like decision making may also be established if a single cascade level shows a very steep dose-response curve. Such a localized switch has been reported for the yeast mating pathway The switch-like dose-response at the terminal level . In this Section, we demonstrate that switching and invariance can be combined if the time window of variability suppression by negative feedback can be separated from the time window of switch-like decision making.de novo protein biosynthesis, thereby affecting signal transduction with a delay. Thus, a time window exists early after stimulation where the steepness of the dose-response curve is unaffected by transcriptional feedback. Yet, transcriptional feedback may promote robustness of the dose-response curve if it operates under basal conditions before stimulation as observed for the yeast mating pathway Feedback loops in mammalian signaling commonly involve transcriptional regulation, and the signaling cascades typically induce the expression of their own inhibitors We model basal transcriptional feedback by implementing a negative feedback loop in the single-switch cascade. The phosphatase at the second level is transcriptionally induced by the active terminal kinase   and alsoBasal signaling was implemented by assuming a low chronic level of the stimulus We performed cell-to-cell variability simulations of this system assuming a high Hill coefficient  . The bas . The sim , which i : TranscrThe invariance due to basal feedback is restricted to a certain range of activation resistances in the cascade, because increasing the expression of several phosphatases in the cascade shifts the system from complete switching of the whole cell population to heterogeneous switching of only a fraction of cells . In FiguRecent experimental evidence supports that Msg5, a basal transcriptional feedback regulator of the yeast mating pathway, suppresses the signaling variability upon pheromone stimulation Signaling networks commonly involve coherent feedforward loops where an upstream kinase controls a downstream target by two parallel pathways. Fus3, the yeast MAPK that mediates the shmooing response, is controlled by two pheromone-dependent pathways, both of which are required for full activation We model coherent feedforward regulation by extending the model of the five-step signaling cascade with a switch at the terminal level (Eqs. 16 and 17). The downstream signaling species tion of  . The effThe threshold In line with previous reports, we find that coherent feedforward regulation increases the ultrasensitivity of the dose-response curve, as the stimulus now enters with the exponent Explicit cell-to-cell variability simulations using Eq. 20 confirm that the threshold variability of the feedforward pathway  is less We conclude that joint regulation of Fus3 by Ste7 and Ptc1 may reduce the cell-to-cell variability of pheromone signaling. However, the simulated signaling thresholds still span a ∼50-fold range of stimulus concentrations , implyinA key question in biology is how cellular systems function robustly in face of internal and external fluctuations. We comprehensively characterized the determinants of cell-to-cell variability in protein kinase signaling cascades, and summarized our main findings in In this paper, we made a central simplifying assumption to study the behavior of protein kinase cascades: it was assumed that the individual levels of a protein kinase cascade function as isolated modules. Based on this assumption, we described the local dose-response behavior of each cascade level by Michaelis-Menten or Hill equations (Eqs. 3 and 14), and studied their behavior in tandem to gain insights into the global dose-response behavior of the five-step cascade. Depending on the protein concentrations and kinetic parameters in the cascade, the modularity assumption may be violated, and explicit simulations of all enzyme-substrate binding and dissociation events in the cascade may be necessary: strong sequestration of upstream kinases by highly abundant downstream substrates affects the phosphorylation state of the upstream kinase, thereby leading to retroactivity in the cascade Negative feedback is known to suppress the variability of biological systems and to reduce the steepness of signaling dose-response curves In this paper, we analyzed the steady state behavior of negative feedback circuits, but did not focus on their dynamical properties such as sustained oscillations One way to reduce noise in biological signaling systems is to correlate the expression fluctuations of antagonizing enzymes Gradual signaling systems transduce information quantitatively and faithfully report the stimulus concentration in the extracellular milieu. We therefore assumed that the maximal pathway activation and the pathway sensitivity of a gradual system should be invariant. However, recent experimental work revealed that the absolute signaling activities of a mammalian MAPK cascade pathway are highly variable, while the stimulus-induced fold-change in the signal is invariant between cells www.sympy.org). Numerical simulations were performed using the odeint function of the scipy.integrate package (www.scipy.org). The details of the model implementation process can be found in the figure captions and in the Supplemental All simulations were done in Python. Analytical solutions were obtained using the open-source python package SymPy . The IQ Ratio is a dimensionless number that reflects the fold difference between cells with high and low levels, while excluding (extreme) outliers. To further support our findings, we show in the Supplemental Figures S1, S2, S3, S4, S5, S6 that very similar results are obtained when using the coefficient of variation (CV = mean/standard deviation) as a measure of variability.Various methods were employed to characterize the cell-to-cell variability of the signaling dose-response curves. In vely see , and 6B.vely see , and 6C,Figure S1Cell-to-cell variability of a gradual kinase cascade quantified using the coefficient of variation. Concepts similar to (PDF)Click here for additional data file.Figure S2Cell-to-cell variability of gradual kinase cascades with negative feedback regulation quantified using the coefficient of variation. The concepts in panels A and B are similar to (PDF)Click here for additional data file.Figure S3Cell-to-cell variability of ultrasensitive kinase cascades with distributed switching quantified using the coefficient of variation. The concepts are similar to (PDF)Click here for additional data file.Figure S4Cell-to-cell variability of kinase cascades with ultrasensitive switching at a single step quantified using the coefficient of variation. The concepts are similar to (PDF)Click here for additional data file.Figure S5Cell-to-cell variability of ultrasensitive kinase cascades with basal transcriptional feedback quantified using the coefficient of variation. The concepts are similar to (PDF)Click here for additional data file.Figure S6Cell-to-cell variability of ultrasensitive kinase cascades with feedforward regulation quantified using the coefficient of variation. The concepts are similar to (PDF)Click here for additional data file.Figure S7Numerical stability analysis of the upstream feedback cascade with low activation resistances reveals that high feedback cooperativity is required for sustained oscillations. The eigenvalues of the Jacobi matrix at the steady state were calculated numerically for different feedback cooperativities (PDF)Click here for additional data file.Figure S8Numerical stability analysis of the upstream feedback cascade with high activation resistances reveals that sustained oscillations are not possible even for very strong feedback cooperativity. The concepts are similar to (PDF)Click here for additional data file.Protocol S1Implementation of model variants.(PDF)Click here for additional data file.Table S1Parameter values used for simulations. The index (PDF)Click here for additional data file.Text S1Analytical derivations and numerical analysis of steady state stability.(PDF)Click here for additional data file.


Genomic markers that reflect responsiveness to a relaxation response (RR) intervention have been detected in healthy volunteers, but have not been examined in clinical conditions for which RR is effective . This study aimed to explore the impact of an RR intervention on gene expression in hypertensive individuals.We performed transcriptional profiling analyses on the Peripheral Blood Mononuclear Cells (PBMCs) obtained before (Pre RR) and after (Post RR) 8 weeks of intervention from a pilot group of 4 hypertensives. Transcriptional profiling used Affymetrix HT HG-U133+ PM arrays, containing >47,000 transcripts corresponding to 38,500 genes. The arrays were normalized using the robust multi-chip analysis (RMA) algorithm. After normalization and preprocessing, differentially expressed genes were identified using the random variance model based t-test. To understand the underlying biological mechanisms associated with RR regulated genes, we performed Gene ontology (GO), pathways and geneset enrichment analysis (GSEA).A total of 474 transcripts were significantly differentially expressed (p<0.05) between the Pre and Post RR conditions. Hierarchical clustering of the top differentially expressed genes demonstrates striking segregation between Pre and Post RR conditions. The GO analysis identified significantly affected categories (p<0.05) that include “mRNA metabolic process,” ”positive regulation of RNA metabolic process,” “Calcium signaling pathway” and “programmed cell death.” The pathway analysis identified the over-representation (p<0.05) of differentially expressed genes to “cell cycle G/M checkpoint regulation,” “P53 signaling,” “Inositol metabolism signaling,” “Apoptosis Signaling,” “MIF Regulation of Innate Immunity,” and “Cardiac β-adrenergic Signaling.” Furthermore, GSEA analysis identified upregulation of 263 genesets (p <0.05) in Post RR including phosphatidylinositol signaling system, IFN-γ endothelial up, B cell receptor signaling pathway, FAS pathway, VEGF signaling pathway, Cardiac EGF pathway and MAPK pathway. Additional results to follow.These results suggest specific biochemical pathways on which to expand future research in the study of mind/body therapies in clinical populations.


S-glutathionylation). The abundance of glutathione in cells and the ready conversion of sulfenic acids to S-glutathione mixed disulfides supports the reversible protein S-glutathionylation as a common feature of redox signal transduction, able to regulate the activities of several redox sensitive proteins. In particular, protein S-glutathionylation is emerging as a critical signaling mechanism in cardiovascular diseases, because it regulates numerous physiological processes involved in cardiovascular homeostasis, including myocyte contraction, oxidative phosphorylation, protein synthesis, vasodilation, glycolytic metabolism and response to insulin. Thus, perturbations in protein glutathionylation status may contribute to the etiology of many cardiovascular diseases, such as myocardial infarction, cardiac hypertrophy and atherosclerosis. Various reports show the importance of oxidative cysteine modifications in modulating cardiovascular function. In this review, we illustrate tools and strategies to monitor protein S-glutathionylation and describe the proteins so far identified as glutathionylated in myocardial contraction, hypertrophy and inflammation.The perturbation of thiol-disulfide homeostasis is an important consequence of many diseases, with redox signals implicated in several physio-pathological processes. A prevalent form of cysteine modification is the reversible formation of protein mixed disulfides with glutathione ( In stress conditions, ROS levels increase and, because of their high reactivity, participate in a variety of chemical reactions. They are involved in cell damage, necrosis and apoptosis via oxidation of lipids, proteins and DNA and also provoke endothelial dysfunction, infiltration and activation of inflammatory cells . ROS genProtein glutathionylation is becoming increasingly recognized as playing a causative role in cardiovascular disorders (CVDs). Indeed, it regulates numerous physiological processes involved in cardiovascular homeostasis and/or perturbed in disease, including myocyte contraction, oxidative phosphorylation, protein synthesis, vasodilation, glycolytic metabolism and response to insulin. Therefore, perturbations in protein glutathionylation status may contribute to the etiology of cardiovascular diseases, such as myocardial infarction, cardiac hypertrophy and atherosclerosis. In this review, we discuss the glutathione-related modifications underlying redox signaling in CVDs focusing on myocardial contraction, metabolism, proliferation, hypertrophy and inflammation.2.S-glutathionylation is emerging as a critical signaling mechanism in cardiovascular diseases, because it regulates numerous physiological processes involved in cardiovascular homeostasis, including myocyte contraction, oxidative phosphorylation, protein synthesis, vasodilation, glycolytic metabolism and response to insulin /[NADH] ratio during caloric restriction activate the enzyme and can regulate gene expression and apoptosis. Using the purified protein, Zee et al. [S-glutathionylation. The authors identified Cys67 as the site of S-glutathionylation by Linear Ion Trap-Orbitrap MS [482 has been found to be modified by hydroxynonenal on MALDI-TOF-TOF analysis [Sirtuins are a highly conserved family of histone/protein deacetylases and have been shown to participate in biological functions related to the development of heart failure, including regulation of energy production, oxidative stress, intracellular signaling, angiogenesis, autophagy and cell death/survival. Mammals possess seven sirtuins. Sirtuin 1 (Sirt-1) is an NAD and p53 . Sirt-1 e et al.  demonstritrap MS . In addianalysis .4.2.5.+ oxidoreductase, AR) is an aldehyde metabolizing enzyme that catalyzes the NADPH-dependent reduction of aldo sugars and a variety of aldehydes. Aldose reductase is an NADPH-dependent monomeric protein that belongs to the aldo-keto reductase superfamily [298[Aldose reductase  beta chain has been known for more than 20 years and is associated with inhibition of Hb S-polymerization, increased oxygen affinity and a reduced alkaline Bohr effect [93 of the metHb beta chain and GSSG; and a disulfide bond between the other cysteine residues of metHb alpha chain and/or metHb beta chain and GSSG [et al. [93, Hb glutathionylation occurs also at Cys112 of the β-chain, providing a new potential GSH source hitherto unknown. They found that CO treatment of whole blood increases the GSH concentration in red blood cells cytosol, and this is linked to a significant Hb deglutathionylation. This process does not activate glycolytic metabolism, boosts the pentose phosphate pathway, increases glutathione reductase activity and decreases GSSG concentration, thus evidencing a CO signaling in human RBC driven by Hb glutathionylation. Hb is also a target for S-nitrosylation [The binding of glutathione to the Cysr effect ,135. At and GSSG ,137. Rec [et al.  found thsylation , and thisylation ,141. Ovesylation  and a go4.3.et al. [S-glutathionylation of p21Ras at Cys118. The GSH-adduct and the resulting activation of the p21Ras-MEK-ERK pathway were reversed by overexpression of catalase, glutaredoxin-1 or dominant negative p47Phox. In cardiac tissue, thioredoxin regulates pressure-overload-induced cardiac hypertrophy. Compared with wild-type animals, those without thioredoxin had greater cardiac hypertrophy, while overexpression of the protein resulted in attenuated cardiac size. The mechanism appeared to be due to modulation of p21Ras at Cys118, as demonstrated by in vitro models of strain and alpha-adrenergic receptor-stimulated cardiac hypertrophy in cardiomyocytes [118 and supports a mechanism of p21Ras S-nitrosation, followed by an exchange reaction, which leaves p21Ras S-glutathionylated.p21Ras is one major regulator in growth factor signaling in the vasculature. It is known to have four exposed redox-sensitive cysteines ,144. Adaet al.  have demmyocytes ,146. The4.4.4.4.1.S-glutathionylation inhibits the NFkB pathway at multiple points, including p50 [In vivo Glrx gene deletion sensitizes cells to oxidative inactivation of IKK-β and dampens TNFα-induced IKK and NFkB activation, resulting in varied responses to inflammation [The transcription factor, NFkB, a central regulator of immunity, is subject to regulation by redox changes . It contding p50 , RelA/p6ding p50  and IKK-ding p50 . NFkB itding p50 ,149. Theding p50 ,151. Oncding p50 . In vivoRecent evidence suggests that signaling by the proinflammatory cytokine, interleukin-1β (IL-1 β), is dependent on ROS derived from NADPH oxidase . Redox s4.4.2.et al. [S-glutathionylation. Upon infection with Sendai virus, IRF3 undergoes deglutathionylation by the cytoplasmic enzyme, glutaredoxin-1 (GRX-1); it becomes phosphorylated, homodimerizes, translocates to the nucleus, binds to target genes and activates transcription by interacting with CBP/p300 co-activators. In virus-infected GRX-1 knockdown cells, phosphorylation, homodimerization and nuclear translocation of IRF3 do not occur; consequently, its transcriptional activity and the expression of interferon-β (IFNβ) are severely reduced. Taken together, these findings reveal a crucial role for S-glutathionylation in controlling the activation of IRF3 and IFNβ gene expression, with IRF3 deglutathionylation essential for transcriptional activation of the interferon genes.Interferon regulatory factor 3 (IRF3) is an essential transcriptional regulator of the interferon genes. IRF3 is constitutively present in a latent conformation in the cell cytoplasm. Prinarakis et al.  reported5.S-glutathionylation is a redox-dependent post-translational modification with growing relevance in signal transduction. Redox signals have been implicated in several physiological processes, including kinase signaling, channel function, apoptotic proteolysis and regulation of transcription; thus, S-glutathionylation may represent a “modulator” of these pathways that often cross-talk with each other. Similarly to phosphorylation, cysteine modification is critical to cellular signaling, and its deregulation has consequences over a number of human diseases, including cardiovascular diseases. Indeed, many proteins undergoing glutathionylation may contribute to the etiology of myocardial infarction, cardiac hypertrophy and atherosclerosis. In this review, we have illustrated tools and strategies to monitor protein S-glutathionylation and describe the proteins so far identified as glutathionylated in myocardial contraction, hypertrophy and inflammation.6.S-glutathionylation affects either phosphatases and/or kinases, thus potentially influencing phospho/dephosphorylation pathways and becoming a nexus between sulfur and phosphorous biochemistry. The cross-talk between glutathionylation and phosphorylation may be critical for the proper functioning of the cardiac proteins. The cardiac ryanodine receptor (RyR2), for instance, is regulated by direct phosphorylation at different sites by cAMP-dependent kinase (PKA) or calmodulin kinase II (CaMKII), and this increases the probability of the RyR2 channel to be open. Furthermore, SERCA2 is indirectly regulated by phosphorylation through the accessory protein, phospholamban (PLN), which binds to and inhibits SERCA2 when dephosphorylated. Even the Na+-K+ ATPase is regulated by the selective phosphorylation of phospholemman in isolated myocytes [Like the phosphorylation/dephosphorylation cycle, glutathionylation represents a mechanism by which glutathione modulates sarcomere functions depending on the tissue redox state. myocytes .As advances in both the methodology and technology accelerate the study of glutathionylated proteins, the critical role they play in CVD is beginning to emerge. Thus, in perspective, these modifications, in combination with more standardized therapies, may offer new promise for drug development and may be used as biomarkers or predictors of cardiovascular pathology.


Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL). Treatment options are limited and prophylactic agents are not available. We have previously demonstrated an essential role for CREB-regulating transcriptional coactivators (CRTCs) in HTLV-1 transcription.In this study we report on the negative regulatory role of LKB1 tumor suppressor and salt-inducible kinases (SIKs) in the activation of HTLV-1 long terminal repeats (LTR) by the oncoprotein Tax. Activation of LKB1 and SIKs effectively blunted Tax activity in a phosphorylation-dependent manner, whereas compromising these kinases, but not AMP-dependent protein kinases, augmented Tax function. Activated LKB1 and SIKs associated with Tax and suppressed Tax-induced LTR activation by counteracting CRTCs and CREB. Enforced expression of LKB1 or SIK1 in cells transfected with HTLV-1 molecular clone pX1MT repressed proviral transcription. On the contrary, depletion of LKB1 in pX1MT-transfected cells and in HTLV-1-transformed T cells boosted the expression of Tax. Treatment of HTLV-1 transformed cells with metformin led to LKB1/SIK1 activation, reduction in Tax expression, and inhibition of cell proliferation.Our findings revealed a new function of LKB1 and SIKs as negative regulators of HTLV-1 transcription. Pharmaceutical activation of LKB1 and SIKs might be considered as a new strategy in anti-HTLV-1 and anti-ATL therapy. Over 20 million people are infected with HTLV-1 worldwide. ATL is a highly aggressive and fatal malignancy of CD4 latency. TreatmeExpression of HTLV-1 provirus is transcriptionally mediated through the viral transactivator Tax, which potently stimulates the activity of long terminal repeats (LTR) by activating the cellular transcription factor CREB and coactivators such as CREB-binding protein (CBP) and CREB-regulating transcriptional coactivators (CRTCs), also known as transducers of regulated CREB activity (TORCs)-9. We haIn addition to a viral LTR, a battery of cellular genes can also be activated by Tax primarily through CREB and NF-κB. Tax is a major viral oncoprotein that plays a crucial role in the initiation of malignant transformation. Tax alsConsistent with Tax playing an essential role in both viral transcription and oncogenesis, counteracting Tax function or eliminating Tax-expressing cells has shown anti-HTLV-1 and anti-ATL effects,15. ThusIn this study, we investigated the regulatory roles of LKB1, AMPKs and SIKs in Tax activation of the HTLV-1 LTR. We demonstrated LKB1 and SIKs to be negative regulators of HTLV-1 transcription. Our work suggests a new model in which LKB1 and SIKs suppress Tax-mediated LTR activation by targeting CRTCs and CREB. Our findings also implicate the utility of small-molecule agonists of LKB1 and SIKs in anti-HTLV-1 and anti-ATL therapy.II, which play a role in viral persistence and transformation[Four lines of analysis prompted us to investigate the role of LKB1 in HTLV-1 transcription. First, chromosomal rearrangements at 19p13.3, where LKB1 is located, have been reported in some ATL cells. Second,ormation,23. ThirTo determine whether LKB1 might inhibit Tax activity, we coexpressed LKB1 and Tax in HeLa cells  in HeLa cells, Tax-mediated activation of LTR was unaffected  and kinase-defective (K56M) mutants were found to be expressed to comparable levels in HeLa cells  and kinase-defective mutants (SIK2-K49M and SIK3-K37M) were expressed in HeLa cells  efficiently depleted its mRNA and protein expression  with increasing doses of metformin resulted in a progressive reduction in the levels of Tax protein  were maintained in RPMI1640 medium supplemented with fetal calf serum and penicillin/streptomycin.HeLa and HEK293T cells were cultured in Dulbecco modified Eagle medium supplemented with 10% fetal calf serum, 2 mM l-glutamine and 1% penicillin/streptomycin at 37°C in a humidified atmosphere of 5% COHeLa and HEK293T cells were transfected using GeneJuice transfection reagent (Novagen). Jurkat and other HTLV-1-transformed cells were transfected using Lipofectamine 2000 (Invitrogen).Reporter plasmid pLTR-Luc and expression plasmids for Tax, A-CREB, CRTC1, CRTC1-S167A, CRTC1-M1, SIK2, SIK3, AMPK and AMPK-T172D have been detailed elsewhere,48. Tax Mutants for LKB1, AMPKα2 and SIKs were generated by Quikchange Site Directed Mutagenesis kit XL (Agilent). DNA sequencing confirmed that all mutations were successfully introduced. The HTLV-1 infectious clone pX1MT has been described previously. Metformt test.The dual luciferase assay and protein analysis were performed as described previously,17. CellHEK293T cells grown in 100-mm petri dish were harvested into 1 ml of immunoprecipitation buffer . Flag-LKB1/SIK1, V5-Tax or GST-SIK2/SIK3 protein was precipitated from the cleared lysate after a 2-hr incubation at 4°C with mouse anti-Flag , mouse anti-V5 (Invitrogen) or glutathione Sepharose 4B . Immunoprecipitates were collected with protein G agarose (Invitrogen). Protein complexes were washed three times with immunoprecipitation buffer and subsequently resuspended in sample buffer . For immunoprecipitation of endogenous Tax, HTLV-1 transformed cells  were harvested in 1 ml of immunoprecipitation buffer. Cleared lysate was then incubated with mouse anti-Tax.HeLa and HEK293T cells were transfected with 100 nM siRNA using Lipofectamine 2000 (Invitrogen). MT2, MT4 and C8166 cells were transfected using TransIT-Jurkat transfection reagent (Mirus). RNAi experiments were performed as described. siRNA s-ΔCt.Real-time RT-PCR was performed as previously described,53. Prim4 cells treated with 10 μl of MTT solution (5 mg/ml) were measured by a microplate reader  at a reference wavelength of 550 nm. Cell viability was calculated as a percentage of the control. In dose–response experiments, cells were treated with 10, 20, 30 and 50 mM metformin for 24 hrs. In time-course analysis, cells were treated with 30 mM metformin for the indicated durations.Cell proliferation was assayed by the 3--2,5-diphenyl-tetrazolium (MTT) method as described. Briefly6/ml and cultured overnight in the presence of escalating concentrations of metformin . Supernatants were collected and ultracentrifuged for 2 hrs at 200,000 × g. Supernatant from cultured Jurkat cells was used as a negative control. Reverse transcriptase activity was measured using a colorimetric method by the use of a reagent kit from Roche.MT-2 cells were seeded at 1 × 10His-Tax, LKB1 trimeric complex and GST-AMPKα2 (1–312) were bacterially expressed and a kinase assay was performed with purified recombinant proteins as described. BrieflyThe authors declare that they have no conflict of interest.HMVT and DYJ designed the experiments, analyzed data and wrote the manuscript; HMVT and WWG performed the experiments and analyzed data; CPC and YTS performed pilot study for the project; CMW and KHK analyzed data and provided advice; and YPC and HT provided reagents and advice. All authors read and approved the final manuscript.in vitro. GST-AMPKα2 (1–312) (5 μg) was incubated with increasing amounts of His-Tax  in the presence or absence of LKB1 trimeric complex (1.5 μg) in an in vitro kinase assay. Proteins were resolved by SDS-PAGE and detected by Western blotting.LKB1 kinase activity was unaffected by Tax Click here for fileNucleotide sequences of RT-PCR primers and siRNAs.Click here for file


Objective(s): Human T Lymphocyte Virus Type one (HTLV-I) is a retrovirus that infects about 10-20 million people worldwide. Khorasan province in Iran is an endemic area. The majority of HTLV-I-infected individuals sustain healthy carriers but small proportion of infected population developed two progressive diseases: HAM/TSP and ATL. The proviral load could be a virological marker for disease monitoring, therefore in the present study HTLV-I proviral load has been evaluated in ATL and compared to HAM/TSP and healthy carriers.Materials and Methods: In this case series study, 47 HTLV-I infected individuals including 13 ATL, 23 HAM/TSP and 11 asymptomatic subjects were studied. Peripheral blood mononuclear cells (PBMCs) were investigated for presence of HTLV-I DNA provirus by PCR using LTR and Tax fragments. Then in infected subjects, HTLV-I proviral load was measured using real time PCR TaqMan method.Results: The average age of patients in ATL was 52±8, in HAM/TSP 45.52±15.17 and in carrier’s 38.65±14.9 years which differences were not statistically significant. The analysis of data showed a significant difference in mean WBC among study groups (ATL vs HAM/TSP and carriers P=0.0001). Moreover, mean HTLV-I proviral load was 11967.2 ± 5078, 409 ± 71.3 and 373.6 ± 143.3 in ATL, HAM/TSP and Healthy Carriers, respectively. The highest HTLV-I proviral load was measured in ATL group that had a significant correlation with WBC count . The proviral load variations between study groups was strongly significant .Conclusion: The present study demonstrated that HTLV-I proviral load was higher in ATL group in comparison with HAM/TSP and healthy carriers. Therefore, HTLV-I proviral load is a prognostic factor for development of HTLV-I associated diseases and can be used as a monitoring marker for the efficiency of therapeutic regime. The latency period is more than 30 years . Since HTLV-I integrated into host genome and has a long period of latency, the percentage of infected cells and proviral load might be good prognostic and monitoring markers for development and therapy efficiency of HTLV-I associated diseases. Consequently, the proviral load seems to be valuable virological marker for disease monitoring, however the impact mechanisms of these factors on disease progression have not been established yet  in ATL group that had symptoms such as organomegaly, cutaneous lesions, hypercalcemia and leukemia with atypical polylobed lymphocytes(flower liked ) .Liver function tests including SGOT,SGPT.ALP and LDH were performed and calcium checked routinely for ATL patients.In HAM/TSP groups Myelopathy proved by electromyography (EMG) and brain radiography. Seropositive activity checked by ELISA commercial kit . PBMCs were isolated from whole blood by Ficoll density gradient . Then cellular DNA was tested for the presence of HTLV-I provirus by conventional PCR using specific primers for Tax and LTR .Study populationA case series study carried out from July 2011 to December 2013 in Mashhad University of Medical Sciences (MUMS) hospitals on 47 HTLV-I infected individuals including 23 HAM/TSP patients; 9 males and 14 females referred by two subspecialists. Thirteen suspected ATL patients investigated, which the disease confirmed in nine individuals according to symptoms and laboratory tests . Six males and five females asymptomatic HTLV-I infected people age matched subjects were selected as healthy carrier. All subjects were seropositive for HTLV-I that then proved by conventional PCR.Proviral load measurement4 and expressed as the number of HTLV-I proviruses per 104 PBMCs  to measure the proviral load of HTLV-I using specific primers and a fluorogenic probe by a Rotorgen Q Real-Time PCR machine . The HTLV-I copy number was reported as an actual amount of cellular DNA by means of quantification of the albumin gene as the reference gene. HTLV-I and albumin DNA concentrations were calculated from two 5-point standard curves. The normalized value of the HTLV- I proviral load was calculated as the ratio of ×1004 PBMCs .Statistical analysisP- value was < 0.05 .Data Analysis was performed by SPSS (version 18 ). Inferential statistical methods including Man Withney, T student and Wilcoxon tests were used to compare the differences between clinical groups and healthy control. Results were statistically significant if Proviral load of HTLV-I investigated in 47 patients, 11 healthy carriers with 38.65±14.9 (CI 95%: 8-48) years old, 6 males and 5 females, 23 HAM/TSP patients with 45.52±15.17 (CI 95%: 38.9 up to 52) years old 9 males and 14 females. Thirteen individual were candidate for ATL, which the disease confirmed in nine individuals according to symptoms and laboratory tests. The average age for ATL group was 52±8 (CI 95%: 45-58) years-old .4(CI 95%:115-23707), in males 17195.67± 11925.36 (CI95%:2368-68076.2) copies/10 4 and in females 8398.2±4797.99 (CI95%:1923-21719.57) copies/104. The percentage of HTLV-I infected PBMCs in ATL group was 116.6±143 (CI 95%: 2.3-236.65), in male 171.31± 118 (CI 95%:137-680) and in female 83.8±47.82 (CI95%: 19.23-21.719).The mean HTLV-I proviral load in ATL patients was 11697.2±5078 copies/10 4, males proviral load average was 363 ± 113.9 (CI95%:3.66-632.3) copies/10 4 and 438.36± 99.9 copies/10 4 in females. In HAM/TSP patients the percentage of HTLV-I infected PBMCs was 3.6±1.14 % and 4.4±1% in male and female, respectively. 11Healthy Carriers HTLV-I proviral load average was 373.6 ± 143.3 (CI 95%:54.2-693) copies/ 10 4 which 3.7± 1.4(CI 95%:0.5-6.9) % of PBMCs were infected. In healthy Carriers HTLV-I proviral load was 427.67 ± 230.825 copies/10 4, in males 427.67 ± 230.825 and in females was 308.8± 176.51. The percentages of infected PBMCs were 4.25± 2.298 % and 3.08±1.76% in male and females, respectively.In HAM/TSP patients mean HTLV-I proviral load was 409 ± 71.3 (CI: 95% 261-556) copies/10 P=0.001). The proviral load variations between study groups was strongly significant .Classified HTLV-I proviral load according to sex and groups of the patients is shown in  Characteristics and proviral load of HAM/TSP, ATL patients and asymptomatic carrier’s subjects are summaDiscussion.+ T cells, age, and gender, routes of transmission , and high antibody titers against HTLV-I and TAX (In this study, HTLV-I proviral load investigated among three groups of HTLV-I infected, HAM/TSP, ATL and healthy carriers in northeast of Iran –Khorasan state. Viral factors, together with host genetics, are associated with an increased risk of developing HAM/TSP, ATL and clinical progression of these diseases that proved in previous studies . The mos and TAX -18. HTLV and TAX .et al reported the Lack of diversity in healthy carriers proviral loads (In the present study, there are significant variations between ATL, HAM/TSP and healthy carriers individuals for HTLV-I proviral load. Independent risk factors for the development of ATL, after adjusting for proviral load are increased age, family history of ATL, and first opportunity to learn HTLV-I infection during the treatment . ATL andal loads .The significant correlation with WBC count and HTLV-I proviral load in ATL patients in the present study could be proven by previous studies that showed decreasing count of T cell lymphocyte and HTLV-I proviral load in ATL patients after treatment -24. Previous studies reported no significant differences in the HTLV-I proviral load by sex and age in asymptomatic HTLV-I carriers , 25; resIn one study, authors found HTLV-I viral load difference in male and female, which might be the result of mode of transmission or acquisition of the virus and the methodology of study . In the Entirely, HTLV-I proviral load is a valuable prognostic factor for development of HTLV-I associated diseases and can be used as a monitoring marker for the progression and efficiency of therapeutic regime.Results of the present study demonstrated that HTLV-I proviral load is a valuable marker for monitoring of ATL and HAM/TSP patients particularly during therapy. Moreover, as HTLV-I proviral load is highest in ATL patients and lowest in healthy carriers, this factor may influence on development and progression of HTLV-I associated diseases and can be used as a prognostic test in HTLV-I infected subjects.