Antiretroviral therapy (ART) with a generic fixed-dose combination (FDC) of stavudine (d4T)/lamivudine (3TC)/nevirapine (NVP) is widely used in developing countries. The clinical data of this FDC among very advanced HIV-infected patients is limited.3 and group B: ≥ 50 cell/mm3).A retrospective cohort study was conducted among ART-naïve HIV-infected patients who were initiated a generic FDC of d4T/3TC/NVP between May 2004 and October 2005. Patients were categorized into 2 groups according to the baseline CD4 (group A: <50 cell/mm3 in group A and 139 (92–198) cells/mm3 in group B. Intention-to-treat analysis at 48 weeks, 71.7% (86/120) of group A and 75.0% (63/84) of group B achieved plasma HIV RNA <50 copies/ml (P = 0.633). On-treatment analysis, 90.5% (87/96) in group A and 96.9% (63/65) in group B achieved plasma HIV RNA <50 copies/ml (P = 0.206). At 12, 24, 36 and 48 weeks of ART, mean CD4 were 98, 142, 176 and 201 cells/mm3 in group A and 247, 301, 336 and 367 cells/mm3 in group B, respectively. There were no differences of probabilities to achieve HIV RNA <50 copies/ml (P = 0.947) and CD4 increment at 48 weeks between the two groups (P = 0.870). Seven (9.6%) patients in group A and 4 (8.5%) patients in group B developed skin reactions grade II or III (P = 1.000). ALT at 12 weeks was not different from that at baseline in both groups (P > 0.05).There were 204 patients with a mean ± SD age of 37.1 ± 8.9 years, 120 (58.8%) in group A and 84 (41.2%) in group B. Median (IQR) CD4 cell count was 6 (16–29) cells/mm3 has no different outcomes in terms of safety and efficacy. FDC of d4T/3TC/NVP can be effectively used in advance HIV-infected patients with CD4 <50 cells/mm3.Initiation of FDC of d4T/3TC/NVP in HIV-infected patients with CD4 <50 and ≥ 50 cells/mm Current antiretroviral treatment guidelines for HIV infection in adults and adolescents in the resource-limited settings recommend using two nucleoside reverse transcriptase inhibitors (NRTIs) plus one non-nucleoside reverse transcriptase inhibitor (NNRTI) as the first-line antiretroviral regimen ,2. RegimAlthough EFV-based ART is an NNRTI-based antiretroviral therapy (ART) of choice according to the recommendation of recent antiretroviral treatment guidelines ,2, NVP-bThere are some potential limitations of NVP-based ART including adverse drug reactions and low genetic barrier. Skin rash is the most frequently observed adverse event from NVP and manifests as a diffuse maculopapular rash or erythematous rash with or without constitutional symptoms. The risk of rash at any severity is greatest in the first six weeks . However3 and those who had CD4 cell counts ≥50 cells/mm3.To date, the data regarding safety and efficacy of the d4T/3TC/NVP FDC among advanced HIV-infected patients with very low CD4 is still limited. Patients in resource-limited settings usually present late with very low CD4 cell counts. We therefore conducted this retrospective cohort study to compare immunological and virological outcomes and adverse events of a generic FDC of d4T/3TC/NVP between HIV-infected patients who had baseline CD4 cell counts <50 cells/mmA retrospective cohort study was conducted among ART-naïve HIV-infected patients who were initiated a generic FDC of d4T/3TC/NVP between May 2004 and October 2005 in Bamrasnaradura Infectious Diseases Institute, Ministry of Public Health, Nonthaburi, Thailand. The patients' identification numbers were identified from annual database of the institute. The data were extracted from the medical records. Inclusion criteria were as follows: (1) HIV-infected individuals ≥ 15 years old, (2) naïve to ART prior to FDC of d4T/3TC/NVP, (3) were initiated with a generic FDC of d4T/3TC/NVP, (4) used separate tablet of NVP 200-mg once-daily lead-in dose during the first 2 weeks, prior to escalation to 200 mg twice daily, (5) followed up at least two clinic visit. Exclusion criteria were as follows: (1) baseline serum creatinine level > 2.0 mg/ml, (2) baseline liver aminotransferase >3 times of upper normal limit, (3) currently active major opportunistic infections (OIs), and (4) receiving medications that have drug-drug interactions with NVP, including rifampicin and fluconazole.3) and group B (CD4 cell counts ≥ 50 cells/mm3). Factors including demographics, previous OIs, CD4 cell counts, plasma HIV RNA were studied and compared between the two groups. Patients were followed up for 48 weeks after initiation of a generic FDC of d4T/3TC/NVP. The parameters including CD4 cell counts, plasma HIV RNA and ALT were assessed at baseline, 12, 24, 36 and 48 weeks of ART. The primary outcome of interest was the proportion of patients who achieved plasma HIV RNA <50 copies/ml after 48 weeks of ART. The secondary outcomes were as follows: 1) the probability to achieve plasma HIV RNA <50 copies/ml, 2) the increment of the number of CD4 cell counts at 48 weeks of ART from baseline value and 3) the incidences of NVP-associated adverse reactions including skin rashes and hepatotoxicity that lead to drug discontinuation.All eligible patients were categorized into two groups according to their baseline CD4 cell counts: group A  and plasma HIV RNA at baseline. Statistical calculations were performed using SPSS program version 11.5 . A two-sided P value of less than 0.05 was considered statistically significant. The study was approved from the institute review board.Mean (± SD), median  and frequencies (%) were used to describe patients' characteristics in both groups. 3 in group A and 139 (92–198) cells/mm3 in group B. Group A had more previous opportunistic infections, higher baseline HIV RNA, ALP and ALT than group B (P < 0.05). Any causes of discontinuation of ART are shown in Table P = 1.000).A total of 204 patients met entry criteria; mean (± SD) age was 37.1 ± 8.9 years and 60.3% were male. There were 120 (58.8%) patients in group A and 84 (41.2%) patients in group B. Table P = 0.947). The Cox proportional hazard model including factors of age, gender, body weight, previous OIs, baseline hemoglobin, baseline CD4 cell counts and baseline plasma HIV RNA showed that patients in group A had similar chance of undetectable plasma HIV RNA with the patients in group B . The other factors were not associated with undetectable plasma HIV RNA after 48 weeks of ART (P > 0.05).The results of the primary outcome are shown in Table 3 and group B patients had a median CD4 of 240, 280, 305 and 331 cells/mm3 at 12, 24, 36 and 48 weeks, respectively  cells/mm3, P = 0.870]. Eleven (9.2%) patients in group A and 12 (14.3%) patients in group B developed NVP-associated skin reactions grade II and III in which lead to the discontinuation of generic FDC of d4T/3TC/NVP (P = 1.000). Mean ± SD ALT when these 23 patients developed skin reaction was 31.5 ± 21.1 U/l. No patients in the present study developed skin reactions grade IV. By repeated measurement analysis, there were no differences of ALT at 12 weeks from baseline value in both groups (P > 0.05). No patients developed clinical hepatitis. Three (2.5%) patients in group A and 5 (6.0%) patients in group B developed stavudine-associated peripheral neuropathy (P = 0.278). Stavudine-associated symptomatic lactic acidosis was observed in 2 patients during the 48-week study period. Four patients in group A and one patient in group B died during the study period. The causes of death of these 5 patients were as follows: disseminated histoplasmosis 1, E. coli sepsis 1, MAC infection 1 and wasting syndromes 2.Group A patients had a median CD4 cell count of 85, 125, 158 and 198 cells/mm3 had similar virological and immunological responses when compared to those who had baseline CD4 cell counts ≥ 50 cells/mm3. This finding can support the use of this generic FDC of d4T/3TC/NVP in very advanced HIV-infected patients in the resource-limited settings. According to the guideline for management of HIV-infected patients in Thailand, ART is recommended for all patients with history of AIDS-defining illness or asymptomatic patients with CD4 cells count less than 200 cell/mm3. Therefore, almost all patients in group B have baseline CD4 cell counts between 50 cells/mm3 and 200 cells/mm3. As expected, group A patients have a higher proportion of previous opportunistic infections and a higher level of plasma HIV RNA.The results from the present study demonstrate that HIV-infected patients who had baseline CD4 cell counts <50 cells/mm3), we found that 71% of patients achieved undetectable plasma HIV RNA after 48 weeks of ART. This number is comparable to the virological response in group B and similar to other studies that conducted in our country and in developed countries [The overall proportion of patients who achieved plasma HIV RNA <50 copies/ml was 73% 149 of 204) after 48 weeks of ART in an ITT analysis. Despite the fact that group A patients were severely immunocompromised  CD4 cell counts increase from 5 (3–7) cells/mm3 to 151 (92–231) cells/mm3 in this subgroup (data not shown). This response would be continued for many years into effective antiviral effect of ART.Overall, CD4 cell counts rise with immune recovery from receiving ART. The increment of CD4 cell counts after 48 weeks of ART is not blunted with very low baseline CD4 cell counts as shown in Figure 3. Furthermore, no factors  were associated with skin rashes in the present study according to the results of logistic regression analysis (data not shown). These findings suggest that there is no clinical factor to predict the occurrence of rash from NVP in patients with very low CD4 cell counts. However, this should be interpreted with caution due to the limitation of sample size. It would be beneficial if there are any factors that can predict this adverse event. Further study to determine immunologic and genetic factors that associated with rash is encouraged.Regarding discontinuation of ART, NVP-associated skin rashes is an important reason for discontinuation. In the present of NVP 200-mg once-daily lead-in for 2 weeks, NVP may cause a mild skin rash in 15 to 20% of patients; 5 to 10% of which discontinue treatment . After 43 at the time of nevirapine initiation when compared with woman with CD4 cell counts of ≤ 250 cells/mm3 (11.0% vs 0.9%). An increased risk was also seen in men with baseline CD4 cell counts >400 cells/mm3 when compare with baseline CD4 cell counts ≤ 400 cells/mm3 (6.3% vs 1.2%) [Although there are limited data regarding the impact on hepatotoxicity, we decided to exclude the patients who concurrently received rifampicin and fluconazole from the study. These drugs may potentially increase incidence of hepatotoxicity -25. The vs 1.2%) -29. The vs 1.2%) . AdditioThe study has demonstrated the satisfactory clinical outcomes, the extent of immunological restoration and virological responses of a generic FDC of d4T/3TC/NVP in very advanced HIV-infected patients. These results provide the evidence of benefit from NVP-based ART in advanced HIV-infected patients. Thus, this may support the physicians to prescribe NVP-based ART regimen for these patients.3. It would be more interesting if we can compare clinical outcomes between these groups. Finally, the sample size may be not large enough to detect small difference of efficacy and low incidence of adverse events, particularly hepatitis.The present study has some limitations. The study design is a retrospective study, which is not the best study design to evaluate the efficacy of antiretroviral regimen. However, this study design is a comparative study that evaluated the efficacy NVP-based ART between the patients who had extremely low CD4 cell counts and those who had moderate level of CD4 cell counts. The results of the present study may provide useful clinical data for caring advanced HIV-infected patients in developing countries. In addition, some clinical data may be underestimated and some possible risk factors may not be included. Our study was based on a tertiary care center for HIV-infected patients. These study populations were cared by infectious diseases specialists and HIV-experienced medical team. Thus, the similar results might not be achieved in the general or community hospital in resource-limited setting. Liver enzymes were not performed during the first few weeks of ART. However, no patients developed clinical hepatitis during this period. Baseline hepatitis B and hepatitis C serology were not routinely performed prior to ART initiation. We did not have reference group of patients with CD4 cell counts greater than 200 cells/mm3 has no different outcomes in terms of safety and 48-week virological and immunological response. Generic FDC of d4T/3TC/NVP can be effectively used in advance HIV-infected patients with CD4 <50 cells/mm3.In conclusion, initiation of a FDC of d4T/3TC/NVP in HIV-infected patients with baseline CD4 cell count of <50 and ≥ 50 cells/mmHAART: Highly active anti-retroviral therapy, HIV: Human immunodeficiency virus, NRTIs: Nucleoside reverse transcriptase inhibitors, NVP: NNRTs: Non-nucleoside reverse transcriptase inhibitors, FDC: Fixed-dose combinations, Nevirapine, EFV: Efavirenz, d4T: Stavudine, 3TC: Lamivudine, ART: Antiretroviral therapy, OIs: Opportunistic infections, AST: Aspartate aminotransferase, ALT: Alanine aminotransferaseThe author(s) declare that they have no competing interests.WM participated in the design of the study and statistical analysis. SC participated in the design of the study. SL participated in the design of the study. SS participated in the design of the study and statistical analysis.


In the absence of tetracycline, replication of vvtetOA12L was inhibited by 80% and this inhibition could be overcome by transient expression of the wild-type copy of the A12L gene. In contrast, mutation of the AG/A site abrogated the ability of the transfected A12L gene to rescue, indicating that A12L proteolysis plays an important role in viral replication. Electron microscopy analysis of the A12L deficient virus demonstrated the aberrant virus particles, which were displayed by the AG/A site mutation. Thus, we concluded that the not only A12L protein but also its cleavage processing plays an essential role in virus morphogenic transition.Like the major vaccinia virus (VV) core protein precursors, p4b and p25K, the 25 kDa VV A12L late gene product (p17K) is proteolytically maturated at the conserved Ala-Gly-Ala motif. However, the association of the precursor and its cleavage product with the core of mature virion suggests that both of the A12L proteins may be required for virus assembly. Here, in order to test the requirement of the A12L protein and its proteolysis in viral replication, a conditional lethal mutant virus ( Proteolytic processing in vaccinia virus (VV) plays an important role in morphogenic transitions during the virus replication cycle. To date, six VV-encoded, proteolytically processed proteins have been reported. They are the gene products of A10L (p4a), A3L (p4b), L4R (p25K), A17L (p21K), G7L, and A12L (p17K) . ExtensiOne of the VV major core proteins, A10L has been shown to be essential in virus replication and its absence in virus assembly resulted in defective virus morphology such as IV-like particles, which lacked granular viral materials and consequently produced the irregular-shaped virus particles . These mOn the other hand, both the G7L and A17L gene products, VV membrane proteins, are required for virus replication and are involved in the early development of IV membranes. G7L, a phosphoprotein in association with the A30L and H5R proteins, is responsible for the correct recruitment and attachment of crescent-shaped membranes to viroplasms . The absVV A12L is a late gene product, which is proteolytically processed from a 25kDa precursor (p17K) into a 17kDa cleavage product (17K) . Its provvtetOA12L), we infected T-REx 293 cells with vvtetOA12L at various concentrations of Tet from 0 to 40 μg/mL  was used to address the requirement of the A12L protein and the AG/A site cleavage for viral replication. To begin with, A12L protein expression was confirmed by immunoblot analysis with A12L specific bands obtained only in the presence of Tet (data not shown). Approximately 80 % reduction of virus titer was observed in the absence of Tet  into ID/I, however, failed to complement the absence of A12L protein, resulting in the similar virus yield to the titer of vvtetOA12L infection in the absence of Tet. Therefore, it is suggested that cleavage at the AG/A site plays an essential role in A12L functionality.The sequence alignment of the A12L open reading frame with other representative orthopoxviruses such as cowpox, variola, and ectromelia viruses has shown highly conserved sequence alignment with more than 95 % identity (data not shown). Thus, it is expected that A12L may be essential for virus replication. An A12L conditional mutant virus  not only confirmed the requirement of A12L in virus replication but also demonstrated that the disrupted A12L proteolysis eliminated its complementing functionality. This is also supported by the electron microscope analysis, which demonstrated the impaired morphological development of IV toward IMV by the failure of AG/A cleavage event.vvtetOA12L mutant virus will lead to the more specific biological function of the A12L protein during VV morphogenic transitions and regulation of A12L proteolysis.The phenotypic defects such as detached viral membrane from the electron-dense virus materials, aberrant shape of IV particles, and disrupted bi-concave core layer of IMV particles suggest that A12L protein and its cleavage events may participate in the viral morphogenesis throughout from the early stage of IV formation to the very last stage of fully matured IMV. The abnormal IV-like particles similarly observed by the A10L deficiency imply that A12L may have a role in correct formation of nucleoprotein complex within the IV . In addiBy demonstrating that A12L protein and its cleavage at an N-terminal AG/A play an important role in viral replication, we were able to conclude that all the VV core precursor proteins, which are proteolytically maturated, are required for the production of infectious progeny. The similar morphological defects observed by the A12L deficiency and single site mutation (AG/A) of A12L give emphasis to the significant participation of VV proteolysis in the viral morphogenic transition.2. For infection of the conditional mutant virus of A12L (vvtetOA12L), T-REx 293 cells (Invitrogen) were grown in Dulbecco's modified Eagle's medium  supplemented with 10% Tet system approved fetal bovine serum (BD Biosciences), 2 mM Glutamax (Invitrogen), and 1% penicillin-streptomycin (Invitrogen), and incubated as described above. Blasticidin  was added to the D-MEM growth media for selection of the pcDNA6/TR plasmid [Monolayer of BSC-40 cells was maintained in Eagle's minimal essential medium  supplemented with 10% fetal calf serum , 2 mM glutamine (Invitrogen), and 10 mM gentamicin sulfate (Invitrogen) at 37°C in a 95% humidified atmosphere containing 5% CO plasmid , which evvtetOA12L). The tetracycline operator (TetO) was inserted in front of the A12L ORF by virtue of two-step polymerase chain reaction (PCR) and amplified with 215 nucleotides (nts) upstream of the A12L ORF and 213 nts downstream of the A13L ORF. The PCR products were cloned into the p7.5:NEO vector [TetOA12L:NEO plasmid. Transfection of the p7.5:TetOA12L:NEO plasmid in concert with VV WR infection induced the first recombination. The Neomycin resistance gene (NEOR) in the p7.5:TetOA12L:NEO plasmid was used as a transient selective marker in the presence of Geneticin G418 sulfate (Invitrogen). The second recombination of NEOR-containing viruses occurred in the absence of Geneticin G418 sulfate, producing a wild type virus and an A12L mutant virus (vvtetOA12L) containing TetO without NEOR. Plaque purifications were performed in concert with PCR screens using the primers specific for TetO and 3' end of A12L ORF to identify pure vvtetOA12L isolates. Experimental infections of vvtetOA12L were carried out in T-REx 293 cell line to control the gene expression, which constitutively provides the Tetracycline repressor.VV WR was used for the construction of the conditional mutant A12L virus  at an MOI of 1 plaque forming unit (PFU)/cell were placed on the cells for 30 min at room temperature. The infection D-MEM containing 5% of Tet-approved FBS, L-glutamax (10 mM), penicillin-streptomycin (10 mM) was then added. Tetracycline  was placed in infection D-MEM media for induction of the A12L protein. Cell extracts were harvested at 24–48 hours post infection (hpi) by centrifugation (750 × g) for 5 min at 4°C, followed by three cycles of freezing and thawing to lyse the cells. Virus titers were conducted on BSC-40 cells, incubated at 37°C for 40 hours, and stained with 0.1% crystal violet solution in 30% ethanol.When T-REx 293 cells were approximately 80% confluent, ATCGACATCTGTGATACAAAATCAAAGAGTTCA-3'. The AG/A site-mutated A12L was inserted in pRB21 vector.In order to rescue the absence of A12L by plasmid-bourn A12L (pA12L), full-length of A12L ORF was placed right after an early/late synthetic promoter in pRB21 [vvtetOA12L at an MOI of 1 PFU/cell for 24 hours. Virus titers were determined as described earlier.For transfection of the plasmids into T-REx 293 cells, infection media of D-MEM medium was placed in new eppendorf tubes and mixed with 2 to 10 μg of DNA and 30 μl of the transfection reagent, DMRIE-C (Invitrogen). After vortexing the mixture, it was placed at room temperature for 20 min. and loaded on 6-well plates of ~ 60% confluent T-REx 293 cells. The cells were incubated at 37°C for 5–6 hours and infected by vvtetOA12L and harvested at 24 hpi by centrifugation (270 × g) at 4°C. The cell extracts were resuspended with 1X PBS, followed by incubation with fixative buffer  for 2 hours at room temperature. Postfixation, ultrathin section, and staining were performed as described [T-REx 293 cells were infected at an MOI of 1 PFU/cell with escribed .vvtetOA12L:VV: Vaccinia virus; IV: Immature virus; IMV: Intracellular mature virus; A12L mutant virus; Tet: Tetracycline; TetO: Tetracycline operator.The author(s) declare that they have no competing interests.


A modified tetrazolium reduction assay (MTT) was used to assess the relation between HLA class I antigen expression on tumour cells and their susceptibility as a target for non-MHC restricted LAK/NK cytotoxicity using interleukin-2 activated peripheral blood mononuclear cells (MNC) from normal individuals. At 20/1 effector/target ratio this ranged from no killing to 77%. The efficiency of killing was dependent on duration of effector cell culture with IL-2, peaking at day 10 and declining thereafter. This killing could be enhanced by addition of other cytokines including interferons alpha, beta and gamma. Study of a panel of 15 tumour cell lines using a single effector showed that there was no statistically significant inverse correlation (using Spearman rank test) between the degree of tumour class I expression and LAK/NK killing at 20/1 (r = 0.23 P = 0.39) and 10/1  and at 5/1 E/T ratio r = 0.47, P = 0.08) respectively. Lack of inverse correlation between these two parameters came from study of one bladder tumour line (FEN), whose absent class I antigens had been corrected by transfection with beta 2 microglobulin gene. At high E/T ratio (20/1) there was an increase in the susceptibility of target cells to lysis , whilst at lower E/T ratios (1/1) there was significantly more killing of the non-transfected cells (10% vs 31%). The addition of anti-class I antibody W6/32 increased killing by 18% but this was non-specific as the same increase occurred with a class II antibody. These data suggest that overall there was not an inverse correlation between class I expression and LAK/NK killing at high E/T ratios, whilst at low (5/1 or lower) E/T ratios this correlation nearly reached statistical significance suggesting that the conflicting literature reports may be due to a threshold levels of effector cells above which the masking effects of MHC antigens disappears.


To date, the earliest stage at which the orientation of the anterior-posterior axis in the mouse embryo is distinguishable by asymmetric gene expression is shortly after E5.5. At E5.5, prospective anterior markers are expressed at the distal tip of the embryo, whereas prospective posterior markers are expressed more proximally, close to the boundary with the extraembryonic region.in situ hybridisation at E5.5 and E6.5 revealed novel expression patterns for known and previously uncharacterised genes, including Peg10, Ctsz1, Cubilin, Jarid1b, Ndrg1, Sfmbt2, Gjb5, Talia and Plet1. The previously undescribed gene Talia and recently identified Plet1 are expressed specifically in the distal-most part of the extraembryonic ectoderm, adjacent to the epiblast, and are therefore potential candidates for regulating early patterning events. Talia and the previously described gene XE7 define a gene family highly conserved among metazoans and with a predicted protein structure suggestive of a post-transcriptional regulative function, whilst Plet1 appears to be mammal-specific and of unknown function.To contribute to elucidating the mechanisms underlying the events involved in early patterning of the mouse embryo, we have carried out a microarray screen to identify novel genes that are differentially expressed between the distal and proximal parts of the E5.5 embryo. Secondary screening of resulting candidates by Our approach has allowed us to compare expression between dissected parts of the egg cylinder and has identified multiple genes with novel expression patterns at this developmental stage. These genes are potential candidates for regulating tissue interactions following implantation. At 5.5 days of development (E5.5) the mouse egg cylinder appears radially symmetrical about its proximo-distal axis with respect to known molecular markers and to the arrangement of its three principle tissue layers – epiblast, extra-embryonic ectoderm and visceral endoderm. However, shortly after E5.5 the first molecular asymmetries that determine the anterior-posterior axis begin to emerge. These involve movement of a subset of visceral endoderm cells, anterior visceral endoderm (AVE), located at the distal tip of the egg cylinder towards the future anterior side -5. SubseThe stages of mouse development between implantation and the gastrulating egg cylinder have been relatively little studied. This is due partly to the relative inaccessibility of embryos within the uterine deciduae during this time, and partly to their relatively poor development in culture compared with preimplantation and gastrula stages. More recently, much attention has been focused on the events preceding gastrulation and their relation to earlier preimplantation development, providing an incentive to identify novel genes with restricted expression patterns during these stages.in situ hybridisation, we identified both known and novel genes with previously unreported differential expression in the early mouse egg cylinder.Several recent microarray screens have focused on stage-specific expression in pre-implantation embryos -11, whilOtx2 . Itss Lefty1 ,38, Cer1y1 [Cer1  and partater AVE .Jarid1b showed expression in epiblast and the outer part of the ectoplacental cone at E5.5, however by E6.5 epiblast expression was weak or undetectable. Jarid1b encodes a nuclear protein that was originally identified in human breast cancer cell lines [ll lines . In normll lines ,42. In Ell lines , with whll lines .Ndrg1, Sfmbt2 and Gjb5 – identified in the screen showed specific expression throughout the extraembryonic ectoderm at both E5.5 and E6.5. Ndrg1 was originally identified by its upregulation in N-myc deficient mice [Drosophila polycomb group of transcriptional repressors, which regulate homeotic and other genes [Sfmbt1 (= Sfmbt) was shown to be highly expressed in adult testis, with a much lower expression in other adult and late embryonic tissues [Sfmbt2 has not been characterised, however database searches of matching ESTs indicate expression in testis and germ cells, suggesting that Sfmbt1 and Sfmbt2 may have related roles.Gjb5, which showed very strong specific expression in the extra-embryonic ectoderm, encodes one of a large family of gap junction proteins. A number of other gap junction proteins also show spatially restricted expression during early post-implantation development, indicative of a role in establishing communication compartments [Gjb5 expression has previously been shown in preimplantation embryos [Three genes – ent mice . While ient mice -48. Sfmber genes -51. The  tissues . Sfmbt2 artments ,54, whil embryos . While tPlet1 and Talia – showed localised expression in the extraembryonic ectoderm that may suggest a role in the interactions between epiblast and extraembryonic ectoderm. Signals from this region of the egg cylinder are believed to regulate proximo-distal patterning of the epiblast via such factors as Nodal, Cripto and Otx2 [Plet1 and Talia is regulated downstream of this signaling, and may have roles in specifying this identity. In particular, the restricted expression of Plet1 from E5.5 appears to be earlier than has been reported for other genes such as Eomes [Bmp4 [Plet1 expression may be directly regulated by this pathway. Recently identified by its trophoblast-specific expression at later stages of mouse, pig and human [Two genes – and Otx2 ,17,56-62and Otx2 ). Thus ias Eomes  and Bmp4es [Bmp4 , suggestnd human ,67, no fTalia/XE7 amongst metazoans further supports an essential role in development.By contrast, we have shown from its predicted tertiary structure that Talia is likely to function as a post-transcriptional regulator due to its similarities with HuD, an RNA-binding protein shown to have a role in specifying neural cell identity . InteresTalia and Plet1, will be of particular interest for further analysis, particularly with respect to possible roles in specifying the identity of extraembryonic ectoderm adjacent to the epiblast or in signaling to the proximal epiblast.This study demonstrated for the first time the application of a microarray strategy for identifying genes that are differentially expressed between dissected parts of the early post-implantation mouse embryo. It successfully identified several genes, both known and previously uncharacterised, with novel expression patterns in the early mouse post-implantation embryo. Some of these, such as Fifty E5.5 embryos from F1 (C57/BL6 × CBA) females crossed with F1 males were collected into M2 medium. After removal of Reichert's membrane, embryos were cut at the embryonic-extraembryonic boundary into proximal and distal halves, using a finely drawn glass needle, respectively pooled, frozen on dry ice and stored at -80C until RNA extraction.Total RNA was extracted using an RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. Target cDNA synthesis and single primer amplification (SPA) followed by labelling with Cy5- or Cy3-dCTP were performed as previously described . LabelleEcoRI and SalI sites of pBluescript II KS(+). Respective forward and reverse primers for the latter were: Jarid1b, 5'-GGAATTCGGGTTGCTTGCTTCTGCTTCTTC and 5'-GCGTCGACATCAGGGGAAACTGGTATCGGC; Gjb5, 5'-GGAATTCCTACCTCTTCCACGCATTCTATCC and 5'-GCGTCGACAGGCATTTGCTCATCGGTGC; Sfmbt2, 5'-GGAATTCGTCTCTGGGGACATCTACTGCTTG and 5'-GCGTCGACTGCTCTGCCTCGGTTCTGTG; Ndrg1, 5'-GGAATTCGAGAGAGAGAGGCAGGAAAGTTGG and 5'-GCGTCGACTACAAACCCAGTCAGCAGGAGG; Cubilin, 5'-GGAATTCAACCTTGCCCGTGTTCTATTCC and 5'-GCGTCGACTGAAGACCCGATTTGATGAAGC; Talia (exon 7), 5'-GGAATTCATCCTGGCACATCAATAATGGC and 5'-GCGTCGACAAGTAACCCCACAGACTGACATCC; Talia (exons 3–4), CATTTTCTGCATAAGGTGGTGTGAGGAC and GCCTGATAGCATCGCTTCTCTGCC;Plet1, 5'-GGAATTCCTGAAAGCAGTGAAGGAGGACG and 5'-GCGTCGACCACGCAGGATGGATGGACTAAG.E5.5 and E6.5 embryos were collected as above and, after removal of Reichert's membrane, fixed in 4% paraformaldehyde in phosphate buffered saline overnight at 4°C. DNA templates were prepared by PCR from plasmid DNA using T3, SP6 and T7 promoter-specific primers. Plasmid templates for each probe were either transcribed directly from NIA clones (corresponding to those used in the array) or were cloned by RT-PCR from E6.5 embryo mRNA into the In situ hybridisation was performed as described by Wilkinson and Nieto [Digoxygenin-labelled antisense RNA probes were prepared using an Ambion MegaScript transcription kit  according to the manufacturer's instructions. nd Nieto .Genomic structure was analysed using the Genomatix web-based sequence analysis software. ESTs weTotal RNA was extracted from adult female mouse tissues and pooled E7.5 egg cylinders using the RNeasy Mini Kit (QIAGEN) according to the manufacturers instructions. Oligo-dT(20)-primed first strand cDNA was prepared from 0.5 μg of total RNA in a 10-μl reaction volume using SuperScript III (Invitrogen) at 50°C for 1 hour according to the manufacturer's instructions. 0.5 μL of template was then used for each 15-μL PCR reaction mixture containing 0.05 Units/μL GoTaq polymerase (Promega), 1× supplied PCR buffer, 0.25 mM dNTPs and 0.5 μM each of forward and reverse gene-specific primers. 35 cycles of PCR were performed comprising 15 seconds denaturation (94°C), 15 seconds annealing (55°C) and 30 seconds extension (72°C). 8 μL of each PCR product was separated by electrophoresis in a 1.5% agarose gel containing ethidium bromide.SF performed all collection and experimental work on mouse embryos and writing of the manuscript. SF, with the crucial assistance of LS, performed the microarray screen and data analysis. AG was involved in facilitating and coordinating the microarray experiments. MZG was involved in the conception and coordination of this project. All authors approved the final manuscript.


Carriers of cystic fibrosis intron-8 5T alleles with high exon-9 skipping could have increased annual lung function decline and increased risk for asthma or chronic obstructive pulmonary disease (COPD).1 decline and risk of asthma or COPD.We genotyped 9131 individuals from the adult Danish population for cystic fibrosis 5T, 7T, 9T, and F508del alleles, and examined associations between 11 different genotype combinations, and annual FEV1 decline, self-reported asthma, spirometry-defined COPD, or incidence of hospitalization from asthma or COPD. In 5T/7T heterozygotes vs. 7T homozygous controls we had 90% power to detect an increase in FEV1 decline of 8 ml, an odds ratio for self-reported asthma and spirometry-defined COPD of 1.9 and 1.7, and a hazard ratio for asthma and COPD hospitalization of 1.8 and 1.6, respectively. Both 5T homozygotes identified in the study showed evidence of asthma, while none of four 5T/F508del compound heterozygotes had severe pulmonary disease. 7T/9T individuals had annual decline in FEV1 of 19 ml compared with 21 ml in 7T homozygous controls (t-test:P = 0.03). 6.7% of 7T homozygotes without an F508del allele in the cystic fibrosis transmembrane conductance regulator gene reported asthma vs. 11% of 7T/9T individuals with an F508del allele (χ2:P = 0.01) and 40% of 7T homozygotes with an F508del allele (P = 0.04). 7T homozygotes with vs. without an F508del allele also had higher incidence of asthma hospitalization (log-rank:P = 0.003); unadjusted and adjusted equivalent hazard ratios for asthma hospitalization were 11 (95%CI:1.5–78) and 6.3 (0.84–47) in 7T homozygotes with vs. without an F508del allele.5T heterozygotes vs. 7T homozygous controls had no increase in annual FEVPolythymidine 5T heterozygosity is not associated with pulmonary dysfunction or disease in the adult Caucasian population. Furthermore, our results support that F508del heterozygosity is associated with increased asthma risk independently of the 5T allele. A putative genetic risk factor for asthma and COPD is the TR) gene -3. This TR) gene . The mosTR) gene ,5.CFTR gene and has mainly been associated with congenital bilateral absence of the vas deferens, a monosymptomatic form of cystic fibrosis [We previously showed that persons heterozygous for a F508del deletion are overrepresented among people with asthma ,6. Anothfibrosis . Howeverfibrosis -14. Becafibrosis ,9-24, cu1-antitrypsin have been studied as modifiers of cystic fibrosis lung disease [Three common alleles are known in the polythymidine tract, 5T, 7T, and 9T. The polythymidine tract is situated in intron-8 near the acceptor splice site for exon-9 ,26. The  disease -37 and c disease ,26.CFTR gene. We combined polythymidine and F508del genotypes [1 decline and risk of asthma or COPD. We also examined whether other common risk factors for lung disease or variations in the genes for mannose-binding lectin and α1-antitrypsin significantly add to risk of lung disease in 5T carriers.We hypothesised that carriers of the 5T allele have increased annual lung function decline and increased risk for asthma or COPD. To test this hypothesis, we genotyped 9131 individuals from the adult Danish population for the 5T, 7T, and 9T alleles in the cystic fibrosis conductance membrane regulator (CFTR) gene. Details of study procedures and some characteristics of non-responders are described elsewhere [Subjects participated in the 1976–78, 1981–83, and/or 1991–94 examination of the Copenhagen City Heart Study, a prospective epidemiological study initiated in 1976–78 . Particilsewhere ,39. MoreParticipants filled out a self-administered questionnaire, which was validated by the participant and an investigator on the day of attendance. Participants reported on long-term occupational exposure to dust or welding fumes, pulmonary symptoms , familial predisposition to asthma (having at least one sibling with asthma), smoking habits , type of smoking and daily tobacco consumption. An estimate of life-time tobacco exposure (in packyears) was calculated as: daily tobacco consumption (g) times duration of smoking (years) divided by 20 (g/pack). If at least once during the study period participants aswered "Yes" to the question "Do you suffer from asthma?", we recorded they had self-reported asthma. Medication for asthma / bronchitis was "Yes" to the question "Do you daily take medication for asthma / bronchitis?" Additional information on hospitalizations due to asthma  and COPD  was drawn from the Danish National Hospital Discharge Register from May 1st 1976 through December 31st 2000. We confirmed in the Danish National Hospital Discharge Register covering all hospital discharges in Denmark, that no participants in the sample were ever hospitalized for cystic fibrosis.1) and forced vital capacity (FVC) were measured with an electronic spirometer  at the 1976–78 and 1981–83 examinations and with a dry wedge spirometer  at the 1991–94 examination. At each examination, three sets of values were obtained, and as a criterion for correct performance of the procedure, at least two measurements of FEV1 and FVC differing by less than 5% had to be produced. The highest set of FEV1 and FVC were used in the analyses as percentage of predicted value using internally derived reference values based on a subsample of healthy never smokers [1 (ml/year) was calculated as FEV1 (ml) obtained at the latest measurement minus the FEV1 value obtained at the first measurement, times 365.25 divided by the number of days between the two measurements (in years-1). Spirometry defined COPD was FEV1<80% predicted and FEV1/FVC<0.7, excluding self-reported asthma [Forced expiratory volume in one second (FEVd asthma .CCGCCGCTGTGTGTGTGTGTGTGTTTTT-3' and 5'GCTTTCTCAAATAATTCCCC-HEX-3' (second step reaction) (mismatch underlined) [CFTR gene [Serine Protease Inhibitor-A1 gene [Mannose-Binding Lectin-2 gene [We amplified the polythymidine tract variants in intron-8 by nested polymerase chain reaction using the primerpairs: 5'-TAATGGATCATGGGCCATGT-3'and 5'-ACAGTGTTGAATGTGGTGCA-3' (first step reaction), and 5'-FTR gene , S and Z-A1 gene , and B, n-2 gene  were iden-2 gene ; expecte, which estimates allele and haplotype frequencies with and without allelic association. The linkage disequilibrium coefficient D was calculated as D = P22 - p2q2, where P22 is the observed frequency of the 9T/F508del haplotype, p2 is the frequency of the F508del allele in the general population and q2 is the population frequency of the 9T allele. The degree of linkage disequilibrium was expressed as D' = D/Dmax × 100%.Linkage disequilibrium between the 9T and F508del alleles was tested by the linkage utility program "EH" 2-test or analysis of variance (ANOVA) was used for overall comparisons between several genotypes; Pearson's or Fisher's Exact χ2-test were used for post-hoc two-genotype comparisons. The most common genotype combination in the population, 7T homozygosity without F508del, was used as reference group for statistical comparisons. We evaluated asthma and COPD prevalences between genotypes using unadjusted and adjusted logistic regression with Wald's test as a measure of significance; the adjusted model included gender, age at study entry (deciles), and packyears at study entry (never smokers and deciles). We evaluated asthma and COPD incidences between genotypes using the log-rank test [1 % predicted at study entry (deciles). We tested possible interactions between the 5T/7T genotype and smoking habits, long-term occupational exposure to dust or welding fumes, familial predisposition to asthma, α1-antitrypsin MS genotype, α1-antitrypsin MZ genotype, or mannose-binding lectin deficiency in predicting FEV1 at study entry in ANCOVA models.Statistical analysis was performed with SPSS; for power calculations, NCSS-PASS and StatMate were used. P < 0.05 on a two-sided test was considered significant. Pearson's χank test -44. Unadcystic fibrosis transmembrane conductance regulator gene, stratified for presence or absence of F508del heterozygosity. Among the 9,131 participants selected randomly from the Danish general population, 352 (3.9%) were 5T heterozygotes and 249 (2.7%) were F508del heterozygotes. Expected numbers of 5T and F508del heterozygotes according to the Hardy Weinberg equilibrium were 349 and 246, respectively. Allele frequencies did not differ from those predicted by the Hardy Weinberg equilibrium . The novel intron-8 polythymidine tract variant, the 6T allele [2-test: P < 0.001).Characteristics of participants are given in Table T allele , was ide1 did not differ between 5T heterozygotes or homozygotes vs. 7T homozygous controls ; eleven percent of 7T/9T individuals with F508del (χ2: P = 0.01) and 40% of 7T homozygotes with F508del (χ2: P = 0.04) had asthma vs. 6.7% of 7T homozygous controls (data not depicted). None of the other genotype combinations differed from 7T homozygous controls.Prevalence of self-reported asthma did not differ between 5T heterozygotes or homozygotes vs. 7T homozygous controls . However, self-reported asthma differed between genotypes overall (χUnadjusted odds ratios for self-reported asthma were 1.7 (95%CI:1.1–2.7) in 7T/9T individuals with F508del and 9.2 (1.5–55) in 7T homozygotes with F508del vs. 7T homozygous controls  and 6.3 (0.84–47) in 7T homozygotes with F508del vs. 7T homozygous controls. None of the other genotype combinations differed from 7T homozygous controls  (data not depicted). Unadjusted and adjusted odds ratios for spirometry defined COPD did not differ between genotypes  and did not differ between genotypes overall  and 0.49 (0.23–1.0) in 5T/7T individuals vs. 7T homozygous controls , α1-antitrypsin MZ genotype (P = 0.47), or mannose-binding lectin deficiency (P = 0.73) in predicting FEV1 % predicted at study entry.There was no interaction between 5T/7T genotype and smoking status (P = 0.78), occupational exposure to dust or welding fumes (P = 0.10), familial asthma (P = 0.37), α1 or risk of asthma or COPD in the adult Caucasian population; these results are independent of age, gender, tobacco smoking, and other potential confounders. Interestingly, however, both 5T homozygotes showed evidence of asthma. Furthermore, our results support that F508del heterozygosity is associated with increased asthma risk independently of the 5T allele.This study shows that polythymidine 5T heterozygosity is not associated with increased annual decline in FEVBecause 1 in 26 carries a 5T allele in this population, it is indeed important that 5T heterozygosity does not increase risk of obstructive lung disease in the population at-large. It appears that the 5T allele causes lung disease only in very rare circumstances -14, leavBecause all 5T/F508del compound heterozygotes were free from severe pulmonary disease, the 5T allele did not appear to explain our previous results ,6 sugges1-antitrypsin S and Z alleles. In line with this, we also observed no increased risk for pulmonary dysfunction in 5T carriers with α1-antitrypsin MS or MZ genotypes.To identify factors in the population that significantly add to risk of lung disease in 5T heterozygotes, we tested for interactions between 5T/7T genotype and potential risk factors for lung disease, but found no significant interactions. Garred  and cowo1 was reduced in 7T/9T individuals and incidence of COPD hospitalization was reduced in 5T/7T individuals. If correction for multiple comparisons was performed, these significant findings become nonsignificant. Therefore, and because reduced COPD risk in 5T/7T individuals is less biologically plausible, the findings are likely due to chance alone rather than representing real phenomena. Misclassification of genotypes is unlikely, because diagnoses were confirmed by sequencing a subsample of different poly-T variants.In the present study, bias caused by investigators' knowledge of disease or risk-factor status seems unlikely, because we selected from a general population and genotyped our sample without knowledge of disease status or lung function test results. Selection bias is possible if severe lung disease in some individuals with 5T genotypes prevented them from participating in our study; however, expected and observed numbers of these genotypes according to the Hardy-Weinberg equilibrium were similar. The 2.7% frequency of F508del heterozygosity found in this study is in accordance with the 2.9% frequency of F508del heterozygosity observed in another previous study of the Danish population . Annual 1 or risk of asthma or COPD in adults in this population-based study; however, both 5T homozygotes showed evidence of asthma. Furthermore, our results also support that F508del heterozygosity may be associated with increased asthma risk independently of the 5T allele.Polythymidine 5T heterozygosity was not associated with increased annual decline in FEVThe author(s) declare that they have no competing interests.Morten Dahl, Anne Tybjærg-Hansen, and Børge G. Nordestgaard carried out the genotyping and statistical analysis. Peter Lange helped collect the data and was involved in the statistical analysis. All investigators participated in designing the study and in writing the paper, and all authors read and approved the final version of the manuscript.


Type 2 diabetes (T2D) is a multifactorial disease in which environmental triggers interact with genetic variants in the predisposition to the disease. A number of common variants have been associated with T2D but our knowledge of their ability to predict T2D prospectively is limited.PPARG (P12A), CAPN10 (SNP43 and 44), KCNJ11 (E23K), UCP2 (−866G>A), and IRS1 (G972R) genes were studied for their ability to predict T2D in 2,293 individuals participating in the Botnia study in Finland. After a median follow-up of 6 y, 132 (6%) persons developed T2D. The hazard ratio for risk of developing T2D was 1.7  for the PPARG PP genotype, 1.5 (95% CI 1.0–2.2) for the CAPN10 SNP44 TT genotype, and 2.6 (95% CI 1.5–4.5) for the combination of PPARG and CAPN10 risk genotypes. In individuals with fasting plasma glucose ≥ 5.6 mmol/l and body mass index ≥ 30 kg/m2, the hazard ratio increased to 21.2 (95% CI 8.7–51.4) for the combination of the PPARG PP and CAPN10 SNP43/44 GG/TT genotypes as compared to those with the low-risk genotypes with normal fasting plasma glucose and body mass index < 30 kg/m2.By using a Cox proportional hazard model, common variants in the PPARG and CAPN10 genes predict future T2D. Genetic testing might become a future approach to identify individuals at risk of developing T2D.We demonstrate in a large prospective study that variants in the  In a large prospective study, Lyssenko and colleagues show that variants in the PPARG and CAPN10 genes can help predict whether a person will develop Type 2 diabetes.  Type 2 diabetes (T2D) is a multifactorial disease in which environmental triggers interact with genetic variants in the predisposition to the disease . T2D is PPARG P12A polymorphism is associated with enhanced insulin sensitivity and protects against T2D [(CAPN10) confer increased susceptibility to insulin resistance and T2D [(KCNJ11) forms together with the sulfonylurea receptor SUR1 (ABCC8), an octamer protein that regulates transmembrane potential and thereby glucose-stimulated insulin secretion in pancreatic β-cells. A E23K polymorphism in KCNJ11 has been associated with T2D [(IRS1) have been shown to have reduced insulin content in pancreatic islets [Although several candidate genes have been associated with T2D –7, many inst T2D ,9–11. Al and T2D . The ATPwith T2D –18. Carrc islets . Althougc islets ,21, two c islets ,23.(UCP2) in the analysis because some studies have associated a polymorphism in the promoter of the gene (UCP2 −866G>A) with increased risk of T2D and impaired insulin secretion [In addition to the genes listed above, we considered it worthwhile to also include the uncoupling protein 2 gene ecretion –27, wherecretion . Increasecretion .In this study, we tested variants in a number of candidate genes for T2D for their ability to predict diabetes in 2,293 individuals without diabetes participating in the Botnia prospective study in western Finland.n = 20) were excluded from the present study. Glucose tolerance was defined according to the current World Health Organization criteria [The Botnia study is a family-based study aiming to identify genes increasing susceptibility to T2D ,31. Detacriteria . All parIR) using a computer-based model [30 min fasting/ΔG30 min fasting); this index is also called the insulinogenic index. The disposition index was used to adjust insulin secretion for the degree of insulin resistance (insulinogenic index/HOMAIR).The participants' weight, height, waist and hip circumference, and blood pressure were measured as previously reported ,31. Bodyed model  and β-ceIRS1 G972R, or using the Multiplex SNaPshot kit  for single base pair extension on ABI 3100 (Applied Biosystems) for CAPN10 SNP43 and 44, or with an allelic discrimination assay-by-design method on ABI 7900 (Applied Biosystems) for KCNJ11 E23K, IRS1 G972R, and UCP2 −866G>A . Genotyping errors are an unlikely explanation for this deviation from equilibrium, because in genotyping 1,880 samples of CAPN10 SNP44 using two different methods  the concordance rate was 99%.Genotyping of SNPs was performed with a polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) method and agarose gel electrophoresis for  −866G>A . By rand2 test was used for comparison of group frequencies. Survival analysis was used to estimate the effect of genetic variants (risk and non-risk genotypes defined from previous studies) on the risk of developing T2D and shown by Kaplan–Meier survival curves as the distribution of age at onset . The risk of developing T2D was expressed as a hazard function  using an age-adjusted Cox proportional hazard regression model [Variables are presented as median (interquartile range). A χon model . The hazPPARG, GG genotype of CAPN10 SNP43, TT genotype of CAPN10 SNP44, EK/KK genotypes of KCNJ11, GR/RR genotypes of IRS1, GG genotype of UCP2). However, the risk TT genotype of CAPN10 SNP44 was in opposite direction compared to other studies [CAPN10 was significantly more frequent in patients with T2D than in control individuals. Therefore, in the present study we refer to the TT genotype of CAPN10 SNP44 as an at-risk genotype. Individuals with missing data were excluded from the analyses.Expected risk genotypes were defined according to earlier reports  defined as insulin secretion (disposition index) and insulin action (HOMAIR) and genotype (G) (1 = risk and 0 = non-risk) on age at onset of T2D were performed using the following Cox proportional hazards model: h(t) = h0(t)exp(β1P + β2G + β3P*G), in which h(t) is the hazard function and h0(t) is the baseline hazard function, with β1 and β2 measuring the univariate effects and β3 measuring the interaction. If there is an interaction (β3 ≠ 0), the HR for carriers and non-carriers of the risk genotype will not be the same. Thus, in different genotype carriers the HR of T2D associated with x units increase/decrease in the phenotypic value (P + x) is HR = exp(β1 + β3)*x for the risk genotype carriers and HR = exp(β1*x) for the non-risk genotype carriers. A logistic regression analysis was applied to explore the relationship between FPG and BMI, with genetic factor (defined as 1 = risk and 0 = non-risk) as dependent variable and FPG, BMI, and an interaction term as covariates. All statistical analyses were performed using Number Crunching Statistical Systems version 2004 , R . Two-sided p-values of less than 0.05 were considered statistically significant.Analyses of interaction between effect of phenotype 2, and 160 (7%) had both elevated FPG and BMI ≥ 30 kg/m2. Of the 2,293 persons included, 132 (6%)  developed diabetes during the follow-up period of 6 y (converters).In total, 2,293 persons  were included in the study . Of themPPARG P12A polymorphism were similar to those previously reported in Caucasians [p = 0.016)  (p < 0.001) estimated by the Cox model , suggesting a steeper increase in FPG for any increase in BMI in carriers of the risk genotype. Furthermore, we observed a significant interaction between the PPARG P12A polymorphism and HOMAIR (p = 0.004), indicating that with increasing insulin resistance [The allele and genotype frequencies of the ucasians , with 73= 0.016) ; Table 3k of T2D , we now < 0.001) . This cosistance  carriersD′ = 0.99, p < 0.001). Fifty percent of the participants had the risk genotype (GG) of SNP43 and 62.1% had the risk genotype (TT) of SNP44. A total of 534 (24%) individuals carried both the GG (SNP43) and the TT (SNP44) genotypes. Seventy (54.3%) of the converters had the GG (SNP43) genotype, while 91 (70.0%) had the TT (SNP44) genotype. While SNP43 had no effect on its own, the SNP44 TT genotype was associated with a moderately increased risk of T2D  (see p < 0.001) (see p < 0.001)  and HOMAIR , showing that the risk conferred by worsening of insulin sensitivity [The G allele of SNP43 and the C allele of SNP44 were in strong linkage disequilibrium (035) see ; Table 3001) see . In the sitivity  increaseUCP2 −866G>A variant. The GG genotype was associated with a modestly increased risk of T2D  (see p = 0.0057) . None of the other tested genotypes predicted significantly earlier onset of T2D.Fifty-eight (44.3%) of the converters were homozygous for the risk genotype (GG) of the 049) see ; Table 3 0.0057) . FurtherIRS1 G972R polymorphism. Whereas the R allele had no independent effect on T2D risk, it increased the risk of T2D in a dominant fashion (RR or RG versus GG) in participants with elevated FPG and BMI ≥ 30 kg/m2 to 9.3  (see Twenty-three (17.6%) converters carried the RR/RG genotypes of the 001) see .KCNJ11 E23K polymorphism, but these genotypes did not increase risk of future T2D, neither alone nor in combination with elevated FPG or high BMI. In line with previous findings of an effect of this variant on insulin secretion [KCNJ11 E23K polymorphism and the disposition index (p < 0.001), suggesting that the risk of T2D associated with a low disposition index [Ninety-six converters (73.8%) had the risk EK/KK genotypes of the ecretion –18, theron index  is increPPARG (PP) and CAPN10 SNP44 (TT), whereas 397 (18.2%) individuals had three risk genotypes: PPARG (PP), CAPN10 (TT), and UCP2 (GG). The effect of both the PPARG PP and CAPN10 SNP44 TT genotypes on the risk of subsequent T2D when present in the same individual was greater  than the individual risks   (see p < 0.001) (p = 0.041).In total, 1,028 (45.9%) individuals carried risk genotypes in both al risks . The eff1.7–6.8)  was incl001) see , with a < 0.001) . Also, tPPARG and CAPN10 genes increased the future risk for T2D, particularly in individuals with other risk factors. A question often raised about genetic association studies of polygenic diseases is whether the information can be used to predict the disease, since the risk conferred by the variant is usually rather modest (odds ratio < 1.2–1.3) [PPARG and SNP44 in CAPN10) [The key finding of the present study was that variants in the 1.2–1.3) . In T2D  CAPN10)  could prs) of developing T2D for members of the Botnia families is about three. However, this risk is greatest in obese (BMI ≥ 30 kg/m2) individuals with FPG above normal (≥5.6 mmol/l) and a family history of T2D [PPARG and CAPN10 variants, and particularly with their combination, gave almost the same strong prediction of subsequent T2D. These genotypes also influenced the relationship between BMI and FPG, i.e., in carriers of the risk genotypes there was a steeper increase in FPG for any given increase in BMI.The relative risk . The Nurse's Health Study also reported reduced risk for developing diabetes in carriers of the A allele [PPARG gene and diet, body weight, and insulin sensitivity in predicting risk of future T2D.The P12A polymorphism in  < 0.001 ,8. The Psitivity , particusitivity . In suppA allele . Most reA allele . These cA allele . This efA allele . It is tA allele . Taken tPPARG gene to predict future diabetes. A 5% misclassification would result in a decrease in HR to 1.31 , 10% to 1.18 , and 20% to 1.08 . Of course, there also could be other factors contributing, such as change in diabetes prevalence  between the time when the cases in the case-control study and the converters in the prospective study were diagnosed. Finally, our study was carried out in a high risk population of first degree relatives of patients with T2D.Notably, the HR in the present prospective study was higher than the odds ratios in previous case-control studies . The samCAPN10 gene explained the linkage to Chromosome 2q in Mexican-Americans represented the first successful positional cloning of a T2D gene [CAPN10 mRNA levels in skeletal muscle, which correlates with more severe insulin resistance [IR and the CAPN10 SNP43 supports this notion. The question arises whether the combined effect of variants in the PPPARG and CAPN10 genes on risk for future T2D can be solely explained by the variants' effect on insulin sensitivity. A combined effect of the two genes on both insulin secretion and action would be more plausible. However, the knowledge of molecular mechanisms by which calpain 10 would increase susceptibility to T2D is limited.The discovery that intronic SNPs in the T2D gene . It alsoT2D gene ,14. In oT2D gene ,14, in tT2D gene . HoweverT2D gene . Carriersistance . Our datUCP2 variant was a strong predictor of T2D with earlier onset. The promoter variant in the UCP2 gene has been associated with increased expression of the gene in adipose tissue [UCP2 mRNA levels in human pancreatic β-cells (which is not known), this could result in increased uncoupling and, in turn, in decreased formation of ATP and impaired insulin secretion.Impaired insulin secretion has been shown to predominate over insulin resistance in individuals with early onset T2D . In linee tissue . If thisKCNJ11 gene have not only been associated with T2D [KCNJ11 E23K variant did not significantly increase the risk for T2D in our study's participants. We have no explanation for this finding other than lack of power (the study had only 52% power to detect an effect of KCNJ11 E23K on risk of developing T2D) or the presence of other unidentified risk factors in the patients with manifest T2D. We did, however, observe an interaction between the EK and/or KK genotypes and impaired β-cell function, supporting a role in insulin secretion.Genetic variants in the with T2D ,18, but with T2D . Whereaswith T2D . The KCNIn conclusion, we show in a large observational prospective study that genetic variants in candidate genes can predict future T2D, particularly in association with conventional risk factors such as obesity and abnormal glucose tolerance. With accumulating data from prospective studies, it should be possible to define whether there will be a future role for these variants in genetic prediction of T2D and whether these variants will influence response to prevention or treatment.Although this study is, to our knowledge, the largest of its kind thus far, it still has limited power to detect an effect of low-frequency alleles. It is therefore obvious that larger studies with longer follow-up are needed to replicate the findings. One such resource will be the Malmö Diabetes Prevention cohort, in which 22,000 individuals have been followed for more than 20 y. They are presently being restudied to obtain DNA and information on whether they have developed diabetes or not. While waiting for these results, it will be important to create consortia to merge data from available prospective studies.Table S1(92 KB PDF).Click here for additional data file.Table S2(93 KB PDF).Click here for additional data file.http://www.ncbi.nlm.nih.gov/gquery/gquery.fcgi) accession numbers for the polymorphisms discussed in this paper are CAPN10 SNP43 (rs3792267), CAPN10 SNP44 (rs2975760), IRS1 G972R (rs1801278), KCNJ11 E23K (rs5219), PPARG P12A (rs1801282), and UCP2 −866G>A (rs659366).The NCBI Entrez  and CAPN10 (which is involved in modifying certain proteins), were associated with people having a higher chance of getting type 2 diabetes. This chance was increased substantially when the participants already had slightly raised blood glucose, and a high body mass index.They studied 2,293 people in Finland who were family members or spouses of people with type 2 diabetes, but who themselves did not have diabetes. They followed these people for up to 12 years, starting in 1990. In total, 132 of these individuals (6%) developed diabetes during this time. They found that changes in two of the genes, In some people, it does seem possible to use certain genes to predict whether a person will develop type 2 diabetes. However, environmental factors are also very important, and any risk is much increased in people who are already overweight.MedlinePlus has many links to pages of information on diabetes:http://www.nlm.nih.gov/medlineplus/ency/article/001214.htmThe Finnish Diabetes Association has information on diabetes in general and more specifically for Finland:http://www.diabetes.fi/english/


SH2D2A, interacts with and modulates Lck activity. Several transcript variants of TSAd mRNA exist, but their biological significance remains unknown. Here we examined expression of SH2D2A transcripts in activated CD4+ T cells and used the SH2D2A variants as tools to identify functionally important regions of TSAd.The activation induced T cell specific adapter protein (TSAd), encoded by ex vivo. Three interaction modes of TSAd with Lck were identified. TSAd aa239–256 conferred binding to the Lck-SH3 domain, whereas one or more of the four tyrosines within aa239–334 encoded by SH2D2A exon 7 was found to confer interaction with the Lck-SH2-domain. Finally the TSAd-SH2 domain was found to interact with Lck. The SH2D2A exon 7 encoding TSAd aa 239–334 was found to harbour information essential not only for TSAd interaction with Lck, but also for TSAd modulation of Lck activity and translocation of TSAd to the nucleus. All five SH2D2A transcripts were found to be expressed in CD3 stimulated CD4+ T cells.TSAd was found to interact with Lck in human CD4+ T cells ex vivo. Alternative splicing of exon 7 encoding aa239–334 results in loss of the majority of protein interaction motives of TSAd and yields truncated TSAd molecules with altered ability to modulate Lck activity. Whether TSAd is regulated through differential alternative splicing of the SH2D2A transcript remains to be determined.These data show that TSAd and Lck may interact through several different domains and that Lck TSAd interaction occurs in CD4+ T cells  When the human genome was sequenced, it was found to contain approximately 35 000 genes . Considein silico analysis of the mouse transcriptome revealed that alternative splicing preferentially adds or deletes domains in transcription factors that are important for DNA binding [Related proteins encoded by the same gene through alternative transcription may have opposing functions in the cell. For instance, transcription factors may exist as isoforms lacking particular DNA- or protein interacting domains. When such isoforms are expressed, they may inhibit the function of the full length protein . An in s binding .The function of a protein encoded by a particular gene may thus be regulated at the level of differential expression of various transcript variants dependent on the tissue or cell type, or the differentiation status and previous history of the cell . One proSH2D2A that encodes the T cell specific adapter protein (TSAd) [Previously, we reported the cloning of the immunoregulatory gene n (TSAd) ,14. Adapn (TSAd) . The funn (TSAd) -18 and hn (TSAd)  as well n (TSAd)  and vascn (TSAd) . Recentln (TSAd) . TSAd man (TSAd) ,23,24, an (TSAd) . Mice lan (TSAd) , but as n (TSAd) .AJ000553) variant encodes full length TSAd [AY763100) and -3 (GenBank:AY763098) variants encodes TSAd proteins shortened by 18 aa and 28 aa in the N-terminus, respectively. The SH2D2A-4 (GenBank:AY763099) variant encodes a 10 aa insertion in the SH2 domain of TSAd, whereas the SH2D2A-5 variant (GeneBank:AA063001) lacks the entire exon 7 [Five naturally occurring transcript variants of human TSAd exist. The SH2D2A-1  was not tyrosine phosphorylated in Jurkat T cells, nor in 293T cells when co-expressed with Lck figure  and 3B. in vivo. Furthermore, phosphorylation of TSAd by Lck is dependent on the presence of one or more of the four C-terminal tyrosines whereas the Lck-SH3 interaction site is dispensable.We previously demonstrated that TSAd interacts with Lck in Jurkat T cells stably expressing HA-tagged TSAd . Here weWe have previously reported that TSAd inhibits early T cell signalling by inhibiting Lck activity ,23. Lck The localisation of TSAd was initially found to be cytosolic , but wasAlternative transcription may extend the repertoire of functionally different proteins expressed from one single gene. In this study, alternative transcript variants of SH2D2A were used as a tool to identify functionally relevant structures of TSAd. We found that TSAd variants displayed different functional features, indicating that differential expression of SH2D2A transcript variants may contribute to the regulation of TSAd function.Our findings are summarised in figure In support of the notion that TSAd aa239–334 is important for TSAd function, a high stringency Scansite search  of possiPrevious studies on TSAd and murine homologue LAD supports the notion that motives in the aa239–334 are functionally important. We previously reported that Lck activity is modulated by the C-terminus of TSAd , and ChoDifferential regulation of the expression of functionally distinct alternate transcripts from a single gene may contribute to the regulation of the gene's function. We found that the alternative TSAd transcripts may constitute up to 15–30% of the total amount of SH2D2A mRNA in the CD4+ T cell. At the protein level, we showed that in CD4+ T cells, although the full length TSAd 52 kDa band dominate, also TSAd of 37 kDa lacking the aa239–334 is expressed. However, we did not reliably observe protein bands representing N-terminally truncated TSAd variants. When expressed in Jurkat T cells or 293T cells, each of the SH2D2A cDNAs produced TSAd molecules of expected lengths figure  and 4B–DSH2D2A gene in anti-CD3 stimulated CD4+ T cells. However, we have only examined expression of TSAd transcripts in one cell type under one stimulatory regimen. Physiological T cell activation also requires the presence of co-stimulatory signals delivered through CD28-B7 interactions. In addition, TSAd mRNA expression may be induced in CD4+ T cells via cAMP dependent signals [We were not able to demonstrate differential regulation of the different alternate transcripts of the  signals . Since t signals ,17, TSAd signals ,22 and eThe importance of the SH2 domain for nuclear translocation of TSAd was previously pointed out by Marti et al . Here wein vitro generated point mutation of the conserved arginine in the SH2 domain (R120K) ([The 10 aa insertion in the SH2 domain of the TSAd protein encoded by the SH2D2A-4 cDNA probably result in a misfolded SH2 domain. This variant behaved like TSAd harbouring (R120K) . Since this clone contains a partial cDNA, the cDNA was fused using mega PCR [The TSAd variants SH2D2A-1-4 were isolated from a cDNA library from activated CD8+ T cells , and clomega PCR  with theThe following monoclonal antibodies (mAbs) were used: anti-Zap-70 , anti-HA , anti-human CD3ε , anti-phosphotyrosine , anti-Lck  and anti-LAP 2 . The following polyclonal antibodies were used: anti-HA, anti-LAT and anti pY319 Zap-70 , and anti-TSAd antibodies raised against synthetic peptides of TSAd ,19. Seco®, Life Technologies™, The Netherlands) and antibiotics. Transfections of 2 × 107 Jurkat T cells in RPMI 1640 with 5% FCS with 5–30 μg plasmid DNA were performed using a BTX electroporator  at 200 V, 70 ms. Transfectants were cultured for 16–24 hours. 293T cells were transfected by Lipofectin (Invitrogen) in Optimem 1 medium (GIBCO BRL®) as described by the manufacturer.Primary resting CD4+ T cells were positively selected from peripheral blood samples from healthy blood donors as described  using an6 CD4+ T cells with 0.05–5 μM SiRNA were performed using the Amaxa electroporator [SiRNA against the human TSAd sequence p690 5'-GGA CCG AAG AAU CAA ACU U tt-3', antisense 5'-AAG UUU GAU UCU UCG GUC C tc-3' (Eurogentec S.A.), was also previously described . SiRNA doporator  and Huma7 cells/IP) were washed in RPMI 1640, resuspended and stimulated with 5 μg/ml anti-CD3 (OKT3) mAbs pr 5 × 107 cells for 2.5 min. Cells were lysed by adding an equal volume of 2 × lysis buffer  or 1% Igepal , 50 mM n-octyl-β-d-glucoside (Sigma-Aldrich), 20 mM Tris (pH 7.5), 100 mM NaCl, 50 mM NaF, 1 mM Na3VO4, 10 μg/ml of the protease inhibitors; leupeptin, pepstatin A, chymostatin and antipain . The 293T cells, Jurkat T cells or primary CD3/CD28 stimulated CD4+ T cells were washed with PBS and lysed in 1 × lysis buffer. Lysates were precleared 2–3× for 45 minutes with protein A/G Sepharose™  or protein G magnetic beads , incubated with the relevant antibodies for 45 minutes followed by protein A/G or protein G beads precipitation for 1 hour. Precipitates were washed 3× in 1 × lysis buffer. Primary CD3 stimulated CD4+ T cells were sonicated in 1 × lysis buffer. Immunoprecipitations or cell lysates were separated by 8–10% SDS-PAGE and blotted onto a polyvinylidine difluoride membrane (PVDF) . Blots were probed with the indicated antibodies in Tris buffered saline  with 0,1% Tween  and 3% bovine serum albumin (BSA)  or 3% skimmed milk (Sigma-Aldrich). Binding of antibodies to the target proteins were detected by secondary HRP-labelled antibodies and Super Signal® West Pico Stable Peroxide Solution , using either Kodak BioMax MR film  or Kodak Image Station 2000R (Kodak). Signal intensity of blots was analysed using Kodak 1D image Analysis software.Primary CD4+ T cells were stimulated using CD3 Dynabeads  or CD3/CD28 beads  when indicated. For immunoprecipitation (IP) experiments, Jurkat T cells , and purified on glutathione Sepharose beads (Amersham Biosciences). Cellular lysates prepared as described above were precleared 3× for 1 hour, each with a 1:1 mixture of GST-glutathion/4B Sepharose. Precleared lysates of 5–10 × 10Total RNA were extracted using Absolutely RNA kit  according to the manufacturers instructions. cDNA was synthesised from total mRNA using random nonamers and MMLV reverse transcriptase as described by the manufacturer .® Universal PCR Master Mix (Applied Biosystems). For the TSAd transcript specific primer/probe sets, each PCR reaction contained 600 nM of the primers and 200 nM of the probe as well as 1 × TaqMan® Universal PCR Master Mix. DNA amplification was performed with the following thermal cycling profile: initial incubation at 50°C for 2 minutes, initial denaturation at 95°C for 2 minutes, 40 cycles of amplification . Fluorescence data were collected during the annealing stage of amplification.Quantitative real-time PCR and subsequent data analysis were performed using the Mx4000 Quantitative PCR system (Stratagene) equipped with version 4.0 software. The custom made primers and probes specific for TSAd transcripts and Zap-70 are shown in table A standard curve in triplicate was constructed for each transcript variant of TSAd and Zap-70. Serial dilutions of plasmids containing an insert with either the relevant TSAd transcripts or Zap-70 were made containing 10000, 1000, 100 and 10 fg/μl of the plasmid. Quantity of TSAd was estimated relative to the quantity of Zap-70. The choice of Zap-70 as a housekeeping gene was based on our previous studies which have shown that the amount of Zap-70 is relatively stable during the various phases of T cell activation ,41. ExpeSG carried out most of the molecular genetic studies, and drafted the manuscript. VSG carried out part of the molecular genetic studies. KZD carried out the immunocytochemistry and the fluorescent microscopy. HSH carried out confocal microscopy. KMK carried out the quantization of alternative transcripts. KH carried out analysis of TSAd-SH2-Lck interaction. TL participated in the design of the study. AS conceived of the study, carried out part of the cloning and sequencing of constructs and participated in its design and coordination. All authors read and approved the final manuscript.


Certain types of potassium channels  are associated with the production of tumours in patients and in animals. We have now studied the expression pattern of the Eag1 channel in a large range of normal and tumour tissues from different collections utilising molecular biological and immunohistochemical techniques.The use of reverse transcription real-time PCR and specifically generated monoclonal anti-Eag1 antibodies showed that expression of the channel is normally limited to specific areas of the brain and to restricted cell populations throughout the body. Tumour samples, however, showed a significant overexpression of the channel with high frequency (up to 80% depending on the tissue source) regardless of the detection method .Inhibition of Eag1 expression in tumour cell lines reduced cell proliferation. Eag1 may therefore represent a promising target for the tailored treatment of human tumours. Furthermore, as normal cells expressing Eag1 are either protected by the blood-brain barrier or represent the terminal stage of normal differentiation, Eag1 based therapies could produce only minor side effects. Ion channels play key roles in cellular functions other than electrical signal transmission . In recein vitro (i.e. it confers loss of contact inhibition and increased growth rate) and increases the speed of growth and the invasiveness of tumours implanted into SCID mice in vivo )To generate a single chain, alkaline phosphatase (PhoA) fused antibody, the antibody cDNA cloning was achieved by first determining the subtype of the murine antibodies (IgG κ2b) using IsoStrip . Total RNA was prepared from the hybridoma cells  and translated into cDNA . Subsequently the genes of the light and heavy chains expressed by the hybridomas were amplified by PCR using Pwo polymerase  with the following primers:GTAACAACGCAGAGTACGCGGG and either TCATTTACCCGGAGACCGG (heavy chain) or CTAACACTCATTCCTGTTGAAGCTC (light chain).The PCR products were subcloned into pBKS+ and sequenced. The variable regions of both chains were identified by sequence comparison  and fusePrimer sequences were:TCTGGAGGTGGAGGTAGTGGGGGAGGAGGTTCAGATGTTGTGATGACCCAAACTCC and CCCACTACCTCCACCTCCAGAGCCTCCCCCTCCTGAGGAGACGGTGACTGAGG (heavy chain) andTCTGGAGGTGGAGGTAGTGGGGGAGGAGGTTCAGATGTTGTGATGACCCAAACTCC and GGCCTAATCGGCCCGTTTGATTTCCAGCTTGGTG (light chain). The fusion was then performed using the first and the last of these sequences.To produce the PhoA fusion protein, the single chain antibody fragment was amplified and a 5' SalI and a 3' NdeI restriction site introduced. This fragment was cloned (blunt end) into an EcoRV-digested pBKS+ vector prior to subcloning  into pQUANTagen(kx) which contains the alkaline phosphatase sequence. Finally, the single chain antibody fragment – PhoA fusion protein was subcloned into the expression vector pASK-iba2 The fusion protein was obtained from inclusion bodies produced in E. coli (triggered by treatment of anhydro-tetracycline 200 ng/ml). The cells were harvested in 100 mM Tris/HCl, 100 mM NaCl, pH 8.0, 8 M urea and sonicated at 0° until lysis was complete and the solution appeared clear. The lysate was stirred overnight at 4°C and then dialyzed (cut-off 30 kDa) against 6, 4, 2, 1 and 0 M urea in extraction buffer. The solution was then centrifuged at 18000 ×g and stored at 4°C with 0.02 % (w/v) sodium azide. The preparation was characterised by SDS-PAGE, PhoA activity and ELISA to determine the relative activity of the fusion protein compared to the whole murine antibody.Transfected cells growing on glass coverslips were fixed , permeabilised with Triton X100 , blocked  and incubated for 2 h with Eag1.62.mAb (1 μg/ml) or Eag1.33.mAb (1 μg/ml). The secondary antibody  was used at a 1:1000 dilution (30 min) and the preparations were examined by confocal microscopy.Two postnatal-day 21 Sprague Dawley rats were anesthetised with a mixture of ketamine HCl  and xylazine  at 0.1 ml/100 g body weight. The animals were transcardially perfused with 4% p-formaldehyde in 0.12 M phosphate buffer (pH 7.2). After perfusion, the brains were removed, fixed for an additional hour at 4°C, rinsed three times with PBS and stored overnight at 4°C. Coronal and sagittal sections (400–500 μm) were cut in cold PBS using a vibratome . Slices were incubated for 1 h with 10% normal goat serum in PBS, then with primary hEag1 antibody  in PBS overnight at 4°C and processed using the avidin biotin-peroxidase system . Antibody binding was visualised using 3'-3-diaminobenzidine tetrahydrochloride . Controls either omitted the primary antibody or incubated the primary antibody with the corresponding fusion protein  at 4°C for 24 h prior to following the procedure as described above. Sections were analysed with a Zeiss Axiophot microscope.For the immunohistochemical detection of Eag1 potassium channels in human tissues, formalin-fixed and paraffin-embedded biopsy specimens from our own archive, samples from the Manitoba Breast Tumor Bank [54] and multiple tissue arrays  were used. Optimal staining conditions and antibody dilutions were determined using formalin-fixed and paraffin-embedded tissue samples from human cerebral cortex. Antigen retrieval was performed in a microwave oven in 10 mM citrate buffer (pH 6.0) at 700 W for at least 15 min. Slides were incubated overnight in a humidified chamber at 4°C with Eag1.62.mAb followed by incubation with the Envision Peroxidase System and DAB . Two operators independently evaluated antigen expression, denoting it either as "negative" (0), "positive" (1) or "strongly positive" (2) as appropriate.For immunodetection of the single chain antibody fragment alkaline phosphatase fusion proteins, the previous protocol was modified to use the BCIP/NBT  substrate and the sample was counterstained with nuclear fast red . Two slides of different multiple tissue arrays were evaluated independently by two researchers using the scoring system described above.Processing of consecutive sections from the same block in different days resulted in identical staining, illustrating the reproducibility of the system.LAP and WS are shareholders of a company interested in the exploitation of ion channels as anticancer targets.All authors participated in the experimental design and interpretation, BH, RMW, FMQ, HK, AS, MER, SM, TS, MJ and LAP performed experiments, and BH, HJR, WS and LAP wrote the manuscript.


Here we provide evidence that at least some tRNA species can be imported into the nucleus of human cells and promote HIV-1 nuclear import.Infection of non-dividing cells is a biological property of HIV-1 crucial for virus transmission and AIDS pathogenesis. This property depends on nuclear import of the intracellular reverse transcription and pre-integration complexes (RTCs/PICs). To identify cellular factors involved in nuclear import of HIV-1 RTCs, cytosolic extracts were fractionated by chromatography and import activity examined by the nuclear import assay. A near-homogeneous fraction was obtained, which was active in inducing nuclear import of purified and labeled RTCs. The active fraction contained tRNAs, mostly with defective 3′ CCA ends. Such tRNAs promoted HIV-1 RTC nuclear import when synthesized in vitro. Active tRNAs were incorporated into and recovered from virus particles. Mutational analyses indicated that the anticodon loop mediated binding to the viral complex whereas the T-arm may interact with cellular factors involved in nuclear import. These tRNA species efficiently accumulated into the nucleus on their own in a energy- and temperature-dependent way. An HIV-1 mutant containing MLV  HIV-1 reverse transcription and pre-integration complexes (RTC) are imported into the nucleus, where they integrate into the cellular DNA; some tRNA species undergo nuclear re-uptake and promote HIV RTC nuclear import. The column was washed with five column volumes of buffer A and eluted with 15 column volumes of a 0 to 1 M NaCl linear gradient in buffer A. Fractions (0.1 ml) were collected, and peak fractions pooled, dialyzed overnight against 4 L of import buffer, and concentrated by centrifugation in Centricon YM-3. After normalization for protein concentration, fractions were tested in the nuclear import assay and analyzed by SDS-PAGE. To obtain the 60S NA, the concentrated active fraction after chromatography on Phenyl-Sepharose column  was treated with Proteinase K (10 μg/ml) at 37 °C, 30 min, and nucleic acids were extracted by phenol/chloroform and ethanol-precipitated.The active fraction was isolated from HSE in the following steps. A saturated solution of AS was added to HSE to a final concentration of 60%. The sample was stirred at 4 °C for 1–2 h and centrifuged for 20 min at 6,000×2, 10 mM DTT, 1 mM ATP, 15% DMSO, 26-U T4 RNA ligase , 10-μCi 5′-[32P]pCp). The reaction was stopped by addition of an equal volume of StopMix (8 M urea/50 mM EDTA) and directly loaded onto a 15% denaturing PAGE. 5′-end radiolabelled (by standard procedure recommended for T4 polynucleotide kinase) pBP322DNA-MspI (New England Biolabs) and oligonucleotide sizing markers  (Amersham) were used as standards. After electrophoresis, the gel was dried and visualised by Storm 860 PhosphoImager .60S NA, total RNA from HeLa cells, and 21mer siRNA were dephosphorylated . The reaction was stopped by phenol/chloroform extraction, and ethanol-precipitated RNA was then 3′-end radiolabelled  or SYBR Gold  staining according to the manufacturer's instructions. Nine bands of different sizes were excised from the gel and eluted overnight at 4 °C into 0.5× TBE, 0.3 M NaCl buffer. The RNA was recovered by ethanol precipitation in the presence of 20-μg/ml glycogen (Roche). All samples were re-suspended in RNAse-free import buffer, normalized for RNA concentration, and tested in the nuclear import assay.2, 0.2 mM ATP, 0.1-mg/ml acetylated BSA, 15% DMSO, 25-U T4 RNA ligase [Amersham]). The ligation reaction was stopped by addition of an equal volume of StopMix and directly loaded onto a long (24 cm) 15% denaturing PAGE. pBP322DNA-MspI (New England Biolabs) and oligonucleotide sizing markers  (Amersham) were used as a size standard. After electrophoresis, the gel was stained with SYBR Gold (Molecular Probes). Ligation efficiency was approximately 50%. The ligation product was recovered from the gel and concentrated by ethanol precipitation. The ligation product was mixed with 100 pM 3′ RT primer (GACTAGCTGGAATTCAAGGATGCGGTTAAA) and heated 2 min at 90 °C. Reverse transcription  in recommended buffer) was followed by RNAse H (Invitrogen) treatment. Synthesis of the first cDNA strand was monitored by a parallel RT reaction in the presence of [32P] dCTP. A poly C tail was added to the 3′ end of the first cDNA strand , and the reaction stopped by heat inactivation at 75 °C, 10 min. Formation of the poly C tail was monitored by a parallel terminal transferase reaction in the presence of Cy5-dCTP. The terminal transferase product was PCR amplified (with Pfu Taq polymerase) using RACE-G primer ) and the 3′ RT primer. The PCR products were gel purified by 7.5% non-denaturing PAGE, eluted, and ethanol precipitated. The PCR products were then directly ligated into pCR2.1-TOPO vector using TOPO TA cloning kit (Invitrogen). Colonies were screened by PCR and directly used for custom sequencing.The small RNAs contained in fraction 2 (Fr2) were cloned using a method modified from Elbashir et al. . A total3 labelled RTCs/cell, recombinant imp7 or small RNAs (concentrations indicated in figure legends), and an energy-regenerating system  in 30-μl final volume. Samples were washed in import buffer, fixed on ice for 5 min with 2% paraformaldehyde in import buffer, and analyzed directly by confocal microscopy. Images acquired by confocal microscopy were analyzed by MetaMorph software version 4.5r4  for quantification. Thresholding was performed to define areas to be analyzed. The threshold value was set so that no objects were visible in negative control samples. Integrated morphometric analysis  was performed on the visible objects in the test samples, and the total grey value divided by the number of nuclei per field , were washed in import buffer and permeabilized for 5 min on ice in import buffer containing 40-μg/ml digitonin . After two washes in import buffer, samples were incubated for 15 min at 37 °C in a tissue culture incubator in the presence of approximately 10er field .Figure S1(A) 15% denaturing PAGE of the 60S NA purified from the indicated cell lines followed by SYBR Gold staining (200 ng/lane). (B) Nuclear import of YOYO-1–labelled RTCs in permeabilized HeLa cells in the presence of 1× energy-regenerating system and 60S NA from the indicated cell line (100 ng) or buffer (ctr −). Scale bar indicates 25 μm.(3.1 KB TIF)Click here for additional data file.Figure S2Nuclear import was performed in permeabilized HeLa cells in the presence of fluorescent-labelled RTCs, 1× energy mix and buffer (ctr−) or 140-nm G3 RNA (test). Samples were fixed, then images were acquired by confocal microscopy and processed by Metamorph software version 4.5r4. The threshold signal of the starting image is adjusted to select all objects of interest (overlaid red cells) in both control and test samples (thresholding). Next, the image is “segmented” so that areas in which the pixel intensities are above the threshold range (red nucleoli and nuclear envelope) will be marked in the image (segmenting). Finally, the software integrates the signal from the areas shown in the segmentation images only (now shown in bright green and present mainly in the nucleoli) and gives the total grey value. This value is divided by the total number of nuclei in the field to give the final reading. Because only the bright green signal is integrated, the background cytoplasmic signal (darker green) is not included in the total grey value.(6.6 KB TIF)Click here for additional data file.Figure S3FISH was performed with an RTC-specific probe following nuclear import in permeabilized HeLa cells in the presence of 1× energy-regenerating system (RTC + E) and 140 nM G3 RNA (RTC + E + G3) or 140 nM B2 RNA (RTC + E + B2). Control experiments in the absence of RTCs (CTR−) were performed to test the specificity of the probe and samples were analyzed by confocal microscopy. Scale bar indicates 12 μm.(2.7 KB TIF)Click here for additional data file.Figure S45 MLV or HIV-1 RTCs/assay and buffer (buffer) or 280 nM G3 (G3) or B2 RNAs (B2). Images were acquired by confocal microscopy using the same settings.Nuclear import assay in permeabilized HeLa cells in the presence of 1× energy mix, approximately 10(2.5 KB TIF)Click here for additional data file.Figure S5(A) Nuclear import assays were performed in permeabilized HeLa cells in the presence of 1× energy mix, 1 μM fluorescent-labelled M9 and 1 μM transportin (M9 + TR) or 100 ng 60S NA (M9 + 60S NA), 100 ng fraction 2 (M9 + Fr2), 140nM (corresponding to 100 ng) G3 RNA (M9 + G3), 140 nM B2 RNA (M9 + B2), 672 nM G3-T RNA (M9 + G3T), 672 nM G3-T RNA, and 1 μM transportin (M9 + TR + G3 T). (B) Nuclear import assays were performed in parallel with experiments shown in (A) in the presence of labelled RTCs and buffer (ctr−) or 140 nM G3 RNA. Note that the settings of the confocal microscope were different between (A) and (B) because M9 produced a stronger fluorescence signal than RTCs.(956 KB TIF)Click here for additional data file.Figure S6(A) Quantification of RTC accumulation in HeLa nuclei following the import assay in the presence of 1× energy-regenerating system, 140 nM G3 RNA, and the mutant G3T RNA (with 3′ C end) at the concentrations indicated. Bars represent mean fluorescent value ± standard deviation of three independent experiments. (B) Same as (A), but the assay was performed in the presence of G3 T (filled triangles) and G3T + 3′ CCA end (empty triangles). Data are representative of two independent experiments. Images were acquired by confocal microscopy and analyzed by MetaMorph software version 4.5r4. Samples were threshold at the same values and the total fluorescence divided by the number of cells per field. Samples containing RTC + buffer were given an arbitrary value of 100%. At least 150 cells were counted per experiment.(1.0 KB TIF)Click here for additional data file.Protocol S1(33 KB DOC)Click here for additional data file.Table S1(38 KB DOC)Click here for additional data file.Table S2(55 KB DOC)Click here for additional data file.


TPH2 gene encodes the enzyme responsible for serotonin (5-HT) synthesis in the Central Nervous System (CNS). Stereotypic and repetitive behaviors are influenced by 5-HT, and initial studies report an association of TPH2 alleles with childhood-onset obsessive-compulsive disorder (OCD) and with autism. GLO1 encodes glyoxalase I, the enzyme which detoxifies α-oxoaldehydes such as methylglyoxal in all living cells. The A111E GLO1 protein variant, encoded by SNP C419A, was identifed in autopsied autistic brains and proposed to act as an autism susceptibility factor. Hyperserotoninemia, macrocephaly, and peptiduria represent some of the best-characterized endophenotypes in autism research.The TPH2 SNPs rs4570625 and rs4565946 were genotyped using the TaqMan assay; GLO1 SNP C419A was genotyped by PCR and allele-specific restriction digest. Family-based association analyses were performed by TDT and FBAT, case-control by χ2, endophenotypic analyses for 5-HT blood levels, cranial circumference and urinary peptide excretion rates by ANOVA and FBAT.Family-based and case-control association studies were performed on clinical samples drawn from 312 simplex and 29 multiplex families including 371 non-syndromic autistic patients and 156 unaffected siblings, as well as on 171 controls. TPH2 alleles and haplotypes are not significantly associated in our sample with autism , with any endophenotype, or with the presence/absence of prominent repetitive and stereotyped behaviors . Also GLO1 alleles display no association with autism , but unaffected siblings seemingly carry a protective gene variant marked by the A419 allele .TPH2 gene variants are unlikely to contribute to autism or to the presence/absence of prominent repetitive behaviors in our sample, although an influence on the intensity of these behaviors in autism cannot be excluded. GLO1 gene variants do not confer autism vulnerability in this sample, but allele A419 apparently carries a protective effect, spurring interest into functional correlates of the C419A SNP. TPH2 and GLO1 were initially assessed despite a relative lack of support by sib-pair analyses for their chromosomal localizations . These initial studies yielded promising evidence supporting TPH2 and GLO1 roles as autism vulnerability genes  and in intron 2 of the ectively . These Sectively , and dueectively ,16. SNPsrs4570625forward primer: ACACTCACACATTTGCATGCACreverse primer: CATTGACCAACTCCATTTTATGTTAATAAGCTreporter 1 (VIC) sequence: CTTGACATATTCTAATTTTreporter 2 (FAM) sequence: ACTTGACATATTATAATTTTrs4565946:forward primer: TCACTCTGCCATCAGCTAGTCAreverse primer: CTGAGGTCCAGATGGGTTAAATGGreporter 1(VIC) sequence: CTTAGCTAAGGCCCCGreporter 2 (FAM) sequence: CTTAGCTAAGACCCCGThe PCR amplification protocol for the TaqMan assays includes denaturation at 95°C for 10 min, followed by 40 cycles at 92°C for 15 sec, 60°C for 1 min, and 72°C for 45 sec, followed by elongation at 72°C for 5 min. The TaqMan assays were then read on a 7900HT Fast Real-Time PCR System and alleles were called using the SDS software .GLO1 SNP C419A was genotyped by PCR amplification of genomic DNA (80 ng) using the following primers GLO1-F [5'-TGTCCAGTACCTGTATTTACTG-3'] and GLO1-R [5'-GCAAACTTACCGAATCCTCG-3']. The PCR amplification protocol includes denaturation at 95°C for 3 min, followed by 35 cycles at 95°C for 30 sec, 57°C for 30 sec, and 72°C for 45 sec, followed by elongation at 72°C for 5 min. The PCR product was cut with BSMAI (5 U) at 55°C overnight and run in 2% agarose or 5% acrylamide gels, yielding different restriction patterns for the C allele (259 bp + 36 bp) and the A allele (198 bp + 61 bp + 36 bp). An alternative restriction pattern was also identified  in 3/171 (1.8%) controls, as well as in one father and in one unaffected sibling.The 2. Head circumference was measured in autistic patients and unaffected siblings by trained physicians using a non-stretchable plastic measuring tape graded in millimeters, placed over the maximum fronto-occipital head perimeter [Blood samples for 5-HT levels were obtained from all family members and centrifuged within 20 min of venipuncture at 140 g for 25 min at 4°C; 1 ml of supernatant  was stored at -80°C and assessed by HPLC, as described . Urinaryerimeter .2 statistics, following randomized selection of one patient per multiplex family. Single-marker and haplotype family-based association analyses were performed applying the transmission/disequilibrium test (TDT) [Hardy-Weinberg analyses were performed using the HWE program of the LINKUTIL software package . Case-cost (TDT)  using thst (TDT) ; only cost (TDT) ,30. All st (TDT) . Since tst (TDT) ,33. Two-TPH2 gene variants marked by these two SNPs also do not seem to influence whether at the time of clinical intake autistic patients display prominent motor or verbal stereotypies (table Both rs4570625 and rs4565946 are in Hardy-Weinberg equilibrium in autistic patients and in their first-degree relatives (data not shown). The two SNPs are in strong linkage disequilibrium , as previously found in other samples . Both TDGLO1 C419A SNP is in Hardy-Weinberg equilibrium in autistic patients and in their first-degree relatives (data not shown). Case-control, TDT and FBAT analyses provide no evidence of association between C419A alleles and autism  declare that they have no competing interests.RS participated in study design and performed the statistical analyses, VP carried out GLO1 genotypings, JH and FR performed TPH2 genotypings and commented on the manuscript, RMoe participated in study design for TPH2. Patient recruitment, clinical assessment and sample collections were performed by RMi and CB in Naples, PC, BM and ST in Rome, CS and RMe in Phoenix, ME in Troina. TP and SPA measured 5-HT blood levels, KLR determined urinary peptide excretion rates, and AMP contributed to study design, coordinated recruitment and data collection, and drafted the manuscript. Authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: