Phlebovirus, family Bunyaviridae) is a mosquito-borne zoonotic pathogen which can cause hemorrhagic fever, neurological disorders or blindness in humans, and a high rate of abortion in ruminants. MP-12 strain, a live-attenuated candidate vaccine, is attenuated in the M- and L-segments, but the S-segment retains the virulent phenotype. MP-12 was manufactured as an Investigational New Drug vaccine by using MRC-5 cells and encodes a functional NSs gene, the major virulence factor of RVFV which 1) induces a shutoff of the host transcription, 2) inhibits interferon (IFN)-β promoter activation, and 3) promotes the degradation of dsRNA-dependent protein kinase (PKR). MP-12 lacks a marker for differentiation of infected from vaccinated animals (DIVA). Although MP-12 lacking NSs works for DIVA, it does not replicate efficiently in type-I IFN-competent MRC-5 cells, while the use of type-I IFN-incompetent cells may negatively affect its genetic stability. To generate modified MP-12 vaccine candidates encoding a DIVA marker, while still replicating efficiently in MRC-5 cells, we generated recombinant MP-12 encoding Punta Toro virus Adames strain NSs (rMP12-PTNSs) or Sandfly fever Sicilian virus NSs (rMP12-SFSNSs) in place of MP-12 NSs. We have demonstrated that those recombinant MP-12 viruses inhibit IFN-β mRNA synthesis, yet do not promote the degradation of PKR. The rMP12-PTNSs, but not rMP12-SFSNSs, replicated more efficiently than recombinant MP-12 lacking NSs in MRC-5 cells. Mice vaccinated with rMP12-PTNSs or rMP12-SFSNSs induced neutralizing antibodies at a level equivalent to those vaccinated with MP-12, and were efficiently protected from wild-type RVFV challenge. The rMP12-PTNSs and rMP12-SFSNSs did not induce antibodies cross-reactive to anti-RVFV NSs antibody and are therefore applicable to DIVA. Thus, rMP12-PTNSs is highly efficacious, replicates efficiently in MRC-5 cells, and encodes a DIVA marker, all of which are important for vaccine development for Rift Valley fever.Rift Valley fever virus . Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic pathogen which can cause hemorrhagic fever, neurological disorders or blindness in humans and a high-rate abortion in ruminants. MP-12 strain, a live-attenuated candidate vaccine, is safe and immunogenic, but lacks a DIVA marker. In this study, we developed and characterized improved MP-12 viruses which encode a DIVA marker by replacing the virulence gene with that of serologically distinct viruses belonging to the same genera. The novel MP-12 variant with such DIVA marker was highly efficacious and replicated efficiently in human diploid cells for vaccine production, and will become alternative candidate vaccines of MP-12 for veterinary applications. Bunyaviridae, genus Phlebovirus, is a zoonotic pathogen transmitted by mosquitoes, and the causative agent for Rift Valley fever (RVF). RVF is characterized by a high rate of abortion and fetal malformation in pregnant ruminants, febrile illness in adult ruminants, and lethal acute hepatitis in newborn lambs Aedes mosquitoes which thus play a role in maintaining RVFV in endemic areas. Other mosquito species are also involved in RVFV transmission if RVFV-infected mosquito population increases subsequent to heavy rain fall or increase in mosquito habitats Rift Valley fever virus (RVFV), which belongs to the family RVFV has a tripartite negative-stranded RNA genome composed of Small (S)-, Medium (M)- and Large (L)-segments. The S-segment encodes N and NSs genes in an ambi-sense manner, the M-segment contains a single open reading frame (ORF) which encodes NSm, 78-kD protein, NSm-Gn, Gn, and Gc proteins from different AUGs and co-translational cleavage; and the L-segment encodes RNA-dependent RNA polymerase Aedes aegypti larvae (Aag2) cells Smithburn vaccine generated by mouse brain passages of RVFV Entebbe strain has been sold as a veterinary vaccine in South Africa, Kenya, Zimbabwe, Namibia, Egypt and Israel Phlebovirus genus consists of the sandfly fever group including serologically distinct Punta Toro serocomplex, Naples serocomplex, Icoaraci serocomplex, Frijoles serocomplex, Sicilian serocomplex, RVFV, and the Uukuniemi group In this study, we aimed to develop MP-12 variants which encode a DIVA marker and replicate efficiently in MRC-5 cells. Although recombinant MP-12 lacking NSs gene in the S-segment encodes a negative DIVA marker VeroE6 cells (ATCC CRL-1586), 293 cells (ATCC CRL-1573), MRC-5 cells (ATCC CCL-171) and MEF cells HpaI and SpeI restriction sites for cloning. The PCR fragments of PTV Adames or SFSV NSs were cloned into pProT7-S(+) The plasmid encoding anti-viral-sense of MP-12 S-segment at the downstream of the T7 promoter, pProT7-S(+), was described previously The recombinant MP-12 encoding NSs truncation or mutation were recovered by using a plasmid combination of pProT7-M(+), pProT7-L(+), pT7-IRES-vN, pT7-IRES-vL, pCAGGS-vG and either of pProT7-S(+)PTNSs or pProT7-S(+)SFSNSs. BHK/T7-9 cells were transfected with those plasmids as described previously Total RNA was extracted from infected or mock-infected cells using TRIzol reagent. Denatured RNA was separated on 1% denaturing agarose-formaldehyde gels and transferred onto a nylon membrane . Northern blot analysis was performed as described previously with strand-specific RNA probes to detect RVFV anti-sense S-segment/N mRNA, mouse IFN-β mRNA, or mouse ISG56 mRNA Western blot analysis was performed as described previously MRC-5 cells were infected with MP-12, rMP12-C13type, rMP12-PTNSs or rMP12-SFSNSs at a m.o.i of 0.01 at 37°C for 1 h, washed cells twice with media, and incubated at 37°C. Culture supernatants were collected at 0 , 24, 48, 72 and 96 hpi, and used for plaque assay The pcDNA3.1mycHisA plasmids encoding CAT in vitro synthesized RNA was performed by using TransIT-LT1 or TransIT-mRNA Transfection Kit  according to manufacturer's instructions, respectively.Transfection of plasmid DNA or 293 cells in 12-well plate were transfected with 1 µg of IFNb-pGL3 plasmids, 4×IRF3-luc plasmid, or pPRDII-luc plasmids in addition to 0.1 µg of pRL-SV40 plasmid. At 24 hours post transfection, cells were mock-infected or infected with 300 µl of SeV (100 U/ml), and mock-transfected or immediately transfected with 1 µg of in vitro synthesized RNA encoding CAT (control) or NSs of MP-12, PTV Adames strain or SFSV. Cells were collected at 16 hpi, and the relative luciferase activity was measured by Dual-Luciferase Reporter Assay System  according to manufacturer's instructions.The analysis of host general transcription suppression was described previously 5 pfu of MP-12 (n = 20), rMP12-NSR173A (n = 10), rMP12-PTNSs (n = 9) or rMP12-SFSNSs (n = 10). Those mice were challenged with 1×103 pfu of wt RVFV ZH501 strain (i.p) at 45 days post vaccination. The challenge experiment was performed at an animal biosafety level 4 facility at the UTMB Shope laboratory. Mice were observed for 21 days after challenge, and the body weight was monitored daily. Sera were collected at 1, 2, 3, and 42 days post vaccination , and at 21 days post wt RVFV challenge (cardiac puncture). Survival curves of mice (Kaplan-Meyer method) were analyzed by Graphpad Prism 5.03 program .For testing the efficacy of MP-12 NSs mutants, 5-week-old female CD1 outbred mice  were inoculated subcutaneously with PBS (mock) (n = 10) or 1×1080 was determined as described previously 80 neutralizing antibody titer.The PRNTE,coli, were coated onto 96-well ELISA plates overnight at 4°C at a concentration of 100 ng/well. After washing 3 times with PBS containing 0.1% Tween 20 (PBS-T), the wells were blocked with PBS-T containing 5% skim milk at 37°C for 2 hours. Then, wells were incubated with serum samples  at 37°C for 1 hour. Wells were washed for 3 times with PBS-T and reacted with HRP-conjugated anti-mouse IgG  at 37°C for 1 hour. After washing with PBS-T for 3 times, ABTS was added to wells. The plate was incubated at room temperature for 30 min, and the optical density (OD) at 405 nm was recorded. The cut-off value, 0.176, was defined as mean+2×standard deviation of 24 normal mouse serum samples (1∶400) for anti-N IgG, while the cut-off value of 0.204 was defined as mean+3×standard deviation of 24 normal mouse serum samples. The highest dilution of sera that made a OD value larger than the cut-off was designated as the anti-N antibody titer. Because the anti-NSs antibody level was low, an OD value of a 1∶100 dilution was used for demonstrating the presence of anti-NSs antibody.His-tagged RVFV N proteins Statistical analyses were performed by using the Graphpad Prism 5.03 program . Unpaired t-test or Mann-Whitney U-test was used for the comparison of two groups. Survival curves of mice were analyzed by log-rank (Mantel-Cox) test.Mouse studies were performed in facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) in accordance to the Animal Welfare Act, NIH guidelines, and US federal law. The animal protocol was approved by the University of Texas Medical Branch (UTMB) Institutional Animal Care and Use Committee (IACUC) (protocol #1007038). All the recombinant DNA and RVFV were created upon the approval of the Notification of Use by the Institutional Biosafety Committee at UTMB. The wt RVFV ZH501 strain was used at the Robert E. Shope BSL4 laboratory at the UTMB in accordance with NIH guidelines and US federal law.In this study, we aimed to develop a modified MP-12 vaccine encoding NSs derived from serologically distinct phleboviruses. We attempted to select the phlebovirus NSs which can inhibit type-I IFN induction. Using reverse genetics for RVFV MP-12 strain, we recovered recombinant MP-12 encoding NSs of PTV Adames strain (rMP12-PTNSs) or SFSV (rMP12-SFSNSs) Fig. 1A.We first tested the replication capability of those viruses in type-I IFN incompetent VeroE6 cells and mouse embryonic fibroblast (MEF) cells Fig. 2A.RVFV NSs promotes the degradation of PKR thus inhibiting the phosphorylation of eIF2α, which promotes viral protein synthesis We found that both the rMP12-PTNSs and rMP12-SFSNSs do not promote the degradation of PKR. To clarify whether the rMP12-PTNSs or rMP12-SFSNSs can induce host general transcription suppression, 293 cells were mock-infected or infected with MP-12, rMP12-PTNSs or rMP12-SFSNSs at a m.o.i of 3, and nascent RNA was labeled with 5-ethynyluridine (EU) We found that rMP12-PTNSs but not rMP12-SFSNSs induces host general transcription suppression. To know whether rMP12-PTNSs or rMP12-SFSNSs can inhibit IFN-β gene up-regulation, type-I IFN-competent MEF cells were mock-infected or infected with MP-12, rMP12-C13type, rMP12-PTNSs or rMP12-SFSNSs at a m.o.i of 3, and total RNA was extracted at 7 hpi. Accumulation of mouse IFN-β, ISG56 mRNA, RVFV antiviral-sense S-segment RNA and N mRNA was analyzed by Northern blot as described previously Renilla luciferase (rLuc) under the constitutively-active SV40 promoter was co-transfected with above plasmids. At 24 hours post transfection, cells were mock-infected or infected with 100 U/ml of Sendai virus (SeV), and immediately mock-transfected or transfected with in vitro synthesized RNA encoding either chloramphenicol acetyl transferase (CAT) (control) or NSs of MP-12, PTV Adames strain or SFSV. Cells were collected at 16 hpi, and the relative luciferase activity was measured. When we defined the FFluc activities obtained from SeV-infected cells transfected with IFNb-pGL3, 4×IRF3-luc or pPRDII-luc as 100%, FFluc activities of mock-infected cells transfected with IFNb-pGL3, 4×IRF3-luc or pPRDII-luc showed 5.5%, 2.8% or 22% of the SeV-infected cells, respectively . On the other hand, rLuc activities of mock-infected cells transfected with pRL-SV40 (control plasmid) were similar or increased compared to those of SeV-infected cells transfected with pRL-SV40 . The results suggest that SeV infection specifically induces the activation of IFN-β gene, IRF-3 and NF-κB. Compared to CAT RNA control , cells transfected with MP-12 NSs RNA reduced the FFluc expression level derived from the IFN-β promoter , IRF-3  and NF-κB . Our results were consistent with the findings that RVFV NSs inhibits the general transcription factor TFIIH and induces general transcription suppression regardless of IRF-3 or NF-κB activation , left panels, yellow bars), IRF-3  and NF-κB . Interestingly, cells transfected with SFSV NSs RNA consistently increased the rLuc activity derived from the constitutively-active SV40 promoter . SFSV NSs reduced FFluc expression level derived from the IFN-β promoter  and IRF-3 , but not that from NF-κB . These results suggest that MP-12 NSs and PTV Adames NSs inhibit the reporter activities derived from the IFN-β promoter, IRF-3 and NF-κB, while SFSV NSs inhibits reporter activities derived from the IFN-β promoter and IRF-3 but not those from NF-κB. In addition, SFSV NSs seems to increase the expression of constitutively active genes through a currently unknown mechanism.To further study these observations, we used dual luciferase reporter assays to analyze the activation of IFN-β promoter and two critical transcription factors for IFN-β gene upregulation; IFN regulatory factor-3 (IRF-3) and Nuclear Factor-Kappa B (NF-κB), in the presence of MP-12, PTV or SFSV NSs 5 pfu of MP-12 (n = 20), rMP12-NSR173A (n = 10), rMP12-PTNSs (n = 9) or rMP12-SFSNSs (n = 10). We used outbred mice to evaluate the immunogenicity and efficacy of mice with different genetic background. Mice were monitored daily, and intraperitoneally challenged with 1×103 pfu of RVFV ZH501 strain at 45 days post vaccination. We performed mouse IFN-α ELISA using mouse sera at 1 day post vaccination as described previously Fig. S1). None of mice vaccinated with rMP12-NSsR173A or rMP12-PTNSs died before wt RVFV challenge. The result suggests that rMP12-SFSNSs retains neuroinvasiveness and neurovirulence similar to those of parental MP-12.Based on the experiments described above, we confirmed that rMP12-PTNSs induces host general transcription suppression, inhibits the up-regulation of IFN-β gene, and does not promote PKR degradation, while rMP12-SFSNSs inhibits the up-regulation of IFN-β gene, but does not induce host general transcription suppression or PKR degradation. Next, we tested the efficacy of MP-12, rMP12-PTNSs or rMP12-SFSNSs against wt RVFV challenge in outbred mice. We also tested the previously reported rMP12-NSsR173A, which encodes mutant MP-12 NSs R173A Fig. S2B), and developed 1∶2,560 of neutralizing antibody at 21 days post wt RVFV challenge (data not shown), while 7 to 20% drop in body weight was observed before euthanasia in 80% of mock-vaccinated moribund mice after wt RVFV challenge .In the challenge study, 90% of mock-vaccinated mice died within 8 days post infection, while 63%, 50%, 78% or 89% of mice vaccinated with MP-12, rMP12-NSR173A, rMP12-PTNSs or rMP12-SFSNSs were protected from wt RVFV challenge, respectively Fig. 7A.80) titer 1∶640 to 1∶2,560, and anti-N IgG titer 1∶800 to 1∶204,800 at 42 days post vaccination, and those mice all survived wt RVFV challenge . On the other hand, 10 (53%) mice did not develop neutralizing antibodies; 3 survived  and 7 died  after wt RVFV challenge. Among the 3 survivors, 2 developed anti-N IgG  and the other (#14-3) did not raise anti-N IgG. The mouse #14-3 showed temporal 16% body weight reduction at 7 days post challenge , and also developed neutralizing antibodies  at 21 days post challenge. On the other hand, rMP12-NSsR173A showed poor efficacy  , while 2 surviving mice (20%) had developed anti-N IgG  without neutralizing antibody . On the other hand, the remaining 5 mice (50%) died without the presence of neutralizing antibodies  . Together with previous observation that rMP12-NSsR173A does not efficiently accumulate viral proteins due to PKR-mediated eIF2α phosphorylation Among the mice vaccinated with MP-12, 9 (47%) mice developed both neutralizing antibodies; plaque reduction neutralizing test . The results suggest that rMP12-PTNSs and rMP12-SFSNSs have slightly higher efficacy than MP-12 and induce neutralizing antibodies at equivalent level to those induced by MP-12 in spite of a lack of PKR degradation function.Compared to vaccination with MP-12 or rMP12-NSsR173A, all mice vaccinated with rMP12-PTNSs or rMP12-SFSNSs developed anti-N IgG , Table 1Fig. S4). As shown in DIVA is important for vaccination of ruminants. Inclusion of negative DIVA marker helps to detect animals exposed to wt RVFV during outbreak. Unfortunately, MP-12 vaccine does not have a DIVA marker, and further improvement of MP-12 is required for an efficient detection of naturally infected animals during RVF outbreak. We determined whether mice vaccinated with rMP12-PTNSs or rMP12-SFSNSs can induce IgG reactive to RVFV NSs or not. For the purpose, we used IgG ELISA using C-terminus RVFV NSs Live-attenuated Smithburn vaccine, generated by serial passage of wt RVFV Entebbe strain in mouse brain, has been used in endemic areas as a veterinary vaccine since 1950s, and its genetic subpopulations have never been reported. Grobbelaar et al. suggested that the use of Smithburn strain in endemic countries could result in the spread of RVFV strain by multiple use of automatic syringes for both viremic and uninfected ruminants during outbreaks, and may have resulted in reassortment of Smithburn strain with circulating wt RVFV during an outbreak MP-12 vaccine strain was generated by serial 12-time plaque passages in MRC-5 cells in the presence of 5-fluorouracil –PTV Adames strain RVFV NSs promotes the degradation of PKR In summary, MP-12 mutants encoding NSs of PTV or SFSV are highly efficacious in mice and encode a DIVA marker, while rMP12-PTNSs also replicates efficiently in MRC-5 cells, which is useful for the vaccine manufacturing process. The immunogenicity and safety profile of rMP12-PTVNSs in ruminants and nonhuman primates will need to be tested to develop this virus as an alternative of MP-12 vaccine for veterinary and human use, respectively.Figure S1Viral antigen distributions in dead mouse vaccinated with rMP12-SFSNSs. Immunohistochemistry with anti-RVFV N antibody in (A) Hippocampus CA2 region, (B) Occipital cortex, (C) Medulla, (D) Liver and (E) Spleen. Cells reacting to anti-RVFV N antibody are indicated by arrows. Tissues of the euthanized mouse were fixed with 10% buffered formalin, and paraffin blocks were generated for pathological evaluation. Sections were incubated for 2 hours with anti-RVFV N rabbit polyclonal antibody (PDF)Click here for additional data file.Figure S2Body weight changes of mice mock-vaccinated or vaccinated with MP-12 or rMP12-NSsR173A. Body weight of mice was monitored from 0 to 21 days post wt RVFV ZH501 challenge. (A) mock-vaccinated dead mice, (B) mock-vaccinated survived mouse, (C) MP-12 vaccinated survived mice with neutralizing antibodies, (D) MP-12 vaccinated survived mice without neutralizing antibodies, (E) MP-12 vaccinated dead mice; neutralizing antibodies (−) and anti-N IgG (+), (F) MP-12 vaccinated dead mice; neutralizing antibodies (−) and anti-N IgG (−), (G) rMP12-R173A vaccinated survived mice with neutralizing antibodies, (H) rMP12-R173A vaccinated survived mice without neutralizing antibodies, (I) rMP12-R173A vaccinated dead mice; neutralizing antibodies (−) and anti-N IgG (+), (J) rMP12-R173A vaccinated dead mice; neutralizing antibodies (−) and anti-N IgG (−). Body weight was normalized (100%) to that at 0 day post wt RVFV challenge.(PDF)Click here for additional data file.Figure S3Body weight changes of mice vaccinated with rMP12-PTNSs or rMP12-SFSNSs. Body weight of mice was monitored from 0 to 21 days post wt RVFV ZH501 challenge. (A) rMP12-PTNSs vaccinated survived mice with neutralizing antibodies, (B) rMP12-PTNSs vaccinated dead mice without neutralizing antibodies, (C) rMP12-SFSNSs vaccinated survived mice with neutralizing antibodies, (D) rMP12-SFSNSs vaccinated survived or dead mice without neutralizing antibodies. Body weight was normalized (100%) to that at 0 day post wt RVFV challenge.(PDF)Click here for additional data file.Figure S4Sensitivity of IgG ELISA using GST-NSs (C-terminus). The sensitivity of IgG ELISA using purified GST-RVFV NSs (C-terminus) or purified Nus-RVFV NSs  to detect anti-NSs IgG by using (A) a mouse serum raised against mouse brain infected with RVFV  or (B) anti-NSs antibody, which was raised against peptide encoding the C-terminus (EESDDDGFVEVD) of RVFV NSs in rabbit (EZ BioLab). Purified GST and Nus were used as control antigens. The 96-well plate was coated with GST, GST-NSs, Nus, Nus-NSs (100 ng per well), and the reactivity of each antibody (2-fold dilutions) was measured by ELISA as described in materials and method.(PDF)Click here for additional data file.


Presence and origin of endemic foci of HTLV-2 infection in Africa remain a matter of debate.To better appreciate the epidemiological and molecular determinants of HTLV-2 infection in Central Africa, we performed a survey in 3903 inhabitants of a South Cameroon forest area, including 1051 Bakola Pygmies, 815 Baka Pygmies and 2037 Bantus living in their neighboring. HTLV-1 and HTLV-2 infection was determined by both specific serological (IFA and WB) and molecular (different generic and specific PCR) methods.HTLV-1/2 prevalence was of 3% (117/3903) with 90 HTLV-1 (2.3%) and 27 HTLV-2 (0.7%). Surprisingly, HTLV-2 infection was restricted to Bakola Pygmies (27/1051 2.5%) with no HTLV-2 infection in any of the 2852 Baka or Bantus individuals. In Bakola Pygmies, HTLV-2 seroprevalence increased with age, reaching 6.5% in the elder persons. Ongoing intrafamilial HTLV-2 transmission was evidenced. Lymphoid T cell lines  producing HTLV-2 antigens, were established from PBMCs cultures of HTLV-2 infected individuals. Sequences of a 672 nucleotide LTR fragment, obtained from 7 HTLV-2 samples, showed a very high degree of homologies among samples (< 1% nucleotide divergence) but also surprisingly with Amerindian HTLV-2 B strains. Complete sequence (8954 bp) of one isolate confirmed a typical HTLV-2 B strain.This study demonstrates clearly a HTLV-2 endemic population, with ongoing transmission, in Central Africa. Furthermore, it gives insights into several central questions regarding the origin and evolution rate of HTLV-2 and the migrations of infected populations.


Improved techniques to assist diagnosis and aid monitoring are required. We prospectively assessed 18FDG-PET/CT at disease activity, except where unstable, and at complete clinical and biochemical remission. 18FDG-PET/CT was evaluated blind to clinical status. Symptoms, C-reactive protein (CRP), HIV viral load (VL) in plasma and KSHV VL in peripheral blood mononuclear cells were assessed. Associations with 18FDG-PET/CT maximal standardized uptake value (SUVmax) were explored using Spearman correlations  or exact Wilcoxon rank sum .Patients enrolled on a natural history study of KSHV-MCD underwent 26 patients  were studied. In 3, we identified intercurrent lymphoma; these were excluded from the primary analysis. The remaining 23 underwent 19 studies during disease activity , and 21 studies at remission.18FDG-PET showed symmetrical hypermetabolic adenopathy  and increased splenic metabolic activity with splenomegaly . Marrow and hepatic abnormalities were less common and mild. In patients with laboratory manifestations only, 2 (66%) had mild splenomegaly and limited adenopathy and 1 (33%) isolated adenopathy.In symptomatic patients, max was 6 (2-8), and was associated with symptom severity (R=0.61 p=0.005), CRP  and KSHV VL , but not HIV VL (p=0.69). Intercurrent lymphomas (2 PEL and 1 diffuse large B-cell) demonstrated intensely hypermetabolic abnormalities involving restricted asymmetrical sites, with median SUVmax 11 (range 7–38). At remission, 11 (53%) had normal 18FDG-PET/CT; 10 (47%) had minor nodal abnormalities and 4 (19%) mildly increased splenic metabolism without splenomegaly. Intercurrent pathologies contributed to some abnormalities. One had progressive increase in splenic and nodal SUVmax over 3 scans  before relapse.During disease activity, median SUV18FDG-PET/CT demonstrated widespread nodal and splenic abnormalities during disease activity, improving with remission. Subclinical disease may also be detectable. Findings were distinguishable from suppressed HIV or intercurrent lymphoma by intermediate metabolic intensity and diffuse anatomic distribution. SUVmax was associated with symptom severity, systemic inflammation, and KSHV burden. 18FDG-PET/CT may be a useful non-invasive adjunct in the diagnosis and monitoring of KSHV-MCD.


Vaccines against DENV are currently unavailable. Guanylate-binding protein 1 (GBP1) is one of the Interferon (IFN) stimulated genes (ISGs) and has been shown important for host immune defense against various pathogens. However, the role of GBP1 during DENV infection remains unclarified. In this study, we evaluated the relevance of GBP1 to DENV infection in Gbp1 was dramatically upregulated in DENV-infected RAW264.7 cells. The intracellular DENV loads were significantly higher in Gbp1 silenced cells compared with controls. The expression levels of selective anti-viral cytokines were decreased in Gbp1 siRNA treated cells, while the transcription factor activity of NF-κB was impaired upon GBP1 silencing during infection.Quantitative RT-PCR (qRT-PCR) and Western blot showed that the expression of mouse Our data suggested that GBP1 plays an antiviral role during DENV infection. Dengue virus (DENV), a member of the mosquito-borne flavivirus family, is an icosahedral, enveloped virus with a single-stranded positive sense RNA genome. Millions of cases of DENV infection occur worldwide each year ,2. DenguInterferons (IFNs) are major antiviral cytokines released by host cells in response to viral and other pathogenic infections and play crucial roles in induction and regulation of both innate and adaptive immune responses . IFNs esin vitro and in vivo models [Gbp1 knockout mice indicated that GBP1 has strong anti-bacterial activity [in vitro models.Guanylate-binding protein 1 (GBP1) is one of the ISGs that most strongly induced by IFNs  and beloo models -15. For o models . Recentlactivity . There aactivity  and viruactivity -21. Howein vitro model for the study of host innate immune response to DENV. By using a quantitative RT-PCR (qRT-PCR) based small cDNA array , we measured the expression profile of genes from Jak-Stat signaling pathway in DENV  infected RAW264.7 cells. A number of genes were found up- or down-regulated upon DENV infection, as reported in our previous work  upon DENV infection . Among nfection , and thiGbp1 or scrambled control (N.C.) were delivered into RAW264.7 cells respectively via electroporation. 24hrs after siRNA transfection, cells were challenged by DENV (MOI=1.0) for another 24hrs. Cells were then harvested and total RNA and cDNA were made according to standard protocols. Gbp1 was silenced efficiently as analyzed by qRT-PCR  in Gbp1 silenced cells compared with control cells. To measure the production of infectious virus from these cells, a plaque assay was performed in 293T cells. The titers of virus in supernatants from Gbp1 silenced cells were about 10- fold higher compared with that from control cells , Interleukin-6 (Il6), Chemokine (C-X-C motif) ligand 1 (Cxcl1), Chemokine (C-X-C motif) ligand 2(Cxcl2), Chemokine (C-C motif) ligand 5(Ccl5) and C-C chemokine receptor type 5(Ccr5). Gene specific primers and probe sets were purchased from Applied Biosystems . After DENV infection, the transcription levels of Ifnβ1, Il6, and Ccl5 in Gbp1 silenced groups were decreased 2 to 3-fold comparing with controls (p<0.05). However, transcription levels of Cxcl1/2 and Ccr5 were not significantly affected  and AP1 (activator protein 1) play crucial roles in antiviral innate immune response through promoting the transcription of numerous antiviral or pro-inflammatory genes [Gbp1 silenced cells after DENV infection, we further measured the activities of NF-κB and AP1 upon GBP1 silencing. 293 cells can be robustly infected by DENV[GBP1 or scrambled control (N.C.), were delivered into 293T cells respectively by transfection using Lipofectamine® LTX & PLUS . 24hrs after transfection, cells were challenged by DENV (MOI=1.0) for another 24hrs, respectively. RNAi efficiency was confirmed by Western blot  ligand 1; Cxcl2: Chemokine (C-X-C motif) ligand 2; Ccl5: Chemokine (C-C motif) ligand 5; Ccr5: C-C chemokine receptor type 5; NF-κB: Nuclear factor kappa B; AP1: Activator protein 1.The authors declare that they have no competing interests.JD, WP and XS designed the experiments and prepared the manuscript. XZ, WP and TF performed all the experiments. All authors read and approved the final manuscript.


Caenorhabditis elegans proteins NID-1/nidogen and PTP-3A/LAR-RPTP function to efficiently localize the presynaptic scaffold protein SYD-2/α-liprin at active zones. Loss of function in these molecules results in defects in the size, morphology and spacing of neuromuscular junctions.The conserved v2-like voltage-gated calcium channel (VGCC) proteins, UNC-2 and UNC-36, and the calmodulin kinase II (CaMKII), UNC-43, function to regulate the size and morphology of presynaptic domains in C. elegans. Loss of function in unc-2, unc-36 or unc-43 resulted in slightly larger GABAergic neuromuscular junctions (NMJs), but could suppress the synaptic morphology defects found in nid-1/nidogen or ptp-3/LAR mutants. A gain-of-function mutation in unc-43 caused defects similar to those found in nid-1 mutants. Mutations in egl-19, Cav1-like, or cca-1, Cav3-like, α1 subunits, or the second α2/δ subunit, tag-180, did not suppress nid-1, suggesting a specific interaction between unc-2 and the synaptic extracellular matrix (ECM) component nidogen. Using a synaptic vesicle marker in time-lapse microscopy studies, we observed GABAergic motor neurons adding NMJ-like structures during late larval development. The synaptic bouton addition appeared to form in at least two ways: (1) de novo formation, where a cluster of vesicles appeared to coalesce, or (2) when a single punctum became enlarged and then divided to form two discrete fluorescent puncta. In comparison to wild type animals, we found unc-2 mutants exhibited reduced NMJ dynamics, with fewer observed divisions during a similar stage of development.Here we show that the CaC. elegans synaptogenesis. UNC-2 has a modest role in synapse formation, but a broader role in regulating dynamic changes in the size and morphology of synapses that occur during organismal development. During the late 4th larval stage (L4), wild type animals exhibit synaptic morphologies that are similar to those found in animals lacking NID-1/PTP-3 adhesion, as well as those with constitutive activation of UNC-43. Genetic evidence indicates that the VGCCs and the NID-1/PTP-3 adhesion complex provide opposing functions in synaptic development, suggesting that modulation of synaptic adhesion may underlie synapse development in C. elegans.We identified UNC-2/UNC-36 VGCCs and UNC-43/CaMKII as regulators of  Cav1 channels localize more broadly and have been associated with events including gene regulation, local translation and dendritic growth . Transgenic animals were generated by germ line transformation as described . All images were collected on either a Zeiss Pascal confocal microscope or an Olympus FV1000 confocal microscope equipped with Fluoview software . Images were acquired using multi-track parameters when necessary (unc-36 and unc-43 rescue), with either a 63X or 60X Plan-apochromat objective, respectively. Animals were anesthetized using 0.5% phenoxy-propanol  in M9 and mounted on 2% agarose pads. Measurements of SNB-1::GFP were as described with minor modifications [t-test, while double and triple mutant combinations were compared within the group using a Kruskal-Wallis test with a Dunn’s Multiple Comparison post hoc test. Circularity is a measure of how close to a perfect circle an object is, where 0 is a line and 1 is a perfect circle. The test relates the area of the observed object to the area of a circle with the same radius:Synapse morphology of D type neurons was visualized by ications . All imaunc-36(e251) defect we generated a PCR product including the putative promoter and endogenous 3′UTR using the following primers (unc-36promF1: 5′-ccacgtacatagaattcggaatc-3′ and unc-36 3′UTR R1: 5′- caaggcagttggaaagtcgac-3′). The PCR product was TOPO cloned into pCRXLII  to generate pBA234. pBA234 was injected at 10 ng/μl into unc-36(e251);juIs1 animals along with pPD118.33 (Pmyo-2::gfp) as a co-injection marker, plus pBA186 (Punc-25::mCherry) to mark the GABAergic motor neurons containing the transgene.To rescue the unc-36 coding region was amplified using the following primers (unc-36promF1: 5′-ccacgtacatagaattcggaatc-3′ and unc-36 3′UTR R1: 5′- caaggcagttggaaagtcgac-3′). The PCR product was T/A cloned into the pCR8/GW/TOPO vector (Life Technologies), and then was recombined using L/R clonase (Life Technologies) into pBA153, creating pEVL404 (Punc-25::unc-36). pEVL404 was injected into unc-36(e251);juIs1 animals at 10 ng/μl along with pPD118.33 (Pmyo-2::gfp) as a co-injection marker. For unc-43, we isolated a full-length cDNA for UNC-43D isoform by RT-PCR from wild type RNA isolated by Trizol using the following primers (unc-43cDNA F1: 5′-atgatgaacgcaagcacca-3′ and unc-43cDNA R1: 5′- ctagaattcagatactgttgtatttgttg-3′). Using the InFusion  enzyme this product was recombined into pEVL387 (Punc-25::mCherry::unc-43 3′ UTR) to generate pEVL400 (Punc-25::mCherry::unc-43E::unc-43 3′UTR). The pEVL400 plasmid was injected into unc-43(e408);juIs1 at 5 ng/μl. Any additional information about sequences or cloning procedures is available upon request.A genomic fragment covering the L4 animals were immobilized on 10% agarose pads in the presence of 5% (w/v) polystyrene beads . Animals were imaged at 5-minute intervals for 1 hour. Animals that died during the acquisition process  were excluded from the analysis. Images were then exported to ImageJ. Z-stacks were produced for each time point, and then times 0 and 60 minutes were thresholded, converted to masks and overlaid as false colored, green and red respectively. This allowed simple determination of spots that were added or removed, grew or shrank or divided during the analysis period. The total number of puncta added/lost during the hour session was determined by comparing initial time and final time points for the appearance or disappearance of puncta.E. coli to achieve the final desired concentration. Using plates with an increasing dose of gabapentin, we found that animals reared throughout development on NGM plates containing 100 μM gabapentin phenotypically resembled unc-36, appearing thin, with poor movement (data not shown), thus we used this concentration for our experiments. Plates were permitted to dry overnight and then 20 L4 animals of each genotype were placed on the media. Animals were imaged 24 hours later (as young adults).Gabapentin  was resuspended in DMSO at 100 mM, and then diluted in 1:10 in PBS and added to standard NGM plates seeded with OP50 CaMKII: calmodulin kinase II; CNS: central nervous system; ECM: extracellular matrix; GABA: γ-amino butyric acid; GOF: gain of function; LOF: loss of function; L4: 4th larval stage; NMJ: neuromuscular junction; S.E.M.: standard error of the mean; Syd: synapse defective; Unc: uncoordinated movement; VGCC: voltage-gated calcium channel; wt: wild type.The authors declare that they have no competing interests.ju493. BDA drafted the manuscript. All authors have read and approved the final manuscript.RCC and BDA carried out the molecular, genetic and cell biological studies and collected and analyzed the data. YJ and BDA designed and conducted the genetic screen to isolate unc-2/unc-36 and nid-1. (A) The egl-19 LOF allele, n582, animals had normal appearing SNB-1::GFP puncta. (B) In egl-19(n582); nid-1(cg119) the puncta appear enlarged and disorganized. (C) A deletion in the Cav3-like channel, cca-1, had no obvious effect on SNB-1:GFP morphology. (D) Removing cca-1 from nid-1 mutants did not suppress the morphological changes, as puncta were observed to be elongated and disorganized. (E) Loss of the second α2/δ subunit, tag-180, had only a modest effect on SNB-1::GFP puncta, but did not suppress the defects found in nid-1 mutants (F). (G) A plot of the puncta area measured by genotype as mean (circle) with the 5 to 95% confidence interval (whiskers). All animals in these micrographs are young adults ). Specificity of interaction between Click here for file


The Wnts are secreted proteins that play important roles in skeletal myogenesis, muscle fiber type diversification, neuromuscular junction formation and muscle stem cell function. How Wnt proteins orchestrate such diverse activities remains poorly understood. Canonical Wnt signaling stabilizes β-catenin, which subsequently translocate to the nucleus to activate the transcription of TCF/LEF family genes.We employed TCF-reporter mice and performed analysis of embryos and of muscle groups. We further isolated fetal myoblasts and performed cell and molecular analyses.in vivo data, constitutive overexpression of Wnt-1 or Wnt-3a inhibited the proliferation of both C2C12 and primary myoblasts. Surprisingly, Wnt-1 and Wnt-3a overexpression up-regulated BMP-4, and inhibition of BMP-4 by shRNA or recombinant Noggin protein rescued the myogenic inhibitory effect of Wnt-1 and Wnt-3a. Importantly, Wnt-3a or BMP-4 recombinant proteins promoted slow myosin heavy chain expression during myogenic differentiation of fetal myoblasts.We found that canonical Wnt signaling is strongly activated during fetal myogenesis and weakly activated in adult muscles limited to the slow myofibers. Muscle-specific transgenic expression of a stabilized β-catenin protein led to increased oxidative myofibers and reduced muscle mass, suggesting that canonical Wnt signaling promotes slow fiber types and inhibits myogenesis. By TCF-luciferase reporter assay, we identified Wnt-1 and Wnt-3a as potent activators of canonical Wnt signaling in myogenic progenitors. Consistent with These results demonstrate a novel interaction between canonical Wnt and BMP signaling that induces myogenic differentiation towards slow muscle phenotype. Skeletal muscles of the trunk and limb, except for some craniofacial and esophageal muscles, are derived from somites during embryonic development -3. Speci+ stem cells to undergo myogenic specification and differentiation [Wnt signaling is also involved in the regulation of postnatal satellite cell function and skeletal muscle regeneration. Satellite cells are muscle resident stem cells responsible for postnatal regeneration of injured muscles. During muscle regeneration, Wnt-5a and Wnt-7a induce muscle resident CD45ntiation . Wnt-7a ntiation ,15. Simintiation . Howeverntiation . Furtherntiation .Adult skeletal muscles contain heterogeneous types of muscle fibers that can be broadly divided into slow- and fast-twitch myofibers . In the Myofibers in postnatal skeletal muscles retain an adaptive capacity to switch between slow- and fast-twitch properties that largely depend on motoneuron activity . Wnt sigIn this study, we used transgenic Wnt-reporter mice and mice with constitutively activated canonical Wnt signaling specifically within skeletal muscle to investigate the function of canonical Wnt signaling in muscle. We found that Wnt signaling is highly activated in prenatal muscle and rapidly declines in postnatal muscle, with some residual activation limited to the slow myofibers. Interestingly, Wnt signaling is highly activated proximal to motor endplates of slow myofibers. Wnt activation induced by stabilized β-catenin inhibits muscle differentiation and promotes slow muscle determination. At the molecular level, canonical Wnt signaling induces BMP-4, which promotes expression of slow MyHC. These novel results demonstrate that interplay between Wnt and BMP signaling regulates skeletal myogenesis and muscle fiber type.Wnts cDNA subcloned from pLNCX-HA-Wnt constructs (kindly gifted from Dr. Jan Kitajewski) to pHAN-puro retrovirus vector by PCR ,28. Wnt-C2C12 cells are purchased from American Type Culture Collection (ATCC)  and cultured in DMEM with 10% FBS and antibiotics. Cells are induced to differentiation upon 80% confluence by serum withdrawal (DMEM with 2% horse serum). Primary myoblasts were isolated from the hind limb of two- to three–month-old wild type mice as previously described . Single Fetal myoblasts were prepared from the legs of Myf5-Cre/ROSA26-YFP embryos at E14.5. The legs are minced and digested by collagenase, dispase and DNaseI, as previously described for satellite cells isolation . After cTo prepare ecotropic retrovirus, Phoenix-eco packaging  cells were transfected with retrovirus vectors using GeneJuice . Viral supernatants were harvested 30 hours post transfection and used to infect C2C12 cells in the presence of polybrene  for 12 hours. Infected C2C12 cells were then washed twice with phosphate-buffered saline (PBS), maintained in growth media and were selected 24 hours post-infection with puromycin . Lentivirus was packaged in 293T cells (ATCC CRL-11268) transfected with the mouse BMP-4 or scramble shRNA pLKO.1-puro plasmids in addition to the pMD2.G and psPAX2 plasmids. Lentivirus was concentrated by ultracentrifuge and resolved in PBS(−) for virus infection. Infected C2C12 and primary myoblast cells were then washed twice with PBS, and maintained in growth media.Total RNAs were prepared from C2C12 and myoblast cells by TRIzol (Life Technologies). RNA samples were reverse transcribed using random hexamer and oligo dT mixed primers with SuperScriptII enzyme (Life Techonologies) according to the manufacturer’s instructions. Reverse transcription reactions were diluted (1:10) with 10 mM Tris, pH 8.0, yielding master samples of reverse-transcribed products. Real-time PCR reactions are previously described . Real-tiMyf5-Cre  heterozyC2C12 cells were incubated at 37°C for two hours with 10 μM BrdU, then washed with PBS(−) and fixed with 2% PFA/PBS. The fixed cells were stained with DAPI for 15 minutes at room temperature, washed with PBS(−), and refixed with 2% PFA for 5 minutes at room temperature. The refixed cells were treated with 2N HCl for 20 minutes at room temperature (RT), neutralized by washing with 0.1 M borate buffer pH 8.5. The cells were permeablized with 0.2% Triton X-100 PBS(−), blocked with broking buffer, incubated with anti-BrdU antibody for 2 to 12 hours. After staining with the primary antibody, cells were washed with PBS(−), stained with anti-mouse IgG1-Alexa488, washed with PBS(−) and mounted on slide glass with Dako mounting buffer. Myoblasts were fixed in 4% PFA/PBS (−) for 5 minutes, blocked with 10% goat serum/PBS (−) for 10 minutes, and stained with MyHC slow, MyHC fast, MyHC pan, anti-mouse IgG1-Alexa568, anti-mouseIgG2b-Alexa648 (Life Technologies) and DAPI (Sigma). Slides were mounted in SlowFade Light antifade Kit Component A (Molecular Probes) and analyzed with a Bio-Rad confocal laser scanning microscope (model MRC-1024) .2) containing 4.5 μl nitro-blue tetrazolium chloride (NBT) and 3.5 μl 5-bromo-4-chloro-3-indolyl phosphate (BCIP) per 1 ml of ALP buffer at 37°C. Isolated whole embryos and tissue were fixed in 2% PFA for 3 hours at 4°C and permeablized with X-gal staining buffer (0.1 M phoshate buffer (pH 7.3), 2 mM MgCl2, 0.01% sodium deoxycholate, 0.02% Nonidet P-40) for 2 hours at 37°C and stained by X-gal staining buffer with 0.1% X-gal, 5 mM potassium ferricyanide and 5 mM potassium ferrocyanide at 37°C for 2 to 24 hours.Cells were fixed 2% PFA and washed with PBS(−), then stained by alkaline phosphatase (ALP) buffer  compound and frozen in deep cold isopentane with ethanol and dry ice. The muscle tissues were cut 16 μm by cryostat and dried in room temperature. The muscle sections were incubated with NADH-TR staining solution (0.8 mg/ml NADH and 1 mg/ml NBT in 50 mM Tris–HCl (pH 7.6) at 37°C. The stained muscle sections were washed with deionized water, unbound NBT was removed by acetone solution, then the sections were re-washed with deionized water and mounted with a coverslip.Myoblasts in 24-well plates were transfected with the plasmids indicated and 50 ng pRL-PGK using Lipofectamine (Invitrogen). Transfected cells were harvested around 24 hours after transfection, and luciferase activities in the cell extracts were measured according to the manufacturer's instructions  in a luminometer . Luciferase activities as indicated by arbitrary unit were normalized by sea urchin luciferase activities in each sample. All experiments were repeated at least three times, and the averages of more than three independent experiments with standard deviations are shown as bars .Infected C2C12 cells were grown in 60-mm dishes, washed twice with PBS and lysed in 100 mL radioimmunoprecipitation assay (RIPA) buffer  containing protease inhibitor cocktail  . Cell exAs the first step to investigate the function of Wnt signaling in myogenesis, we used the TCF-lacZ transgenic reporter mouse to examine the activity of the canonical Wnt signaling pathway in embryonic and adult muscles. The promoter of the LacZ transgene  contains multimerized TCF binding sites , a key d+ myofibers co-expressed the slow MyHC, where only 8% of the β-gal- myofibers co-expressed slow MyHC  transgenic mice in which the exon 3 of β-catenin (Ctnnb) gene flanked by LoxP sites [Ctnnb exon 3, β-cateninΔEx3 is prevented from degradation (stabilized) and, therefore, constitutively active.To confirm the role of canonical Wnt signaling in muscle fiber type specification xP sites . The exoΔEx3 expression in myogenic progenitor cells. Myf5 is an early myogenic commitment marker during embryonic myogenesis [lox(ex3) mice die at E15.5 with extremely reduced muscle mass (data not shown), thus precluding further analysis of myofiber types. We next used MCK-Cre to drive β-cateninΔEx3 expression only in differentiated muscle cells. As expected, we detected increased activation of canonical Wnt signaling in vivo in transgenic mice carrying MCK-Cre, Ctnnblox(ex3) and TCF-LacZ alleles. β-gal activity was detectable in TA muscles of the MCK-Cre/Ctnnblox(ex3)/TCF-LacZ mice, but not the Ctnnblox(ex3) /Tcf-lacZ littermate controls  mice  compared to the same muscle of wild type littermates   staining. Strikingly, the high and middle oxidative myofibers were increased in TA muscles of MCK-Cre/ Ctnnbloxex mice , Wnt-2 and Wnt-10b moderately activated (>20 times increase in luciferase activity), and Wnt-2b and Wnt-4 weakly activated (>2 times increase in luciferase activity) the canonical Wnt reporter  had a significantly decreased cell number after 96 h in culture  that lacks the β-catenin binding site. As expected, DN-TCF-4b suppressed both Wnt-1 and Wnt-3a induced ALP activity in C2C12 cells  given its role in osteogenesis. Indeed, BMP-4 in C2C12 cells. This approach resulted in nearly 80% reduction of BMP-4 transcripts  expression by more than 70% , there were only a few MyHC positive myotubes in the C2C12 cells overexpressing Wnt-1 .Our analysis of canonical Wnt signaling in adult mature muscles reveal several interesting points. First, β-gal activity is only detectable in muscles known to contain slow myofibers. This result confirms our observation in the developing embryonic muscles. Second, in contrast to embryonic muscle, where β-gal activity is evenly distributed within myofibers, the highest β-gal activity was within the slow myofibers was proximal to the motor endplate Figure G-H. ThisPrevious studies demonstrate that Wnt molecules released by motor neurons play key roles in the development of neuromuscular junctions. Specifically, interaction of Wnt and LRP is necessary for clustering of postsynaptic acetylcholine receptors (AchR) . Our newLox(ex3) mice, we found that adult muscles indeed have higher canonical Wnt activity based on the TCF-LacZ reporter assay. This verifies the utility of our mouse model.Using Cre-inducible transgenic mice that express stabilizes β-catenin, we investigated the role of canonical Wnt signaling in embryonic myogenesis and postnatal muscle maintenance. When Myf5-Cre is used as the driver mouse, which is expressed in embryonic myogenic progenitors, we detected abnormal muscle development and perinatal lethality. This observation is consistent to recent studies using Pax7-Cre or Myogenin-Cre to stabilize β-catenin, which also results in lethality at P0 ,41. In tMck-Cre/ctnnbLox(ex3) model bypasses the premature lethality and provides novel insights of canonical Wnt signaling in regulating the oxidative capacity of adult muscles.Importantly, we found the adult fast (TA) muscles exhibited features of slow muscle phenotype as increased oxidative capacity. We also examined myosin heavy chain expression by immunohistochemistry but did not find any overt changes in myofiber types (data not shown). This result suggests that although Wnt signaling affects metabolic properties in the adult muscles, it is not sufficient to switch myosin heavy chain expression in the adult. This is expected since other factors, such as hormones and neural activity, can also influence myosin expression . TogetheThe observation that C2C12 cells overexpressing Wnt-1 and Wnt-3a exhibited reduced proliferation and myogenic differentiation is quite intriguing. It could suggest that canonical Wnt/β-catenin inhibits the proliferation and differentiation of myogenic cell lineages. This possibility would explain the reduced muscle mass phenotypes of the Myf5-Cre, Myogenin-Cre and Pax7-Cre induced stabilized β-catenin mice ,41. AlteInterestingly, embryonic and fetal myoblasts seem to have different responses to canonical Wnt signaling , suggestWe discovered a novel interaction between Wnt and BMP signaling in myoblasts. Bone morphogenetic proteins (BMPs) are multi-functional proteins belonging to the transforming growth factor beta (TGFβ) superfamily. In zebrafish and frogs, BMP signaling inhibits the differentiation of muscle precursors in the dermomyotome and controls the number of myogenic cells ,43. Duriin vivo. We further identify an unexpected role for BMP-4 in promoting slow muscle fate during fetal myogenesis. It is important to mention that low concentrations (1 to 5 ng/ml) of BMP-4 protein were used in our study. Non-physiological, high concentrations of BMP will probably generate completely different effects [Our cell culture results indicate that Wnt signaling induces BMP4, and BMP4 inhibition rescues the inhibitory effect of Wnt-1 and Wnt-3a on myogenesis. This result is in line with the above results  effects . Future Drosophila larval neuromuscular junctions, retrograde BMP signaling controls synaptic growth [Interestingly, in c growth . The musc growth . Taken tin vitro. Strikingly, canonical Wnt-signaling induced low level BMP-4 expression that act to induce slow myofibergenesis. Therefore, we conclude that canonical Wnt and BMP signaling plays a hitherto unappreciated role in myofiber type specification during fetal myogenesis.Our study demonstrates that canonical Wnt-signaling controls the development of skeletal muscles via BMP-4 expression. High concentrations of BMP-4 have been previously established to inhibit myogensis and induce osteogenesis. We found that isolated fetal myoblasts do not normally form slow myofibers during myogenic differentiation BMP: Bone morphogenetic protein; TCF: T-cell factor; LEF: Lymphoid enhancer-binding factor; Pax: Paired box protein; FBS: Fetal bovine serum; bFGF: Basic fibroblast growth factor; PFA: Paraformaldehyde; PGK: Phosphoglycerate kinase; GSK: Glycogen synthase kinase; MCK: Muscle creatine kinase; BrdU: Bromodeoxyuridine.The authors declare no competing interests.KK and MAR designed the research and wrote paper. SK performed the histology and tissue staining, and helped with paper writing. TMM provided the Ctnn1 exon3 floxed mice. All authors read and approved the final manuscript.The sequence of PCR primers for qPCR analysis is listed.Click here for fileA) Relative luciferase activity of C2C12 cells overexpressing various Wnt genes together with the SuperTop Flash reporter plasmid. (B) Graph of C2C12 cells transduced with retroviral Wnt plasmids. (C-E) The Ki-67 antibody staining and (F-H) BrdU incorporation of control, Wnt-1 and Wnt-3a transduced cells. (I) Percentage of Ki-67 positive cells in control, Wnt-1 and Wnt-3a transduced cells. (J) Percentage of BrdU incorporated cells in Control, Wnt-1 and Wnt-3a retrovirus infected C2C12 cells.Canonical Wnt signaling inhibits the growth and proliferation of C2C12 myoblast cells. (Click here for fileA-B) Ki-67 antibody staining of primary myoblasts treated with vehicle control (A) and Wnt-3a recombinant protein (50 ng/ml) (B). (C) Percentage of Ki67 positive cells. (D-E) BrdU incorporation of primary myoblast cells treated with control (D) and Wnt-3a protein (E). (F) Percentage of cells incorporated BrdU.Wnt-3a inhibits proliferation of adult primary myoblasts. (Click here for fileA-D) Immunofluorescence showing sarcomeric myosin heavy chain protein expression in C2C12 cells transduced with retrovirus overexpressing (A) Mock, (B) Wnt-1, (C) Wnt-3a and (D) Wnt-7a after four days of induced myogenic differentiation. (E) Western blotting analysis showing expression of myosin heavy chain (MyHC), myogenin and GAPDH in control, Wnt-1, Wnt-3a and Wnt-7a overexpression C2C12 myoblasts at different time points after induced to differentiate. GAPDH: Glyceraldehyde 3-phosphate dehydrogenase. Scale bar: 100 μm.Overexpression of Wnt-1 and Wnt-3a inhibits the myogenic differentiation of C2C12 myoblasts. (Click here for fileA-F) ALP staining  of C2C12 cells transduced retrovirus expressing empty vector (Mock) or Wnt vectors as indicated. (G) Relative ALP activity. (H) The dominant negative (DN) TCF-4 suppressed ALP activity in Wnt-1 and Wnt-3a expressing C2C12 cells.Canonical Wnts  induces alkaline phosphatase (ALP) activity in C2C12 cells. (Click here for fileA), forelimb (B) and hindlimb (C) of E14.5-15.5 Myf5-Cre/ROSA26-YFP embryo. (D) Strategy for isolating fetal myoblasts by FACS. Whole limbs of Myf5-Cre/ROSA26-YFP embryos were minced and digested by collagenase and dispase. Single cells were stained with alpha7-integrin antibody and selected by YFP and alpha7-integrin expression. The sorted YFP and alpha7-integrin double positive cells were stained with antibody for Pax7 and desmin, makers of fetal myoblasts (nuclei were counterstained by DAPI in blue).Isolation of fetal myoblasts from E14.5 embryos of Myf5-Cre/ROSA26-YFP mice by fluorescence activated cell sorting (FACS). (A-C) YFP expression in whole embryo (Click here for file


Reliable reference genes are a vital prerequisite for any functional study employing quantitative real-time RT-PCR (RT-qPCR) for analyzing gene expression. Yet a proper selection and assessment of the chosen reference genes is only rarely included into a study. To date, no reference genes have been validated for differentiation of THP-1 monocytes. Here we report on the selection of validated reference genes during differentiation of THP-1 monocytes into macrophages induced by phorbol 12-myristate 13-acetate (PMA).q ± SD, 14.1 ± 0.3) and RPL37A  (14.5 ± 0.3) as the most stable genes. While other frequently used reference genes such as GAPDH  (20.8 ± 0.8) or G6PD (glucose-6-phophate dehydrogenase) (16.1 ± 1.0) were found to be not as reliable and were therefore unsuited for use as reference genes. These findings were validated by investigating mRNA expression of macrophage scavenger receptor CD36, known to be regulated during monocyte-to-macrophage differentiation. Using ACTB and RPL37A as reference genes a profound and significant regulation of CD36 could be demonstrated, while use of G6PD resulted in a much less pronounced apparent regulation of CD36.The mRNA expression of 21 preselected potential reference genes was measured by RT-qPCR at several time-points over six days of PMA-induced THP-1 monocyte-to-macrophage differentiation. A ranking according to expression stability was calculated. Calculations were performed using Microsoft Excel-based applets GeNorm, NormFinder and BestKeeper. Our results indicated ACTB (β-actin) (CConsequently, it is recommended to normalize any real-time PCR-based expression data obtained during THP-1 monocyte differentiation using ACTB and RPL37A. Macrophages are the phagocytic cells of the immune system which play a pivotal role in many disease processes . Upon a Although pure human primary monocytes can be obtained either by dextran sedimentation followed by Ficoll density centrifugation , or leukPhorbol esters, such as phorbol 12-myristate 13-acetate (PMA), are frequently used to elicit the differentiation of THP-1 monocytes into macrophage-like cells which mimic many characteristic features of human primary macrophages ,9. The pMinimum Information for publication of Quantitative real-time PCR Experiments) guidelines propose reference gene validation for all RT-qPCR experiments  and GAPDH [20.8 ± 0.8]) . Since distinct model systems and experimental settings require individual validation of reference genes, we considered studies only closely resembling our own experimental setup. Three studies using a monocyte-to-macrophage maturation model involving either THP-1 or primary human cells were available   Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F: Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 2002, 3:research0034.1-0034.11.Click here for fileFigure S3. NormFinder analysis showing logarithmic intergroup and intragroup variances of the 21 preselected reference genes and CD36. NormFinder application was used to calculate inter- and intragroup variances as an estimate of gene stability [Reference [2] Lindbjerg CA, Jensen JL, Ørntoft TF: Normalization of real-time quantitative reverse transcription-PCR data: A model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res 2004, 64:5245-5250.Click here for fileTable S2. 260/280 and 260/230 ratios for assessment of RNA quality. RNA quality was assessed photometrically using an Eppendorf BioPhotometer plus.Click here for file


Hox genes such as HoxA9 is associated with development of myeloproliferative disorders and leukemia and indicates a poor prognosis. To investigate the molecular mechanisms by which HoxA9 promotes immortalization of hematopoietic cells, we generated growth factor dependent myeloid cells in which HoxA9 expression is regulated by administration of 4-hydroxy-tamoxifen. Maintenance of HoxA9 overexpression is required for continued cell survival and proliferation, even in the presence of growth factors. We show for the first time that maintenance of Bcl-2 expression is critical for HoxA9-dependent immortalization and influences the latency of HoxA9-dependent leukemia. Hematopoietic cells lacking Bcl-2 were not immortalized by HoxA9 in vitro. Furthermore, deletion of Bcl-2 delayed the onset and reduced the severity of HoxA9/Meis1 and MLL-AF9 leukemias. This is the first description of a molecular link between HoxA9 and the regulation of Bcl-2 family members in acute myeloid leukemia.Deregulated expression of  The Hox genes are a family of highly conserved transcriptional regulators with critical roles in normal development and hematopoiesis -3. ElevaHoxA9 overexpression maintains the self-renewal capacity of leukemic stem cells and blocks differentiation ,12, induMyb and Flt3 [Chromatin-immunoprecipitation and sequencing (ChIP-seq), combined with expression microarrays, have mapped potential HoxA9-regulated genes ,18. Thesand Flt3 .HoxA9−/− mice [Several lines of evidence indicate that HoxA9 regulates pathways that control cell survival. For example, increased apoptosis is observed in the fetal thymus of −/− mice  and sile−/− mice , suggestTo investigate how HoxA9 regulates cell viability, we used a lentiviral expression system to express HoxA9 in myeloid progenitor cells under the control of a 4-hydroxy-tamoxifen (4-OHT) inducible promoter. Downregulation of HoxA9 expression induced apoptosis in the majority of cells and was correlated with the loss of Bcl-2 expression. Moreover, deletion of Bcl-2 expression in HoxA9 factor-dependent cells induced apoptosis. In HoxA9/Meis1 and MLL-AF9-dependent AML, deletion of Bcl-2 dramatically altered clinical features, resulting in longer survival, diminished peripheral white cell counts and more destructive splenic disease. Together our data show that HoxA9 regulated Bcl-2 expression is important for the survival of myeloid progenitors and contributes to the oncogenic effects of HoxA9 overexpression.+/lin− myeloid progenitor cells with lentivirus encoding a Flag-tagged HoxA9 under the control of a 4-OHT-inducible promoter to generate HoxA9 Factor Dependent Myeloid (FDM) cells. For simplicity, we refer to the 4-OHT-induced expression of HoxA9 as HoxA9on, and the absence of 4-OHT as HoxA9off. Consistent with previous results [on and HoxA9off cells cultured in GM-CSF. In the HoxA9off state, a proportion of cells differentiated, as described by others [HoxA9 overexpression immortalizes myeloid progenitor cells dependent on cytokines such as IL-3 or GM-CSF. To explore the consequences of varying HoxA9 expression, we infected c-kit results , overexpy others , indicatoff cells were refractory to the proliferative signals transduced by IL-3 or GM-CSF, resulting in declined cell viability after 4 days in HoxA9off conditions , β common chain (βc or CD131), CD135 (Flt3) and reduced CD34 expression . We detef Figure . The expoff state, whereas Mcl-1 and BclxL expression remained unchanged and A1 expression was increased. Consistent with the changes in protein expression, Bcl-2 mRNA decreased in HoxA9off cells , Bcl-2+/− and Bcl-2−/− mice. After transduction with HoxA9 lentivirus and selection for infection, equal cell numbers of Bcl-2+/+, Bcl-2+/− and Bcl-2−/− cells were seeded in liquid culture containing cytokines and cell numbers were assessed over 9 days. The numbers of wild-type and Bcl-2+/− cells increased steadily, consistent with HoxA9-dependent immortalization  and counted over 56 days. Nup98-HoxA9 immortalized wild-type and Bcl-2−/− cells with similar efficiency (p15:p15) translocation, which fuses the nucleoporin a Figure  however,9 Figure . Howeverd Figure .L, Mcl-1 or Bcl-2 alleles flanked by LoxP sites [L, Mcl-1Bcl-x or Bcl-2 and viability was determined  -28. To eRecently, Valk et al studied the gene expression profile in 285 human AML patient samples , which win vivo, hematopoietic progenitor cells from C57BL6/Ly5.2 Bcl-2+/+(wt) or Bcl-2−/− embryos were transduced with retrovirus expressing HoxA9/Meis1, MLL-AF9 or GFP and then transplanted into irradiated C57BL6/Ly5.1 recipients. All mice that received Bcl-2+/+ cells transduced with HoxA9/Meis1 or MLL-AF9 developed rapid and aggressive leukemia with an average latency of 40 days  and Gr-1  were, to greater or lesser degrees, killed by ABT-737, as has been previously characterized . Howeverironment , one migDespite the complexity of genetic lesions that will invariably be present in any population of patients with AML , we obseBcl-2−/− blasts compared to those resident in the spleen and bone marrow may result from cell autonomous differences between these cell populations. Mcl-1 is absolutely required for the survival of the population of leukemic cells that permit serial transplantation of AML [The diminished numbers of peripheral n of AML , the so-n of AML ,35. The Bcl-2 expression has been previously observed. HoxA9−/− mice have reduced numbers of bone marrow myeloid and lymphoid progenitor cells [HoxA9−/− mice [Bcl-2. However many other transcription factors, including c-Myc and Myb are also known to promote Bcl-2 expression [Bcl-2 expression indirectly via these transcriptional regulators. Our results and others [Bcl-2 would be among the differently regulated genes.The association between HoxA9 and −/− mice . Definitpression ,37, and pression . Thus Hod others  show thad others . It willIn conclusion, we have identified Bcl-2 as a key protein mediating HoxA9-dependent immortalization. This adds Bcl-2-regulated apoptosis to the pathways that contribute to the oncogenic effects of HoxA9 overexpression. Deciphering the molecular links between HoxA9 and the regulation of Bcl-2 family members will greatly expand the potential therapeutic strategies that may be used in malignancies in which HoxA9 overexpression plays a key role.Bcl-2−/−, Bax−/−;Bak−/−, Mcl-1fl/fl, Bcl-xfl/fl, Bcl-2fl/fl and Rosa26-Cre ERT2) from which IL-3- or GM-CSF-dependent cell lines were derived, have been previously described [2 humidified atmosphere in DMEM low glucose media (Invitrogen) supplemented with 10% fetal calf serum, 50nM 4-OHT or 1ug/ml Doxycycline (Sigma) and 0.5ng/ml IL-3 and/or 2.5ng/ml GM-CSF (Peprotech). 293T cells and fibroblasts (Ψ2) cells were cultured in DMEM/10% FCS.Mice . The hexapeptide-mutant Flag-HoxA9 was cloned into the lentiviral inducible vector pF 5xUAS SV40 puro GEV16. MSCV-Nup98-HoxA9 retroviral vector was previously described [escribed  as was Hescribed . RetroviIL-3- or GM-CSF-dependent myeloid cell lines overexpressing Flag-HoxA9, HoxB8 or Nup98-HoxA9 were generated by retroviral or lentiviral transduction of hematopoietic progenitors from E14.5 fetal livers or bone marrow stem cells with the different constructs using a Retronectin-based protocol. MLL-AF9 tetracycline-repressible cells were generated by retroviral transduction of myeloid progenitor cells from E14.5 fetal livers with pMSCV-IRES-AtTA and pSIN-TREtight-Red2-ires-MLL-AF9 (a kind gift from Luke Dow and Scott Lowe). Three weeks after infection dsRed cells were sorted in culture in IMDM media containing 10%FCS and 5ng/ml IL-3. Infection and selection of cell lines are detailed in supplemental material and methods.2 humidified atmosphere in IMDM media supplemented with 10% fetal calf serum and 5ng/ml IL-3.All in vivo experiments were conducted in accordance with the guidelines of The Walter and Eliza Hall Institute Animal Ethics Committee. HoxA9/Meis1 retroviral construct and MLL-AF9 retroviral construct were previously described ,43. ViraCell viability was determined by flow cytometry analysis of Annexin V-FITC/PI stained cells, as previously described . Cell cyL pAb (R&D Systems). pAb anti-Bid, anti-Bmf and anti-A1 pAb were a kind gift from Andreas Strasser, Lorraine O'Reilly and Marco Herold respectively. ABT-737 and ABT-199 was a kind gift from David Huang supplied by AbbVie [Cells were lysed in sodium dodecyl sulfate (SDS) buffer  and boiled for 10 min. The antibodies used were; Anti-mouse Bcl-2 mAb (BD Biosciences), anti-Actin mAb and pAb anti-Bax and anti-Bak (Sigma), anti-Bad pAb , anti-Bim pAb (Stressgen), anti-Puma pAb (ProSci), anti-HoxA9 pAb and anti-MLL clone N4.4 mAb (Millipore), anti-Mcl-1 pAb (Rockland) and anti-Bcl-xy AbbVie ,47.Clonogenic assays were performed as previously described .Detailed methodology is described in supplemental Material and Methods.


Bcl-x levels are elevated in maturing erythroblasts, but not in their progenitors; truncated EPOR alleles that lack a major p85/PI3K recruitment site nonetheless promote polycythemia; and Erk1 disruption unexpectedly bolsters erythropoiesis. To discover novel EPO/EPOR action routes, global transcriptome analyses presently are applied to interrogate EPO/EPOR effects on primary bone marrow-derived CFUe-like progenitors. Overall, 160 EPO/EPOR target transcripts were significantly modulated 2-to 21.8-fold. A unique set of EPO-regulated survival factors included Lyl1, Gas5, Pim3, Pim1, Bim, Trib3 and Serpina 3g. EPO/EPOR-modulated cell cycle mediators included Cdc25a, Btg3, Cyclin-d2, p27-kip1, Cyclin-g2 and CyclinB1-IP-1. EPO regulation of signal transduction factors was also interestingly complex. For example, not only Socs3 plus Socs2 but also Spred2, Spred1 and Eaf1 were EPO-induced as negative-feedback components. Socs2, plus five additional targets, further proved to comprise new EPOR/Jak2/Stat5 response genes (which are important for erythropoiesis during anemia). Among receptors, an atypical TNF-receptor Tnfr-sf13c was up-modulated >5-fold by EPO. Functionally, Tnfr-sf13c ligation proved to both promote proerythroblast survival, and substantially enhance erythroblast formation. The EPOR therefore engages a sophisticated set of transcriptome response circuits, with Tnfr-sf13c deployed as one novel positive regulator of proerythroblast formation.Certain concepts concerning EPO/EPOR action modes have been challenged by in vivo studies:  As committed erythroid progenitors transit through a CFU-e stage, proerythroblast formation becomes dependent upon key signals transduced by EPO's cell surface receptor (EPOR). Interest in better understanding EPO effects (and EPOR action mechanisms) recently has intensified. This is based, in part, on the clinical emergence of new EPO orthologues and mimetics Erk1 is disrupted With regards to action mechanisms, the EPOR occurs pre-assembled with Jak2 kinase (as apparently paired dimer sets) Stat5 disruption per se yielded disparate results for erythropoietic roles Stat-5a and -5b loci, however, has since been shown to markedly compromise erythropoiesis Bcl-x previously was proposed to comprise one important EPO/EPOR- response factor whose anti-apoptotic actions might largely explain EPO's effects Bclx connection, and instead point to roles for Bcl-xL within maturing late-stage erythroblasts A third central EPOR signaling route involves a Jak2-plus-Stat5 axis which has been shown to be important for EPO-dependent erythropoiesis during anemia Podocalyxin as a proposed mediator of erythroblast adhesion/migration Cyclin G2 as an EPO/EPOR- repressed cell cycle inhibitor Serpina-3g as an EPO- induced candidate erythropoietic factor Tnfr-sf13cpos erythroblasts. Overall findings are discussed in the contexts of unique response circuits that the EPOR regulates within primary bone marrow progenitors to sustain the balanced production of red blood cells at steady-state, and over a dynamic range of rates during anemia.Towards advancing insight into EPO/EPOR action, our laboratory recently has applied basic gene profiling approaches to initially identify select EPO-modulated targets, and for these few factors has generated basic evidence for functional significance. Examples include Unless otherwise indicated, C57BL/6 mice (Jackson Laboratory) were used at age 8–14 weeks as a source of bone marrow erythroid progenitor cells (EPC's). Mice harboring the knocked-in minimal EPOR alleles EPOR-H, and EPOR-HM were as characterized by Menon et al Bone marrow cells were prepared from femurs and tibiae as previously described 6 cells were incubated  with 5 ug of rat IgG in PBS, 0.5% BSA (0.2 mL assay volumes). PE-Ter119, FITC anti-CD71, and APC-anti-Kit antibodies  then were added . Washed cells were analyzed by flow cytometry . In assays of cell survival, Annexin-V or YoPro3 were used as recently detailed In analyses of stage E1, E2 and/or E3 cells (and per 200 uL assay), 10In analyses of EPO/EPOR response genes, purified stage E1 CFUe- like progenitors were cultured in the absence of EPO for 5.5 hours in HEPES buffered IMDM supplemented with 10 µg/mL transferrin, 15 ng/mL insulin, 0.1 mM 2-mercaptoethanol, 0.5% BSA, 1.5mM glutamine. Cells were then exposed to EPO (4 U/mL) or carrier . At 90 minutes of exposure, cells  were lysed directly in Trizol reagent. RNA was then isolated and used to prepare biotinylated probes for array hybridizations. Gene profiling utilized Affymetrix 430 2.0 arrays, GeneChip 3000 scanning and initial GCOS software (Affymetrix) analyses of hybridization signals. Subsequent bioinformatics analyses utilized GeneSpring GX 11.0. Microarray data were assessed for background signals, normalized, and probe-summarized using a GC-RMA algorithm. Significantly expressed genes were identified using a Benjamini-Hochberg FDR filter. For EPO modulated genes, a p-value cut-off of 0.05 was used. K-means clustering utilized a Euclidean distance matrix. For significance testing between [−] EPO vs [+] EPO samples, Student's T-testing was used (single tailed). Analyses of transcription factor binding sites represented within K-means clusters #1 – #4 of EPO- modulated genes was via DiRE Reverse transcription (standard 1 ug of RNA per reaction) was with Superscript III (and included pre-treatment with DNAse I) (Invitrogen). Quantiative PCR utilized Sybr-green reagents and MyIQ single color real-time PCR detection system (BioRAD). Primer pairs were from SuperArray, and are defined in supplemental materials .Epo, or the EpoRposCD71highTer119neg progenitors (termed “stage E1”). posCD71highTer119neg cells . With regards to ligand concentration, EPO levels during anemia can increase several hundred-fold Pilot experiments next served to assess time-courses for EPO-induction of four known response genes as Matr3, Chac1, Ccrn4l, Socs2, Tnfr-sf13c and Rpl12. To critically assess the candidate nature of these targets as EPO/EPOR/Stat5 response genes, independent quantitative RT-PCR analyses were performed using CFUe-like progenitors isolated from mice harboring knocked-in EPOR-HM and EPOR-H alleles. The minimal EPOR form EPOR-HM is coupled to JAK2 but lacks all cytoplasmic PY sites, while EPOR-H retains a single PY343 site for Stat5 recruitment nd of 10 in indicated importance) . To inform further, all EPO- modulated genes within each cluster are listed in Cyclin-d2 was represented for five probe sets, and T-cell receptor-gamma for three probe sets (for example), data not shown. In addition, cluster #4 contained a number of EPO- modulated genes that previously have been implicated as STAT5 targets . STAT5a elements, however, were enriched in DiRE analyses of cluster #3   . As poinIn keeping with broadening concepts for EPO's effects Pim1 S/T kinase has previously been described. Pim3 (like Pim1) acts via an mTOR-like pathway Gas5, a non-coding RNA that supports mTor expression also was induced (2.6-fold). Lyl1 also comprised a novel EPO/EPOR target, and recently has been shown to act  to promote hematopoietic progenitor cell survival Bim was EPO/EPOR regulated . Finally (and as reported recently) Serpina-3g and the pseudokinase Trb3 each were strongly EPO-induced. Each has potential anti-apoptotic activities, and each comprises a singular EPO-modulated orthologue within multi-member families  (data not shown). Notably, modulation of the above EPO/EPOR-response genes  Cyclins G2 and D2, global transcriptome analyses revealed four additional EPO-modulated cell cycle regulators (Btg3 as an E2F-1 regulator p27/Kip1 as a repressed target (and CDK2 inhibitor) together with two regulators of phase G2 progression as Cdc25a and a Cyclin B interacting protein, Cyclin B-IP1. EPO therefore appears to engage a select collection of cell cycle regulators which overall are proposed to promote G1 and G2 phase progression within rapidly dividing CFUe-like progenitors. For the above cell cycle regulators . Tnfr-sc13c binds BAFF ligand, and as studied to date in B-cells Lrp8, a receptor for apolipoprotein-E ligands also was induced 1.9 – to 2.9 – fold. Interestingly, roles for Lrp8 in platelet function and neural cell migration recenty have been described. In stage-E1 EPC's, EPO also induced the expression of three cytokines: Cmtm6, Gdf3 and Oncostatin-m. Cmtm6 is a chemokine-like factor superfamily member with presently unknown function, while Gdf3 can act as a TGF/BMP antagonist (and may therefore counter inhibitory effects of TGFbeta on erythropoiesis). Oncostatin-m is a known EPO response gene, but recently has been shown to induce hepatocyte hepcidin expression, and to consequently decrease serum iron levels Next, with regards to an extended category of EPO/EPOR targets as additional signal transduction factors (STF's), global analyses revealed 22 such factors to be significantly modulated. Two STF sub-categories were cytokines plus receptors, and negative feedback factors  guiding as PMIDsSpred2, Spred1 and Eaf1. SOCS factors act as E3 ubiquitin ligases, and exert feedback effects on activated EPOR/Jak2 complexes Socs-3 but also Socs-2 in this feedback loop. Spreds are adaptor proteins that have been characterized as negative feedback factors in RTK systems EPO/EPOR STFs also included five negative feedback factors  . Tnfr-sf13c expression then was induced by exposure to EPO (2U/mL) for 4 hours. Cells were washed thrice, and replated in the presence or BAFF (as Tnfr-sf13c's ligand). In parallel, cells were exposed to EPO . At a subsequent 15 hour time point, possible effects on survival and/or Ter119pos erythroblast formation were assessed (by flow cytometry). Notably, BAFF ligand proved first to cytoprotect erythroid progenitors with an efficiency approaching that of EPO    . CFUe formation may be only modestly affected by BAFF-ligand based on apparent actions at a post-CFUe (pro)erythroblast stage. These experiments employing primary bone marrow erythroblasts provide direct evidence for BAFF-ligated Tnfr-sf13c in promoting erythroblast development, and therefore the actions of EPO. This especially applies to developing proerythroblasts with heightened Tnfr-sf13c levels . Factors indicated to regulate Tnfrsf13c also were limited. Cytokines included SDF1, IL4 and IL2 while transcription factors included STAT5b, Zfp3L2 and Pu1  . (For exiciency) . In colo Pu1 see . For eacPim, Socs, Spry, Spred and Bcl2 factors; no modulation of PodxL) (data not shown). Using a serum-free short-term ex vivo expansion system plus optimized rapid and efficient cell immunoadsorption procedures, sufficient primary stage-E1 bone marrow EPC's presently were prepared in sufficient numbers for robust quadruplicate profiling studies.Since the cloning of EPO and its receptor, the EPOR system has served well as a paradigm for HGF-R action Pias3) were confirmed. Second, for the >2-fold modulated set considered, PCA and clustering analyses did not reveal outlying factors. Third, when cluster-4 was independently analyzed, all EPOR targets tested were validated. In addition  six new EPOR-PY343/Stat5 response genes were independently confirmed including Tnfr-sf13c, Ccrn4l, Rpl12, Socs2, Chac1 and Matr3 as novel EPO-regulated factors. Confidence in our presently reported EPO/EPOR- regulated transcriptome is also reinforced via heat-map and p-value distribution analyses.One related basic point for consideration concerns the choice of stage-E1 CFUe-like cells as a target for EPO-action analyses. Previously, we have shown that with regards to survival effects, EPO can also cytoprotect stage E2 proerythroblasts and stage E3 erythroblasts, but less effectively than for highly-dependent E1 cells Gas5, and Lyl1. Gas5 is of interest in ways that relate to Pim-1 and -3. Specifically Gas5 can act as a non-coding RNA to regulate mTOR levels Bim (a BH3-only factor) is in-keeping with Bim's role as a Bclx antagonist EPO is best known to promote EPC survival, and expansion In maturing CFUe, cell cycle regulation is an atypical affair in that cell division rates accelerate markedly, become synchronous, and then sharply decrease as erythroblasts transition to reticulocytes Socs-3 as well as Socs-2 are shown to be induced by EPO. Spreds-1 and -2, as also induced by EPO, may well affect not only EPOR complexes, but also RTK's such as Kit and/or EphB4, each of which play erythropoietic roles In a broad category of additional signal transduction factors, one interesting subset of EPO/EPOR- regulated STF's is negative feedback factors. Among these, suppressors of cytokine signaling Socs-3 previously has been implicated as an EPOR/JAK2 complex inhibitor Cmtm6 as a CMTM cytokine Gdf3 as a candidate inhibitor of TGFbeta Oncostatin M as a newly discovered regulator of hepatic hepcidin and therefore of iron availability Tnfr-sf13C proved to comprise a strong EPO/EPOR response factor in EPC's and (as discussed below) this has proven to be of clear functional significance.By extended considerations, select EPO-induced transcriptional events also may result in erythroid cell extrinsic EPC effects. Examples are given by EPO- induction of Tnfr-sf13c is a major EPO-response gene in primary bone marrow EPC's, and ligation of Tnfr-sf13c results in not only cytoprotective effects, but also appears to bolster the formation of stage-E3 erythroblasts from stage E1 and E2 progenitors. Our profiling analyses of E1, E2 and E3 EPC's further indicate that these cells are not a significant source of BAFF as compared directly to primary bone marrow stromal cells (data not shown). Therefore, a novel functional connection is implicated between EPC's (via Tnfr-sf13c) and bone marrow stromal cells. It is of potential clinical interest to suggest that BAFF may comprise a rationale anti-anemia agent for possible use in combination with EPO. Via MGI Gene Expression Atlas, twenty-four cited experiments which include Tnfrsf13c indicate skewed expression among nine non-hematopoietic tissues (with 7 of 24 studies in brain).Tnfr-sf13c to date has been studied in B-cells and (together with its BAFF ligand) is essential for B-cell survival and development Eklf3 therefore might contribute to late erythroid development . Mllt3, a transcription factor recently shown in Mllt3-null and transgenic mice to regulate early stage erythro- and megakaryopoiesis Usp12 and Fbxw7. Usp12 is a deubiquitinating enzyme previously implicated in Fanconi anemia, while Fbxw7 (a substrate binding component of Ub ligase complexes) can act as a tumor suppressor in part by regulating Mcl1 levels Tim-9 and Tim-10 which act coordinately to facilitate transport of hydrophobic proteins to inner mitochondrial membranes Slc40a1, an iron transporter that may correspond to erythroid ferroportin A final set of observations that merit discussion involves additional functional subsets of targets presently discovered to be modulated by EPO. This includes up to 99 EPO modulated genes see , S7, S8.Figure S1Transcription factor binding site representation among EPO regulated genes within murine bone marrow- derived CFUe-like progenitors. For the EPO- modulated genes in K-means clusters #1 – #4 Click here for additional data file.Figure S2EPO- modulated survival factors, and cell cycle regulators in CFUe- like EPC's. Quantitative RT-PCR data for this sub-set of EPO- modulated transcripts are illustrated in bar-graph format.(TIF)Click here for additional data file.Figure S3EPO- modulated cytokines/receptors, and negative feedback factors in CFUe- like EPC's. Quantitative RT-PCR data for this subset of EPO- modulated transcripts are illustrated in bar-graph format.(TIF)Click here for additional data file.Figure S4BAFF inhibition of the apoptosis of primary bone marrow (pro)erythroblasts. Primary bone marrow erythroid progenitors were expanded (SP34ex culture). At day-3, EPC's (erythroid progenitor cells) were washed thrice, and returned to culture for 15 hours in the absence of SCF and EPO, but presence of BAFF at moderate [500ng/ml (+)], low [50 ng/ml (++)], or 0 ng/ml [-]. Frequencies of apoptotic EPC's then were determined by Annexin-V staining and flow cytometry. For gated Ter119pos erythroblasts, primary staining profiles are shown. .(TIF)Click here for additional data file.Table S1Quantitative Pcr Primer Pairs.(PDF)Click here for additional data file.Table S2Summary Of Epo- Modulated Genes Within K-Means Clusters #1 – #4, Including Candidate Transcription Factor Binding Sites (As Predicted Via Dire).(PDF)Click here for additional data file.Table S3Epo Modulated Transcription Factors.(PDF)Click here for additional data file.Table S4Epo/Epor Modulated Metabolism.(PDF)Click here for additional data file.Table S5Epo/Epor Modulated Cancer Biology.(PDF)Click here for additional data file.Table S6Epo/Epor Modulated Ribosome Biosynthesis.(PDF)Click here for additional data file.Table S7Epo/Epor Modulated Rna Processing.(PDF)Click here for additional data file.Table S8Epo/Epor Modulated Other.(PDF)Click here for additional data file.Table S9Candidate Regulators Of Tnfrsf13c Expression.(PDF)Click here for additional data file.


Respiratory navigated, ECG-gated, 3D-isotropic steady state free precession (3D-SSFP) is increasingly utilized in pediatric aortopathy. This is particularly true in adolescents as acoustic windows become limited. The agreement between echocardiography (ECHO) and 3D-SSFP obtained measurements could be influence by imaging plane, timing (systole versus diastole) and image quality. With 3D-SSFP the image quality is generally superior when acquisition is timed to diastole. As diastolic measurements are utilized as standard practice in adult aortopathy decision-making, whereas systolic diameters are standard in pediatric practice, examining the agreement between diastolic 3D-SSFP and ECHO (both systole and diastole) has merit. This study examined this question retrospectively.Echocardiographic and CMR data from 21 consecutive subjects referred for CMR, with and without heart disease were retrospectively reviewed. Subjects with >6 months between examinations (N=1) or unavailable ECHO data (N=5) were excluded. A standard 3D-SSFP sequence was utilized. A single, experienced observer used multiplanar reformatting to define a CMR plane that mirrored a standard parasternal long-axis ECHO plane. The maximum inner diameters at three levels ) were measured offline from 3D-SSFP and ECHO imaging data and analyzed by Bland-Altman method of agreement.The median age was 15.3 years (range 8-26 years). The median (range) for the diameters was 2.34 (1.86-3.35), 3.18 (2.59-4.18) and 2.48 (2.09-3.65) cm for annulus, root and STJ, respectively. Table Given the current definition of rapid progression as >5mm per year, acceptable agreement was found for 3D-SSFP versus ECHO in this retrospective cohort with a mean difference near zero and tight limits of agreement. Careful attention to matching the imaging plane utilizing 3D multiplanar reformatting is key to maximizing the agreement between the two imaging modalities.