D ~3–4 nM) and direct interaction between eEF1A and reverse transcriptase (RT). Biolayer interferometry analysis of cell lysates with titrated levels of eEF1A indicates it is a predominant cellular RT binding protein. Both the RT thumb and connection domains are required for interaction with eEF1A. A single amino acid mutation, W252A, within the thumb domain impaired co-IP between eEF1A and RT, and also significantly reduced the efficiency of late reverse transcription and virus replication when incorporated into infectious HIV-1. Molecular modeling analysis indicated that interaction between W252 and L303 are important for RT structure, and their mutation to alanine did not impair heterodimerisation, but negatively impacted interaction with eEF1A. Didemnin B, which specifically binds eEF1A, potently inhibited HIV-1 reverse transcription by greater than 2 logs at subnanomolar concentrations, especially affecting reverse transcription late DNA synthesis. Analysis showed reduced levels of RTCs from HIV-1-infected HEK293T treated with didemnin B compared to untreated cells. Interestingly, HIV-1 with a W252A RT mutation was resistant to didemnin B negative effects showing that didemnin B affects HIV-1 by targeting the RT-eEF1A interaction. The combined evidence indicates a direct interaction between eEF1A and RT is crucial for HIV reverse transcription and replication, and the RT-eEF1A interaction is a potential drug target.Reverse transcription is the central defining feature of HIV-1 replication. We previously reported that the cellular eukaryotic elongation factor 1 (eEF1) complex associates with the HIV-1 reverse transcription complex (RTC) and the association is important for late steps of reverse transcription. Here we show that association between the eEF1 and RTC complexes occurs by a strong and direct interaction between the subunit eEF1A and reverse transcriptase (RT). Using biolayer interferometry and co-immunoprecipitation (co-IP) assays, we show that association between the eEF1 and RTC complexes occurs by a strong , an abundant cellular protein, directly and strongly binds to the viral enzyme reverse transcriptase (RT). The biological relevance of the association is supported by mutational analysis of RT and by treating cells with the small molecule didemnin B that specifically binds eEF1A. Mutation of RT or treatment of cells with didemnin B resulted in significantly decreased efficiency of reverse transcription. Didemnin B treatment of cells disrupted HIV-1’s ability to maintain the viral machinery necessary for reverse transcription. However, an HIV-1 mutant, which does not interact with eEF1A, was resistant to didemnin B negative effects on early viral replication, showing that didemnin B affects HIV-1 by targeting the RT-eEF1A interaction. Altogether, this study demonstrates that eEF1A is an integral component of the viral reverse transcription complex and that the RT-eEF1A interaction is a possible new drug target to inhibit HIV-1 replication. Lys3, which anneals to a viral primer binding site sequence so that DNA synthesis can initiate. Reverse transcription can also be regulated by viral proteins such as Tat , and 0.5 mM β-mercaptoethanol) using a Dounce homogenizer. The lysate was cleared by centrifugation in a microfuge . The supernatant was incubated with anti-eEF1A rabbit antibody or rabbit IgG coupled Dynabeads Protein G  for 2 h at 4°C. The immunoprecipitate was washed three times with S100 buffer plus 0.1% Triton-x100 followed by western blot analysis using anti-FLAG, anti-V5 antibodies or a mouse anti-eEF1A antibody (Santa Cruz) as indicated. Where indicated western blots were probed with anti-HIV-1 immunoglobulin  and HIV-1 RT antibody (39/4.12.2) (Santa Cruz). The Agilent 2100 system and Protein 230 kits were used to evaluate protein lysate used in the study according to the manufacturer’s instructions. Some western blots were analyzed using ImageJ software [Co-IP was performed as elsewhere described . Brieflysoftware  followinsoftware .The plasmids containing wild type or mutant RT p66 in pDEST 40 vector  were transformed into BL21 AI bacterial cells . The cells were cultured to the density of OD600 ~0.4 and the protein expression was induced by the final concentration of 1mM IPTG and 0.2% L-arabinose . The protein purification was carried out using the HisLink protein purification system according the product protocol . The purified proteins were dialyzed using MINI dialysis unit  in PBS and examined by SDS PAGE and Coomassie blue staining.NL4.3 or HIV-1NL4.3-pol-W252A  at 4°C for 2 h to allow virus attachment. Heat-inactivated virus  were used as a negative control. The cells were then incubated at 37°C for 4 h to permit virus fusion with the cells and reverse transcription. Cells were fixed using 4% paraformaldehye followed by permeabilization using acetone. Duolink proximity ligation assays were performed using a mouse monoclonal antibody to detect HIV-1 RT in conjunction with a rabbit antibody to eEF1A (Santa Cruz). Otherwise PLA reactions were performed according to the manufacturer’s instructions (Sigma-Aldrich). DAPI stain was used to visualize the nuclei. Cells were visualized using a DeltaVision Core imaging system. Maximum-intensity projections of deconvolved images were analyzed using Duolink Image Tool software. More than 200 cells from at least 20 fields were analyzed for each experiment.PLA assay was performed as elsewhere described . Briefly−20. A total of 406 equivalent or related structures/domains were retrieved and the X-ray structure with the highest resolution among them (PDID B ID: 4G1Q) [A BLAST search  was undeD: 4G1Q)  that matin silico with the individual p51 and p66 subunits, and the p51/p66 heterodimer, independently. The ‘RepairPDB’ option followed by the ‘Mutate residue’ option of FoldX was used to calculate the stability change with default parameters . All resulting energies are expressed in kcal/mol. The result is the delta Gibbs free energy (ΔΔG) of mutation expressed in kcal/mol; average of 3 calculation runs is reported. The error margin of FoldX is ~0.5 kcal/mol, so changes in that range are considered insignificant. The prediction decision on whether the mutation stabilises or destabilises the structure is based upon [The empirical forcefield FoldX ,48 undersed upon ,48 wherehttp://www.pymol.org) was used for visualization and analysis.Inter and intra-protein residue interactions were determined using the Protein Interactions Calculator (PIC) . ASAView15 template following the manufacturer's instructions.HIV-1 CA in the culture supernatant was measured by using a RETROtek HIV-1 CAp24 antigen ELISA , according to the manufacturer's instructions. For RT analysis, after the addition of detergent to virions, an RT assay (Roche) was used to measure RT activity released from virions using a poly(A):oligo(dT)Did B was kindly provided by the Natural Products Branch, NCI , and CHX was purchased from Sigma Aldrich. Both chemicals were dissolved in DMSO for stock. To monitor translation of proteins, Jurkat cells were transfected with pCMV-Gluc 2 plasmid and seeded in 24-well plate. At 5 h post-transfection, serially diluted stocks of Did B and CHX were added at concentrations of 0.032, 0.16 and 0.8 nM. The luciferase activity in culture supernatant was measured after 8 h and 24 h of treatment using Biolux Gaussia luciferase Flex Assay kit (NEB). Cell proliferation was monitored using CellTiter 96 AQueous One Solution Cell Proliferation assay (Promega), as instructed by the manufacturer.TZM-b1 or Jurkat cells were treated with nevirapine at 1 μM/ml for 2 h incubated at 37°C and with HIV-1 (wild type or mutant) for 2 h at 4°C to facilitate virus attachment and then at 37°C for 2 h to initiate fusion and entry. Cellular nucleic acids were collected using Trizol reagent (Life Technologies) and the RNA fraction was column purified using a Direct-zol mini-prep kit (Zymo Research) and treated on the column with DNase I prior to elution. The purified RNAs were used in RT-PCR reactions both with and without SuperScript III reverse transcriptase (Life Technologies) using random hexamer oligonucleotides for the first strand DNA synthesis. Second strand DNA and PCR was performed using oligonucleotides specific for full length viral mRNA as previously described [The interaction of HIV RT p51 and p66 subunits was performed using MAPPIT system as previously described with modification ,20. BrieNL4.3 or HIV-1 NL4.3-pol-W252A stock for 2 h at 4°C with polybrene (80 μg/mL) and then incubated at 37°C for 4 h. Heat-inactivated virus were used as a negative control. The RTCs were prepared as previous described [Purification of HIV-1 RTCs was carried out as previously described , with miescribed  and placescribed  and by Et-test from at least three independent experiments or measurements. Statistical significance was set at p < 0.05.Statistical analyses were performed using the Student’s S1 FigBiotinylated (A) eEF1A1, (B) eEF1B, (C) eEF1D and (D) eEF1G were immobilized on biosensors. The association and dissociation with various concentrations of HIV RTp66/p51 were measured respectively on the OctetRed system. Data are representative of three independent experiments.(PPTX)Click here for additional data file.S2 FigBiotinylated eEF1A1 was immobilized on biosensors. The association and dissociation of eEF1A with various concentrations of HIV RT subunit (A) p51, (B) p66, (C) p66/p51 heterodimer and (D) Gag were measured with the OctetRed system. Data are representative of three independent experiments.(PPTX)Click here for additional data file.S3 FigFLAG-tagged RT51 was expressed in HEK293T cells by plasmid transfection. Cell lysates were prepared 24 h post-transfection and subjected to immunoprecipitation using an anti-FLAG antibody. The detection of eEF1A (A), eIF3A (B) and RT (C) in cell lysate (left column) and the co-immunoprecipitated eEF1A  were detected by western blot. A lysate from mock-transfected cells was used as a control.(PPTX)Click here for additional data file.S4 FigThe W252 is located in the loop region just before the first alpha-helix of the thumb domain and is shown in sphere representation (red) within each subunit.(PPTX)Click here for additional data file.S5 FigPurified wild type RT p66 and mutant RT W252A, L303A mutants were analyzed using SDS-PAGE followed by Coomassie blue staining (A). In BLI assay, purified eEF1A was immobilized on the biosensors and incubated in solutions containing purified RT. The association maximums of 90 nM of wild type RT and mutants was compared (B). The data is presented as a mean value ± standard deviation from 3 independent experiments. *p<0.05.(PPTX)Click here for additional data file.S6 FigA leptin receptor deficient for STAT3 recruitment (red) is C-terminally to a bait protein (orange), in this case either RTp66 or RTp51. A flexible spacer (green) separates the fused proteins. A prey protein (purple) is N-terminally fused to a gp130 chain with four functional STAT3 recruitment sites. In the presence of ligand, interaction between the bait and prey leads to complementation of STAT3 signaling and activation of a reporter luciferase gene expressed by the rPAP1 promoter.(PPTX)Click here for additional data file.S7 FigThe dimerization of RTp66 and RTp51 was investigated by MAPPIT. The pXP2d2-rPAP1-luciferase reporter plasmid was co-transfected with combinations of wild type or mutant, W252A (252A) and T253A (253A), RTp51 bait and RTp66 prey expression plasmids into HEK293T cells as shown. A bait construct having the myeloid differentiation primary response protein 88 (hMvD88) and a prey construct having SV40 large T antigen (SVT) were used as negative controls. Leptin (100 nM) was added to the cells at 20 h post-transfection and the cellular lysate was prepared 24 h later and used in luciferase assays. The data is presented as a mean value ± standard deviation from at least 3 independent experiments.(PPTX)Click here for additional data file.S8 Fig(A) The plasmid pCMV-Gluc2 was transfected into Jurkat cells for luciferase expression and Did B or CHX were added at concentrations as indicated. The levels of luciferase in culture supernatant were measured 24 h after treatment. (B) Jurkat cells were incubated with concentrations of Did B and CHX as indicated for 24 h at 37°C and then incubated with CellTiter 96 AQueous One Solution Cell Proliferation solution for 2 h at 37°C. The absorbance was measured at 490nm in a 96-well plate reader. The data is presented as a mean value ± standard deviation from at least 3 independent experiments. *p < 0.05(PPTX)Click here for additional data file.S9 FigIn vitro RT activity in the presence of Did B, CHX and nevirapine (NVP). RT (0.5 ng) was incubated with concentrations of Did B, CHX and NVP using the Roche Reverse Transcriptase Assay (colorimetric). The data is presented as a mean value ± standard deviation from at least 3 independent experiments.(A) The copy number of COXII in DNA samples extracted from Did B and CHX treated cells. Jurkat cells were incubated with various concentrations of Did B and CHX followed by HIV-1 infection. The cytoplasmic nucleic acids were extracted from cells and COXII levels were measured by qPCR. (B) (PPTX)Click here for additional data file.


Detection of advanced fibrosis (Metavir F≥3) is important to identify patients with a high urgency of antiviral treatments vs. those whose treatment could be deferred (F≤2). The aim was to assess the diagnostic value of novel serological extracellular matrix protein fragments as potential biomarkers for clinically significant and advanced fibrosis.Specific protein fragments of matrix metalloprotease degraded type I, III, IV and VI collagen  and type III and IV collagen formation (Pro-C3 and P4NP7S) were assessed in plasma from 403 chronic hepatitis C patients by specific ELISAs. Patients were stratified according to Metavir Fibrosis stage; F0 (n = 46), F1 (n = 161), F2 (n = 95), F3 (n = 44) and F4 (n = 33) based on liver biopsy.Pro-C3 was significantly elevated in patients with significant fibrosis (≥F2) compared to F0-F1 (p<0.05), while the markers C3M, C4M, C6M and P4NP7S were significantly elevated in patients with advanced fibrosis (≥F3) compared to F0-F2 (p<0.05). C1M showed no difference between fibrosis stages. Using Receiver Operating Characteristics analysis, the best marker for detecting ≥F2 and ≥F3 was Pro-C3 with AUC = 0.75 and AUC = 0.86. Combination of Pro-C3 and C4M with age, BMI and gender in a multiple ordered logistic regression model improved the diagnostic value for detecting ≥F2 and ≥F3 with AUC = 0.80 and AUC = 0.88.The Pro-C3 protein fragment provided clinically relevant diagnostic accuracy as a single marker of liver fibrosis. A model combining Pro-C3 and C4M along with patient’s age, body mass index and gender increased the diagnostic power for identifying clinically significant fibrosis. Chronic hepatitis C (HCV) affects approximately 170 million people worldwide, and represents a major healthcare problem due to the high risk for fibrosis development and consequently cirrhosis and hepatocellular carcinoma , 3. ConvVarious candidates have been proposed as biomarkers for liver fibrosis either alone or in combination, including FibroTest , HepaScoStructural changes in the extracellular matrix (ECM) as a reflection of matrix turnover are however especially attractive to stage liver fibrosis as the remodeling results in release of protein products related to both synthesis and degradation of the ECM –13. IndeWe hypothesized that measurement of both degradation and formation markers of ECM remodeling could provide an alternative to existing non-invasive serum markers to assess disease stage in a large cross-sectional study of patients with chronic HCV. The primary aim was to evaluate a panel of Protein Fingerprint markers to identify patients with a high urgency of antiviral treatments (Metavir Fibrosis ≥F3) and those whose treatment could be deferred (Metavir Fibrosis ≥F2) in patients with HCV compared to the traditional markers alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and the algorithms FIB-4 and AST to platelet ratio index (APRI). A secondary aim was to evaluate whether the Protein Fingerprint markers could differentiate patients with fibrosis (≥F1) from those without. The study found that a marker of true type III collagen formation (Pro-C3) provided clinically relevant diagnostic accuracy as a single marker of liver fibrosis. A model combining both formation and degradation markers along with patient’s age, body mass index and gender increased the diagnostic power for identifying clinically significant fibrosis.In a cross-sectional study-design we included chronic HCV infected patients referred to Westmead Hospital, Westmead, Australia, between July 1991 and August 2010 for the evaluation of HCV infection. Patient baseline characteristics have been reported as part of a recently published study . HCV infLiver biopsy was performed for assessment of severity of inflammation and fibrosis. The stained biopsies were evaluated by one expert pathologist (DM) blinded to patient clinical characteristics and serum measurements and scored according to the Metavir Scoring system : F0, no 9/L) and FIB-4 = age (years) x AST (UI/L)/(platelet count (109/L) x (ALT (IU/L)½). Body mass index (BMI) was calculated as BMI = (weight (kg)/height (m)2).APRI and FIB-4 were calculated as previously described , 23: APR2SO4 as stopping solution and measured at 450 nm with 650 nm as reference. A calibration curve was plotted using a 4-parametric mathematical fit model.The Protein Fingerprint markers of matrix metalloproteinase (MMP) degraded type I, III, IV and VI collagen , and type III and IV collagen formation (Pro-C3 and P4NP7S) were assessed in plasma from 403 HCV patients as previously described , 20, 24.Data were logarithmically transformed for normality and symmetry of variance. Comparisons between the mean marker levels were performed using one-way analysis of variance (ANOVA) test with Tukey’s multiple comparisons test using each group as fixed factor. The correlation between individual markers and Metavir Fibrosis scores was analyzed by Spearman’s rank correlation. We performed multiple ordered logistic regression analyses with the Metavir Fibrosis score as dependent variable and protein fingerprint markers as well as other continuous variables as independent variables. In Model 1 we hypothesized that combination of structural markers and liver function markers would improve the liver fibrosis staging. Thus we included the protein fingerprint markers, AST, and ALT using stepwise elimination with a significance level of 0.1 as cut-off. In Model 2 we investigated the impact of demographic risk factors in combination with protein fingerprint markers for liver fibrosis. Based on a multiple ordered logistic regression model with Metavir Fibrosis score as dependent variable and protein fingerprint markers as independent variables using stepwise elimination with a significance level of 0.1, we selected the protein fingerprint markers most strongly associated with fibrosis. Thereafter we included age, gender, and BMI with direct entry of all variables. Thus Model 2 included Pro-C3, C4M, age, gender, and BMI. The diagnostic power of individual markers and combination models was investigated by the area under the receiver-operating characteristics (ROC) curve (AUC) with 95% confidence interval (CI). Sensitivity and specificity were determined for appropriate cut-off values based on the ROC curves. Pair-wise comparison of ROC curves were evaluated using the method of Delong et al .Data are shown as geometric mean with 95% CI unless otherwise stated. P-values <5% were considered significant. The multiple ordinal logistic regression models were calculated in STATA version 12.0 . All other statistical analyses were calculated in MedCalc version 12 . Graphs were designed using GraphPad Prism version 5 .2 with no significant differences between Metavir F stages. We observed a significant difference in ALT and AST levels with increasing severity of fibrosis (p<0.001).Patient characteristics are shown in Mean plasma levels of the Protein Fingerprint markers were stratified according to Metavir fibrosis stages . All marFurther, ALT was significantly elevated in patients with ≥F2 compared to F0 (p<0.05), whereas AST was the only marker significantly increased in patients with ≥F1 compared to patients without fibrosis (F = 0) (p<0.05) (data not shown).The area under the ROC curves were used to evaluate the diagnostic values for detecting F≥2 and F≥3 . The besTo improve the diagnostic power for detection of liver fibrosis, we performed multiple ordered logistic regression analyses with Metavir Fibrosis score as dependent variable and protein fingerprint markers as well as other continuous variables as independent variables. Model 1 was built on the hypothesis that structural changes of ECM as well as liver tests are associated with fibrosis. In this model the parameters Pro-C3, C4M, AST, and ALT were significantly associated with Metavir Fibrosis score (p<0.0001–0.026) resulting in an algorithm with the following equation: -0.72 x logALT + 1.69 x logAST + 3.81 x logProC3 + 1.53 x logC4M .Model 2 was built on the hypothesis that a combination of structural changes and demographic/clinical risk factors might improve liver fibrosis staging. Thus model 2 included Pro-C3, C4M, age, BMI, and gender resulting in an algorithm with the following equation: -0.18 x logBMI + 0.34 x gender + 5 x logAge + 4.87 x logProC3 + 2.11 x logC4M .Both models detected patients with ≥F2 and ≥F3 with AUCs = 0.76–0.88 . FurtherA pair-wise comparison showed that the established FIB-4 index provided similar diagnostic power as Model 1 and 2 for detecting patients with ≥F2 and ≥F3. Compared to APRI, Model 2 was significantly better for detecting ≥F2 (p = 0.046), while Model 1 was significantly better for detecting F≥3 (p = 0.023) . FurtherIn the present study we investigated novel Protein Fingerprint biomarkers for liver fibrosis staging in HCV patients. The principal findings were: 1) Pro-C3 provided clinically relevant diagnostic accuracy as single marker of significant (≥F2) and advanced (≥F3) liver fibrosis; 2) a combination of the markers Pro-C3 and C4M with age, BMI, and gender improved the diagnostic accuracy; 3) a multimarker model was able to separate F0 from F1.et al (AUC = 0.77 and 0.88) and Saitou et al (AUC = 0.75 and 0.79) [Non-invasive markers such as serum markers or FibroScan may be used to aid and/or replace the need for liver biopsy to stage liver fibrosis. Most non-invasive markers are able to diagnose extreme stages of fibrosis however the “grey area” between Metavir F1 and F2 still remains a clinical challenge . We havend 0.79) , 31 is snd 0.79) . In our formation of type III collagen was up-regulated earlier in patients with chronic HCV than degradation of type III collagen reflected by a significant increase in Pro-C3 in patients with Metavir F≥2 compared to C3M, which was not significantly increased until Metavir F≥3. Similarly, the basement membrane remodeling markers, P4NP7S and C4M, were not up-regulated until advanced fibrosis stages, indicating that development of a basement membrane-like structure in the Space of Disse occurs later in fibrosis development than remodeling of interstitial matrix, reflected by C3M and Pro-C3. By contrast, C1M did not show any relevance to liver fibrosis in these patients. This might indicate that degradation of type I collagen does not occur until cirrhosis which is supported by our previous study showing significantly up-regulated C1M in cirrhotic patients with portal hypertension [Interestingly, rtension . Furtherrtension . This inrtension , 36. Thiet al. who found a mean APRI AUC for significant fibrosis of 0.84 [Several composite algorithms of non-invasive tests have been evaluated for the diagnosis of fibrosis in HCV patients. These tests are either based on serological markers alone or in combination with FibroScan. We proposed two models for diagnosing significant fibrosis in HCV infection. Model 1 correlated to Metavir Fibrosis stages however the diagnostic performance was only slightly better than using Pro-C3 as an individual marker. Model 2 was significantly better than Pro-C3 and APRI for detecting ≥F2 with an AUC = 0.80. Several studies have evaluated the diagnostic performances of APRI and FIB-4 as non-invasive algorithms for detecting significant and/or advanced fibrosis –40. They of 0.84 . Common  of 0.84 , which iThe major strength of the study is the large group of treatment naïve well-characterized biopsy proven HCV patients covering the entire spectrum. A limitation of the study is the missing comparison to novel elastography techniques, such as FibroScan or MR elastography, as they fulfill many of the requirements of an ideal non-invasive marker of fibrosis. However elastographic techniques carry some limitations as well, including requirement for expensive equipment, considerable expertise, and lack of standardized cut-off for diagnosis of fibrosis stages . Thus thIn conclusion we assessed a panel of specific protein fingerprint markers for detection of clinical significant and advanced fibrosis in patients with chronic HCV. Pro-C3 proved useful as single diagnostic marker; however the multimarker models improved the diagnostic value for detecting significant and advanced fibrosis. Prospective studies are needed to evaluate the prognostic utility of the Protein Fingerprint markers for deciding on earlier antiviral therapy and as efficacy markers.


K98E,E145A,L169F with three substitutions in the VP1 protein—K98E, E145A and L169F—productively infected both mouse cell lines for at least three passages of the virus in murine cells. Moreover, the virus gained the ability to utilize the mSCARB2 protein to infect murine cell lines. These results demonstrate that the three VP1 residues cooperate to effectively interact with the mSCARB2 protein on murine cells and permit the virus to infect murine cells. Gain-of-function studies similar to the present work provide valuable insight into the mutational trajectory required for EV71 to infect new host cells previously non-susceptible to infection.Enterovirus 71 (EV71) is a neurotrophic virus that causes hand, foot and mouth disease (HFMD) and occasional neurological infection among children. It infects primate cells but not rodent cells, primarily due to the incompatibility between the virus and the expressed form of its receptor, scavenger receptor class B member 2 (SCARB2) protein, on rodent cells (mSCARB2). We previously generated adapted strains (EV71:TLLm and EV71:TLLmv) that were shown to productively infect primate and rodent cell lines and whose genomes exhibited a multitude of non-synonymous mutations compared with the EV71:BS parental virus. In this study, we aimed to identify mutations that are necessary for productive infection of murine cells by EV71:BS. Using reverse genetics and site-directed mutagenesis, we constructed EV71 infectious clones with specific mutations that generated amino acid substitutions in the capsid VP1 and VP2 proteins. We subsequently assessed the infection induced by clone-derived viruses (CDVs) in mouse embryonic fibroblast NIH/3T3 and murine neuroblastoma Neuro-2a cell lines. We found that the CDV:BS-VP1 Enterovirus A; genus Enterovirus; family Picornaviridae)1 is a small non-enveloped RNA virus closely related to polioviruses. It is the major causative agent of hand, foot and mouth disease, an illness often associated with mild fever but that can also escalate to a severe disease with neurological involvement.3 Cellular infection by EV71 can be divided into three major stages: cellular entry, expression of the viral proteins and hijacking of the host translational machinery, and production of viable viral progeny. Cellular entry, the first stage, is dependent on the EV71 capsid protein. The capsid is comprised of 60 identical units (protomers) assembled from four structural proteins (VP1–VP4).4 Interaction of the capsid with its cognate receptor on host cells triggers a series of events in viral uncoating: capsid expansion (to form the 135 S or A particle), extrusion of the VP1 N-terminus and VP4 from the capsid junction, which has a fivefold axis of symmetry, into the endosomal membrane and release of the viral genomic RNA into the host cell cytoplasm.8 Once in the cytoplasm, the viral genome is translated via the cap-independent protein synthesis machinery of the host cell to generate 11 mature viral proteins that are required to shut off translation of the host proteins, inhibit cellular apoptosis during the early stages of infection, form an efficient viral genome replication complex, evade the host cell antiviral system and induce cell death in the later stages of infection.16 Finally, after the viral genome has been sufficiently replicated and the viral structural proteins have been formed, the viral progeny are assembled. The four structural capsid proteins (VP1–VP4) self-assemble around the genomic RNA, and the virion undergoes a series of structural transitions to form mature, viable and infectious viral progeny.17Enterovirus 71 37 were susceptible to clinical isolates of the virus. Rodent cell lines that ectopically express the human SCARB2 (hSCARB2) protein, however, could be successfully infected with EV71.38 Transgenic mice that express hSCARB2 also exhibited more severe signs of EV71-induced disease.39 Thus, the incompatibility of EV71 with murine SCARB2 (mSCARB2) governs the restriction of EV71 infection of murine cells, and circumventing this incompatibility leads to an environment more conducive to infection.The main EV71 receptor that triggers viral uncoating has recently been identified as scavenger receptor class B member 2 (SCARB2).40 Previous viral RNA transfection studies have demonstrated that EV71:BS infection of murine cells is blocked at an early stage during cellular entry, and the adapted strains (EV71:TLLm and EV71:TLLmv) overcame this, presumably by adaptive mutations. Genome sequence comparison of the three strains revealed that EV71:TLLm and EV71:TLLmv accumulated multiple non-synonymous mutations in the capsid-encoding (P1) region, suggesting a possible correlation between the capsid mutations and the novel ability to infect rodent cell lines. In this study, we identified the capsid amino acid substitutions responsible for enabling EV71:BS to productively infect murine cell lines. By using reverse genetics and standard mutagenesis techniques, we synthesized plasmid cDNA clones of EV71:BS; incorporated site-specific mutations into the genome; and investigated the role of these mutations in productive infection of mouse cells.In an attempt to overcome the natural incompatibility of EV71 and mSCARB2, our group adapted EV71 to grow in a cell line derived from mouse embryonic fibroblasts . Through serial passage of a clinical isolate (EV71:BS) in this murine cell line, we generated 2 strains—EV71:TLLm (60 infection cycles) and EV71:TLLmv (100 infection cycles)—that productively infected a range of rodent cell lines.The plasmid pACYC177 , which is a low-copy plasmid , was use40Three strains of EV71 were used in this study. The clinical isolate EV71:BS (GenBank Accession NO KF514878) and the adapted strains EV71:TLLm (GenBank Accession NO KF514879.1) and EV71:TLLmv (GenBank Accession NO KF514880.1) have been previously described.40African green monkey kidney Vero (CCL-81), mouse neuroblastoma Neuro-2a (CCL-131) and mouse embryonic fibroblast NIH/3T3 (CRL-1658) cells were purchased from the American Tissue Culture Collection . Cells were propagated and maintained as previously described.All primers used to construct the plasmid clones are listed in We used a two-step cloning procedure to construct pACYC-BS, which is the pACYC plasmid containing the full-length cDNA of EV71:BS . To intr41 CDVs were inoculated onto cells seeded overnight at an multiplicity of infection (MOI) of 1 by pre-adsorption, as previously described.40 We assessed the cellular infection phenotype by observing the progression of virus-induced lytic cytopathic effects (CPE) for several days post-inoculation (p.i.) and immunofluorescence staining of viral proteins as previously described.42 Culture supernatants of infected cells were also harvested by centrifugation  and subjected to 10-fold serial dilutions with viral disaggregation using either chloroform or 1% sodium deoxycholate. Viral titers were determined using the Reed and Muench method.41The constructed pACYC-BS clones were co-transfected with pCMV-T7pol into Vero cells using Lipofectamine 2000 reagent  following the manufacturer's protocol. Supernatants from transfected cells containing the CDVs were harvested 7–10 days after transfection, and viral titers were determined using the Reed and Muench method.43 We incubated the virus at an MOI of 100 with various concentrations of soluble mouse SCARB2 (mSCARB2) proteins (200 ng/μL stock solution) at 1000 ng, 500 ng, 250 ng and 0 ng for 2 h at 37 °C on a shaking platform. The virus was inoculated onto NIH/3T3 cells (6000 cells per well) seeded overnight and incubated at 37 °C. Cellular infection was assessed by observation of CPE and immunofluorescence detection of viral antigens at 48 h p.i.These experiments were adapted from previously published procedures.in vitro induction of viral uncoating, we incubated 106 median cell culture infective doses (CCID50) of the virus with 200 ng soluble mSCARB2 protein at 4 °C on a shaking platform. The mixture was digested with 100 mg/mL RNase A  for 10 min at room temperature (RT) to degrade susceptible RNA molecules, and samples were subsequently treated with 10 U of reaction RiboLock RNase inhibitor  for 10 min at RT to inactivate the RNases. Genomic RNA was extracted from intact viruses using an EZNA Viral RNA Kit  following the manufacturer's protocol, and eluted RNA samples were stored in −80 °C until further use. In similar experiments, virus at an MOI of 10 was incubated with various concentrations of mSCARB2 protein  or 200 ng BSA as a non-specific protein (NSP) control at 4 °C overnight. The treated virus was inoculated onto seeded NIH/3T3 cells (105 cells per well) for 1 h at 4 °C, and then cells were washed 3 × with sterile, cold PBS and incubated in DMEM (1% FBS) for 2 h at 37 °C. Total cellular RNA was extracted from the inoculated cells using an AxyPrep Multisource Total RNA Miniprep kit  following the manufacturer's protocol, and eluted RNA samples were stored in −80 °C until further use. The For 43 NIH/3T3 cells seeded overnight in 96-well plates (1 × 104 cells per well) were incubated with twofold serial dilutions (1:20 to 1:640) of anti-mSCARB2 rabbit sera for 1 h at 37 °C. Cells were subsequently inoculated with virus (100 MOI) for 1 h at 37 °C. Cells were washed 2 × in PBS and incubated in DMEM (1% FBS) for 1 h at 37 °C. Cellular infection was assessed by detection of CPE and measurement of viral titer in cell culture supernatants harvested three days p.i.These experiments were adapted from previously published procedures.5 cells per well) and incubated with various concentrations  of anti-mSCARB2 rabbit serum in a rocking platform at 4 °C for 1 h. Alternatively, cells were treated with rabbit serum against Saffold virus L protein (2 μg) as a control serum against a NSP. Treated cells were subsequently washed with cold, sterile PBS 3 × and inoculated with virus at an MOI of 10 at 4 °C for 1 h on a rocking platform. Cells were then washed 3 × in cold PBS and incubated with DMEM (1% FBS) at 37 °C for various durations (1–4 h). Total cellular RNA was extracted using the AxyPrep Multisource Total RNA Miniprep kit following the manufacturer's protocol, and eluted RNA samples were stored in −80 °C until further use.In another set of experiments, NIH/3T3 cells were seeded in 12-well plates  following the manufacturer's protocols. Primers and gene-specific probes targeting a conserved region of the EV71 5′ untranslated region (EV5′NC) were used to detect the virus, while primers and probes targeting an exon of human β-actin were used as internal controls. Primer and probe sequences are listed in 6 CCID50 of virus at day 0. Mock-infected mice were inoculated with equal volumes of DMEM (1% FBS). Inoculated mice were observed twice daily for signs of infection, and body weights were measured once daily. The general criteria for euthanasia followed previously established guidelines35 and included (i) loss of >20% maximum recorded body weight, (ii) paralysis persisting >48 h, (iii) absence of feeding or inability to feed, (iv) inability to self-right, and (v) altered state of consciousness presenting as either stupor or coma. On appearance of these disease manifestations, mice were killed by I.P. injection of pentobarbitone (100 mg/kg). Animals that survived the 28-day observation period were also killed by pentobarbitone. Blood samples were terminally collected by cardiac puncture for subsequent serum analysis for neutralizing antibodies. Serum neutralization tests are described in the Procedures for handling and infection of mice were approved by the Institutional Animal Care and Use Committee of Temasek Lifesciences Laboratory , which follows the guidelines specified by the National Advisory Committee on Laboratory Animal Research (NACLAR) of Singapore. Groups of eight 6-day-old BALB/c pups were inoculated via the intraperitoneal (I.P.) route with 10www.graphpad.com). When choosing the appropriate statistical tests, the normal distribution of data was first confirmed with QQ plots and Kolmogorov–Smirnov tests. Comparisons of mean values for normally distributed data with unequal variance were performed using t-tests with Welch's correction, while comparisons of medians for non-normally distributed data were performed with Mann–Whitney U-tests.All graphs and statistical analyses were performed using GraphPad Prism ver. 6.01 for Windows  of pACYC-BS, which is the plasmid containing the full-length cDNA of EV71:BS, with equivalent sequences from EV71:TLLm to produce a chimeric plasmid pACYC-BSΔM-P1 . We tran progeny .M-P1 induced full CPE and viral protein expression in inoculated Vero cells. In contrast, only CDV:BSM-P1 induced significant CPE and viral antigen production in NIH/3T3 and Neuro-2a cells (P=0.0055) and CDV:BSM-P1 (P=0.0005)  and Neuro-2a (P=0.0051) cells inoculated with CDV:BSM-P1  to productively infect murine cells reside within the capsid protein.CDV:BS and CDV:BS2a cells . Viable =0.0005) , confirmV:BSM-P1 . These r40 To determine which of these could facilitate entry into murine cells, we selected several candidates based on the assumption that these molecular determinants should be (i) common to both EV71:TLLm and EV71:TLLmv and (ii) either surface-exposed or located near the capsid canyon region to potentially interact with the viral receptor on host cells. We therefore focused on five amino acid substitutions in VP1 and VP2 that are found in both adapted strains. VP1-K98E and E145A were selected because of their surface exposure, and L169F was selected because of its proximity to the canyon region46 that participates in binding to the viral receptor  were located in the capsid-encoding (P1) region.receptor . Similarptor  and Neuro-2a cells (P=0.0391) compared with the respective CDV:BS titers  and Neuro-2a cells (P=0.039) . These findings demonstrate that simultaneous introduction of VP1-K98E and E145A into EV71:BS leads to productive infection of NIH/3T3 and Neuro-2a cells. In contrast to CDV:BS-VP1K98E or CDV:BS-VP1E145A, CDV:BS-VP1K98E,E145A not only entered and replicated in the infected cells, but it also produced viable viral progeny. Furthermore, combining VP1-K98E, E145A and L169F together enhanced the infectivity of the resulting CDV in murine cells. In NIH/3T3 cells, higher titers were observed for CDV:BS-VP1K98E,E145A,L169F compared with CDV:BS-VP1K98E,E145A-inoculated cells (P=0.0136) (P<0.0001). These findings suggest that the three amino acid residues in VP1  may constitute the minimum determinants for mouse cell adaptation necessary to permit productive infection of EV71:BS in murine cell lines.We also introduced various combinations of the VP1 and VP2 substitutions into the pACYC-BS plasmid by site-directed mutagenesis . Severaled cells . In contP=0.039) . Similar=0.0136) . SimilarM-P1, CDV:BS-VP1L169F, CDV:BS-VP1K98E,E145A and CDV:BS-VP1K98E,E145A,L169F. To evaluate whether these CDVs induce stable productive infections of murine cells, we performed three viral passages at an MOI of 1 in NIH/3T3 and Neuro-2a cells to simulate a genetic bottleneck. Of the four CDVs, only CDV:BSM-P1 and CDV:BS-VP1K98E,E145A,L169F exhibited sustainable productive infection of Neuro-2a cells for three cycles and of NIH/3T3 cells for two cycles. Viral antigens were detected in Neuro-2a cells at both P1 and P2, while antigens were observed in NIH/3T3 cells only at P1 , and the P2 titers were higher than P1 (P=0.0031)  (We identified four mutant CDVs that productively infect NIH/3T3 and Neuro-2a cells—CDV:BSly at P1 . Compare=0.0031) . For CDV=0.0032) .K98E,E145A,L169F (M-P1 (data not shown). These findings demonstrate that the infection phenotypes induced by CDV:BS-VP1K98E,E145A,L169F and CDV:BSM-P1 are sustainable in Neuro-2a cells for at least three passages and would induce sustained infection in these cells. Moreover, the VP1 mutations are stable in the viral genome after the third passage, suggesting that they may persist even in later passages. In contrast, both CDVs did not achieve sustainable infection in NIH/3T3 cells, and instead the population collapsed as expected for populations with unestablished fitness subjected to a genetic bottleneck.49 This suggests that further genetic alterations are necessary to establish stable infection in mouse fibroblast cells, as reflected in the multitude of non-synonymous mutations observed in the non-structural proteins of both EV71:TLLm and EV71:TLLmv.40To confirm the genomic stability of the three VP1 substitutions, we sequenced the VP1 gene region of the virus derived from passage P2 in Neuro-2a cells. The introduced nucleotide mutations A2734G, A2876C and C2947T resulting in amino acid substitutions VP1-K98E, E145A and L169F, respectively, were evident in CDV:BS-VP15A,L169F  and CDV:19 To determine whether EV71:TLLm and EV71:TLLmv also utilize SCARB2 to infect murine cells, we performed several assays. In the viral competitive entry experiment, we pre-incubated EV71:TLLmv with various concentrations of soluble mSCARB2 protein prior to inoculation onto NIH/3T3 cells. We assessed viral infectivity by immunostaining for viral antigens in cells at 48 h p.i. and counting the number of infected cells. Pre-incubation of the virus with soluble mSCARB2 reduced viral infectivity in a dose-dependent manner, and fewer viral antigens were detected in cells inoculated with virus pre-incubated with 500 ng or 1 μg soluble mSCARB2 (P=0.0004) and 1 μg (P<0.0001) of soluble mSCARB2 , 2 h (P=0.0036) and 3 h (P=0.0141) p.i. (P=0.0084) but not in samples incubated with the lower doses of mSCARB2 or with a NSP (P=0.0003), 100 ng (P<0.0001), or 200 ng (P<0.0001) of mSCARB2 protein (P=0.0157), 50 ng (P=0.0142), 100 ng (P=0.0096) or 200 ng (P=0.0006) of mSCARB2 resulted in a similar reduction in EV71:TLLm RNA detected in inoculated cells  p.i. . Comparith a NSP . Further protein . In addied cells . This reed cells . In conted cells .Escherichia coli and native SCARB2 proteins from mammalian cell lysates (P<0.0001), which was not observed in cells pre-incubated with anti-NSP sera  resulted in a dose-dependent reduction of viral titers compared with mock-incubated controls (P<0.0001), 4 μg (P=0.0010) or 2 μg (P=0.0257) of anti-mSCARB2 serum resulted in lower amounts of EV71:TLLmv RNA detected in inoculated cells, which was not observed in cells pre-incubated with anti-NSP sera   .All these results demonstrate that the interaction between EV71:TLLmv (or EV71:TLLm) and mSCARB2 is crucial for the infection of murine cells. Blocking this interaction by incubating cells with anti-mSCARB2 sera reduced cellular infection. Similarly, virus pre-incubation with soluble mSCARB2 led to a reduction in viral infectivity, suggesting that the interaction of the virus with mSCARB2 resulted in either viral sequestration or alteration of the capsid structure to effectively disable it from infecting murine cells. Thus, we confirmed that EV71:TLLmv and EV71:TLLm use mSCARB2 to infect murine cell lines.M-P1 and CDV:BS-VP1K98E,E145A,L169F utilize mSCARB2 to infect murine cells. In viral uncoating experiments, we incubated the CDVs with soluble mSCARB2 protein for 2 h and quantified the total RNA extracted from the intact virus. Compared with untreated controls, incubation of CDV:BSM-P1 with 200 ng (P=0.0365) or 100 ng (P=0.0172) of soluble mSCARB2 significantly reduced the amount of intact viral RNA  or 100 ng (P=0.0352) of soluble mSCARB2 (M-P1 treated with 25 ng (P=0.0349), 50 ng (P=0.0004), 100 ng (P<0.0001) or 200 ng (P<0.0001) of mSCARB2 contained lower amounts of viral RNA  or 200 ng (P=0.0003) of mSCARB2 , 4 μg (P=0.0004) or 8 μg (P<0.0001) of anti-mSCARB2 serum prior to inoculation with CDV:BSM-P1 showed lower amounts of viral RNA compared with mock-incubated cells (P=0.0015), 4 μg (P=0.0002) or 8 μg (P<0.0001) serum and inoculated with CDV:BS-VP1K98E,E145A,L169F  of anti-mSCARB2 serum exhibited lower CDV:BS1 titers  but not F titers . Furthered cells . Similar5A,L169F . Togethe6 CCID50 (median CCID) of virus via the i.p. route and observed the animals for 28 days. No significant signs of disease were observed in all mice inoculated with EV71:BS, CDV:BS-VP1K98E,E145A,L169F or CDV:BSM-P1, and all mice survived throughout the study  or mock-infected controls   . All ino=0.0057)  despite M-P1) to productively infect NIH/3T3 and Neuro-2a cells. Second, we introduced individual substitutions into the VP1  and VP2 (S144T and K149I) regions of the capsid protein by site-directed mutagenesis and determined which ones supported EV71:BS entry into murine cells by assaying the expression of viral proteins. All assayed CDVs were able to enter murine cells; however, only the VP1-L169F substitution permitted the resulting virus to produce viable progeny. We also generated infectious clones containing multiple substitutions in the VP1 and VP2 proteins and found that only CDV:BS-VP1K98E,E145A and CDV:BS-VP1K98E,E145A,L169F productively infected murine cells. These findings demonstrate that three VP1 residues  are the minimum and necessary requirements for EV71:BS to productively infect murine cell lines. However, these substitutions are not sufficient to induce lethal disease in mice, suggesting that other determinants may govern virulence in mice.In this study, we investigated the molecular determinants that enable the adapted strains EV71:TLLm and EV71:TLLmv to productively infect the mouse cell lines NIH/3T3 and Neuro-2a. First, we demonstrated that the restriction in EV71:BS infection of murine cells is at the stage of cellular entry and is due to the capsid protein. Replacement of the EV71:BS capsid with complementary sequences from EV71:TLLm enabled the resulting chimeric virus  into EV71:BS resulted in productive infection of murine cell lines, and the infection phenotype was sustainable for at least three viral passages in Neuro-2a cells. These experiments are similar to previous studies by Yamayoshi et al.,22 where they replaced exon 4 of the mouse SCARB2 (mSCARB2) gene with exon 4 of human SCARB2 (hSCARB2) in the L929 mouse cell line and demonstrated that this was sufficient to facilitate entry and replication of EV71 clinical isolates. These findings led us to hypothesize that EV71:TLLm and EV71:TLLmv could productively infect rodent cell lines because they have evolved to effectively utilize mSCARB2. To explore this possibility, we designed several viral entry/uncoating competitive assays and demonstrated that pre-incubation of the virus with soluble mSCARB2 protein reduced its cellular infectivity. Similarly, pre-incubation of NIH/3T3 cells with anti-mSCARB2 sera blocked viral infection. These experiments conclusively demonstrate that in contrast to EV71:BS, EV71:TLLm and EV71:TLLmv could utilize mSCARB2 to infect NIH/3T3 and Neuro-2a murine cell lines.As a prerequisite to cellular entry, the viral capsid must recognize and interact with the SCARB2 protein on host cells. SCARB2 interacts with the virus at the canyon region,M-P1 and CDV:BS-VP1K98E,E145A,L169F utilize mSCARB2 to infect murine cells. This led us to further hypothesize that the three VP1 substitutions may have been selected in the course of serial adaptation of EV71 to NIH/3T3 cells due to the improved interaction with mSCARB2 on murine cells. Circumstantial evidence indicates that this is a likely scenario. VP1-169 lies within the capsid canyon, and previous studies reported that mutations around the canyon of the poliovirus and rhinovirus capsids resulted in altered viral-binding kinetics with their receptors.53 It is also part of the SP55 peptide, which can elicit EV71-specific neutralizing antibodies.47 VP1-98 lies within a major immunodominant VP1 linear epitope that exhibits significant immunoreactivity with EV71-positive sera,54 and VP1-169 and VP1-145 reside within the region that contributes to the strength of VP1 self-association and interaction.55 The VP1-145E residue was also reported to increase cytotoxicity to Neuro-2a cells,56 while VP1-98 and VP1-145 are markers for viral binding to primate PSGL-1 proteins on leukocytes.58 Nishimura et al.57 previously surveyed 1702 VP1 protein sequences from EV71 clinical isolates published in GenBank. Analyzing this database of sequences, which can be found in the Supplementary Materials and Methods,57 we observed that the amino acids VP1-98E and 145A appeared in the same genome in only 5 out of 1702 isolates (0.3%), and VP1-169F was not observed in the database at all. These observations suggest that the amino acid combination of VP1-98E, 145A and 169F is novel and has not been identified in any EV71 isolate, supporting our theory of the relevance of these three substitutions during the adaptation process in NIH/3T3 cells. It would be of scientific interest to test these five strains  for their ability to productively infect NIH/3T3 and Neuro-2a cell lines, especially following introduction of the 169F substitution.We also demonstrated that CDV:BSK98E,E145A,L169F using agarose beads coated with recombinant soluble mSCARB2 proteins. However, this experiment to demonstrate sustained binding of the virus to mSCARB2 was not successful despite multiple attempts. In similar control experiments, the mSCARB2-coated beads could not pull down EV71:TLLm or EV71:TLLmv , but only SCARB2 has so far been shown to induce viral uncoating.50In an effort to provide further support for our hypothesis that the three VP1 substitutions enabled EV71 to more efficiently interact with mSCARB2, we performed pull-down assays on CDV:BS-VP171:TLLmv , suggest23 and access to this tunnel is regulated by a cap structure that maintains the tunnel in a ‘closed conformation' at neutral pH and an ‘open conformation' at acidic pH.50 Interestingly, this cap structure is formed by part of exon 4 (aa 142–164) of the protein, which is known to be highly divergent between mSCARB2 and hSCARB2 and is important for EV71 binding and cellular infection.22 Furthermore, in this proposed model, protonation of His150 of hSCARB2 at an acidic pH within the endosome triggers the structural rearrangement required to open the lipid tunnel, which subsequently sequesters the sphingosine displaced from the virus canyon. In molecular homology models of the mSCARB2 protein, His150 is replaced by threonine, and the tunnel interior is more hydrophobic compared with hSCARB2. This suggests that acidification is not sufficient to open the lipid tunnel, and another trigger—perhaps a specific interaction with the virus—may be required. Further investigations are necessary to explore this scenario in mSCARB2-dependent EV71 infection of murine cells, and our adapted strains provide opportunities for potential future collaborations to address these unsolved questions.This is the first study that successfully identified specific amino acid substitutions in the capsid that enabled EV71 to productively infect non-susceptible mouse cell lines, specifically NIH/3T3 and Neuro-2a. Identifying the mutations required for EV71 to productively infect murine cells could provide information on the interactions with mSCARB2 that are necessary for the virus to ‘evolve' or develop to utilize the specific receptor. We also emphasize that EV71:TLLm and EV71:TLLmv are the first reported EV71 strains that successfully utilize the physiologically expressed mSCARB2 protein to infect murine cells. Previous attempts to enable EV71 infection of murine cells relied on ectopic expression of the hSCARB2 protein. Although we still do not fully understand why or how these three VP1 residues enable productive infection of murine cells, the recently solved three-dimensional structure of hSCARB2 may reveal the underlying mechanisms of why EV71 cannot utilize mSCARB2 to infect murine cells, and consequently provide clues for how the combination of VP1-98E, 145 and 169F enable EV71 to utilize mSCARB2. A large cavity or tunnel, which can accommodate sphingosine and other lipid molecules, traverses the length of the protein,


MCSFR promoter through antagonizing PU.1's ability to bind to DNA. Exogenous E47 expression in hematopoietic cells inhibits myeloid differentiation. Our data suggest that E2A antagonism of PU.1 activity contributes to its ability to commit multipotential hematopoietic progenitors to the lymphoid lineages.Antagonistic interactions between transcription factors contribute to cell fate decisions made by multipotent hematopoietic progenitor cells. Concentration of the transcription factor PU.1 affects myeloid/lymphoid development with high levels of PU.1 directing myeloid cell fate acquisition at the expense of B cell differentiation. High levels of PU.1 may be required for myelopoiesis in order to overcome inhibition of its activity by transcription factors that promote B cell development. The B cell transcription factors, E2A and EBF, are necessary for commitment of multipotential progenitors and lymphoid primed multipotential progenitors to lymphocytes. In this report we hypothesized that factors required for early B cell commitment would bind to PU.1 and antagonize its ability to induce myeloid differentiation. We investigated whether E2A and/or EBF associate with PU.1. We observed that the E2A component, E47, but not EBF, directly binds to PU.1. Additionally E47 represses PU.1-dependent transactivation of the E2A−/−, Ebf1−/−, and Pax5−/− pro-B cells all overexpress the myeloid cytokine receptor gene MCSFR (macrophage-colony stimulating factor receptor). Hematopoietic expression of MCSFR is dependent on the Ets family transcription factor PU.1 as demonstrated by the lack of detectable MCSFR mRNA in PU.1−/− hematopoietic cells . EB. EB20]. α antibody prebound to protein A agarose beads. E47 was detected in PU.1 immunoprecipitates, but not in anti-GMCSFR immunoprecipitates  lacking the basic-helix-loop-helix (bHLH) DNA binding domain could bind to PU.1 . This suTo identify the domain of PU.1 that interacts with E47, in vitro translated deletion products of PU.1 were incubated with purified GST-E47bHLH protein. Deletion of the transactivation domain (Δ33–100) and the PEST region (Δ118–160) of PU.1 had no effect on binding to E47. However, a deletion construct lacking the Ets DNA binding domain (Δ201–272) was unable to bind to E47 . These d GCSFR and MCSFR promoters [ MCSFR and GCSFR promoter constructs over 10-fold  induced transcriptional activity. Cells cotransfected with an E2A-reporter plasmid ((μE3)4-Luciferase) and E47 expression plasmid resulted in an almost 50-fold enhancement of luciferase activity over cells transfected with the E2A-reporter alone. Coexpression of PU.1 with E47 did not repress transcriptional activity from the reporter construct , these cells remain undifferentiated. In the presence of 100 nM OHT, PUER cells differentiate into macrophages. These cells were superinfected with either an E47-IRES-GFP virus or a GFP only virus. Cell lines expressing high levels of GFP were generated and their ability to differentiate into macrophages was assayed. After 8 d of OHT treatment, PUER GFP cultures became adherent and expressed the macrophage specific marker F4/80  but not a similar probe containing a mutated PU.1 binding site (lane 4) from the extracts expressing only PU.1. This complex was ablated by addition of a PU.1 antibody (lane 5). In nuclear extracts coexpressing PU.1 and increasing amounts of E47 we observed a decrease in the PU.1/MCSFR complex . Expression of PU.1 and E47 in the 293 T extracts is shown by immunoblot  by a PU.1 antibody (lane 3). Additionally this complex was induced by OHT treatment as it was not present in extracts from untreated PUER GFP cells (lane 6). Significantly the PU.1 containing complex was not detectable in PUER E47 OHT-treated cells (Lane 9).To determine if E47 abrogates PU.1 DNA binding in hematopoietic cells we performed EMSAs with GFP− and E47− expressing PUER cell nuclear extracts. As shown by immunoblot E47-infected cells expressed substantially more E47 in the presence or absence of OHT, than GFP-infected PUER . As doneUsing PUER GFP cells and two independent lines of PUER E47 cells, chromatin immunoprecipitations (ChIP) with anti-PU.1 were performed to determine if E47 would antagonize PU.1 DNA binding in cells. Anti-PU.1 efficiently precipitated the MCSFR promoter, whereas the promoter was not detected in normal rabbit serum immunoprecipitates. Consistent with our findings with the EMSA assays, less MCSFR promoter was precipitated with anti-PU.1 from chromatin isolated from both PUER lines expressing E47 . The res MCSFR promoter activity and inhibited myeloid differentiation. The E47 bHLH domain mediates the interaction with PU.1, so potentially other bHLH proteins may also be able to bind to PU.1 and affect its activity. Preliminary results demonstrated that another E2A related molecule HEB binds to PU.1 (data not shown). However, there is specificity to the binding of PU.1 to bHLH proteins as the bHLH PAS domain protein, ARNT, cannot bind to PU.1 (We have demonstrated that the E2A protein E47 binds to PU.1 and blocks its ability to bind to DNA. This inhibition of DNA binding resulted in repression of to PU.1 . The Ets E2A−/− mice showed that the lack of E2A results in increased development of monocytes and granulocytes from these progenitors [E47 inhibited macrophage differentiation of PUER cells and myeloid differentiation of primary hematopoietic cells suggesting that E47 may play a role in cell fate decisions. This may explain why high levels of PU.1 are required for inducing myeloid differentiation . In an ugenitors . Similargenitors .Consistent with our observations Bhalla et al. previously observed that E2A component E12 could induce B cell differentiation of fetal liver MPPs . E2A actOur data demonstrates that E2A and PU.1 directly bind to each other, which results in antagonism of PU.1's DNA binding activity. Previous inhibitory binding partners of PU.1 such as Pax5, GATA1, and C/EBP alpha have either recruited in transcriptional corepressors or displaced coactivators without affecting association with DNA. The observations support the hypothesis that during hematopoiesis E47 can antagonize PU.1 activity to direct B cell development. If PU.1 levels are high though, PU.1 overcomes this antagonism and directs myeloid differentiation.


A majority of facioscapulohumeral muscular dystrophy (FSHD) is caused by contraction of macrosatellite repeats called D4Z4 that are located in the subtelomeric region of human chromosome 4q35. Sequencing the FSHD locus has been technically challenging due to its long size and nearly identical nature of repeat elements. Here we report sequencing and partial assembly of a BAC clone carrying an entire FSHD locus by a single molecule real time (SMRT) sequencing technology which could produce long reads up to about 18 kb containing D4Z4 repeats. De novo assembly by Hierarchical Genome Assembly Process 1 (HGAP.1) yielded a contig of 41 kb containing all but a part of the most distal D4Z4 element. The validity of the sequence model was confirmed by an independent approach employing anchored multiple sequence alignment by Kalign using reads containing unique flanking sequences. Our data will provide a basis for further optimization of sequencing and assembly conditions of D4Z4. About half  and possNearly identical long repeats such as D4Z4 cannot be assembled by aligning sequence reads shorter than the repeat length. In addition, D4Z4 has been hard to sequence due to its high GC content –6. The Shttp://www.ncbi.nlm.nih.gov/projects/genome/assembly/grc/human/) still contains 14 open gaps including one in the chromosome 4. Here we report sequencing and partial assembly of a BAC clone spanning an entire D4Z4 repeat region of the human chromosome 4.The latest human genome assembly GRCh38.p5 released on September 15, 2015 .BAC DNA was first purified by MoBio PowerClean Kit, sheared by centrifugation through g-Tube, and then concentrated by AMpure. SMRT bell libraries were constructed using DNA Template Prep Kit v2.0 and then analyzed by PacBio RS using DNA/Polymerase Binding Kit P4, Sequencing reagent v2.0, and a single SMRT cell v3 (P4C2) –14. AddiHGAP.1 pipeline was employed for de novo assembly of CH16-242C23 and RP11-242C23 SMRT sequence data each from a single cell. Briefly, sequence errors were first corrected by mapping short subreads (< 500 bp) to longer subreads (> 500 bp) using Basic Local Alignment with Successive Refinement (BLASR), and then assembled by Celera Assembler v.7.0 to obtain contigs –14. To fhttps://www.qiagenbioinformatics.com/) Create Pairwise Comparison. Dot plot representation of sequence similarities was generated by Create Dotplot program in CLC Main Workbench.A query sequence containing entire D4Z4 repeats and both upstream and downstream 500 bp unique flanking sequences was derived from the published RP11-242C23 sequence (CT476828.7). NCBI blastn  was usedhttp://www.ncbi.nlm.nih.gov/Traces/trace.cgi) by searching center_project = 'bA242C23' and center_name = 'SC'. Five hundred reads containing D4Z4 were identified by NCIB blastn and arranged in the order of their 5' ends, grouped into four overlapping subsets , and clustered by CLC Main Workbench Create Alignment algorithm with Gap open cost 10.0 and Gap extension cost 1.0. CLC Main Workbench Create Tree was used to generate trees with Neighbor Joining algorithm, Jukes-Kantor distance, and 1000 bootstrap analysis.Sanger sequences of RP11-242C23 were obtained from the NCBI Trace Archive Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S6 Fig(TIF)Click here for additional data file.S7 Fig(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S1 Data(ZIP)Click here for additional data file.


EAChm is a negatively charged acidic protein retaining aspartate and glutamate. Furthermore, we identified that Chameau and EAChm stimulate transcription in vitro together with purified general transcription factors. In addition, RNA-seq analysis of Chameu KD and EAChm KD S2 cells suggest that Chameau and EAChm regulate transcription of common genes in vivo. Our results suggest that EAChm regulates gene transcription in Drosophila embryos by enhancing Acetyltransferase Chameau activity.Acetylation of nucleosomal histones by diverse histone acetyltransferases (HAT) plays pivotal roles in many cellular events. Discoveries of novel HATs and HAT related factors have provided new insights to understand the roles and mechanisms of histone acetylation. In this study, we identified prominent Histone H3 acetylation activity  The eukaryotic genome is packaged into a higher-order nucleoprotein structure called chromatin that can be replicated and segregated appropriately during the cell cycle. Transcriptional regulation from chromatin is a dynamic and precise process through its condensation and de-condensation accompanied by histone modification . The basRecently, diverse posttranslational modifications of nucleosomal histones have been identified, including methylation, phosphorylation, ubiquitylation, acetylation and sumoylation –6. AmongAmong A-type HATs, the largest histone acetyltransferase family is the MYST family comprised of three subgroups; the Moz /Qkf, Tip60/Mof, and Hbo1/ Chameau subfamilies , 13. MYSDrosophila embryo extracts. We purified its activity and showed that prominent histone H3 acetylation activity composed of MYST histone acetytransferase Chameau and Enhancer of Acetyltransferase Chameau (EAChm), stimulated transcription in vitro.In this study, we found prominent nucleosomal histone H3 acetylation activity in Histone acetylation in the nucleus plays pivotal roles in chromatin assembly and gene transcription, yet the mechanisms that regulate the acetyltransferase activity and HAT complex assembly have not been fully determined. Since we identified prominent histone H3 acetylation activity in nuclear extracts made from 0–12 hour Drosophila embryos, we started to purify the activity to uncover its regulation mechanisms. To monitor histone acetylation activity, we added [3H] acetyl-CoA to the reaction as well as salt dialyzed chromatin and extracts or fractions. As depicted in Drosophila CG5229 gene product that is known as the histone acetyltransferase, Chameau (Drosophila CG13463 gene product (http://flybase.org). Since CG13463 gene product was the only peptide which shows a high Xcorr score and proper molecular weight in the top fraction of glycerol gradient sedimentation, we tentatively named this gene Enhancer of Acetyltransferase Chameau (EAChm).To further purify the HAT activity, the fractions from the Blue Sepharose chromatography were applied to glycerol gradient sedimentation and subsequently analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining . Fig 2B  Chameau . There w product . CG13463Drosophila CG13463 gene product is an acidic protein consisting of 211 amino acids containing glutamate and aspartate in its c-terminus as shown in Drosophila CG13463 gene product is EAChm and it stimulates acetyltransferase activity of native Chameau.Drosophila melanogaster homologue of human Hbo1 and belongs to the Hbo1 subfamily of the MYST family of histone acetyltransferases . The dialyzed sample was applied to a column of 120 ml of SP sepharose F.F. (Pharmacia) and eluted stepwise by 0.25 M KCl, 0.4 M KCl and 1.0 M KCl HEG buffer. H3 acetylation activity was confirmed by HAT assay (described below). H3 acetylation activity was mainly eluted in the 0.4 M KCl fraction. Then, the 0.4 M KCl fraction was diluted to 0.1 M KCl HEG. The Diluted sample was subsequently subjected to 120 ml Q sepharose F.F(Pharmacia) column chromatography and eluted by a 10 column volume linear gradient from 0.1 M to 1.0 M KCl HEG buffer. H3 acetylation activity was mainly eluted in the 0.25 M KCl fraction. The 0.25 M KCl fraction was then diluted to 0.1 M KCl HEG. The diluted sample was subjected to 8 ml POROS Heparin (Roche) column chromatography and eluted by a 20 column volume linear gradient from 0.1 M to 2.0 M KCl HEG buffer. H3 acetylation activity was mainly eluted in the 0.8 M KCl fraction. The 0.8 M KCl fraction was then diluted to 0.1 M KCl HEG. Whereupon the diluted sample was subjected to 2 ml Blue sepharose F.F. (Pharmacia) column chromatography and eluted using a 20 column volume linear gradient from 0.1 M to 2.0 M KCl HEG buffer. We repeated this Blue sepharose chromatography step three times. During the first Blue sepharose chromatography step, H3 acetylation activity was mainly eluted in the 0.9 M KCl fraction. After which the 0.9 M KCl fraction was diluted to 0.1 M KCl HEG. Whereupon the diluted sample was subjected to a second Blue sepharose chromatography step and eluted in 0.8 M KCl HEG. At the third Blue sepharose chromatography step, H3 acetylation activity was mainly eluted in the 1.0 M KCl fraction and dialyzed against 50 mM KCl HEG. The dialyzed fraction was then subjected to 20–50% glycerol gradient sedimentation . Active fractions from the glycerol gradient sedimentation were analyzed using a Thermo LCQ ion trap mass spectrometer.Histone H3 acetylation activity was purified from 4HCO3. The gel was dried and reduced in 10 mM DTT/ 25 mM NH4HCO3 at 50°C for 1 hr. Subsequently, the gel was alkylated in 27.5 mM iodacetoamido/ 25 mM NH4HCO3 at room temperature for 20 min. The alkylated gel was washed with acetonitrile, 25 mM NH4HCO3 and with acetonitrile again. Protein in the gel was digested with 10 μg/μl Modified Trypsin (Promega) / 50 mM NH4HCO3 overnight at 37°C. Digested peptides were eluted by adding 0.05% formic acid/distilled water. Trypsin digested peptides were analyzed by reversed-phase liquid chromatography on an EASY-nLC 1000 system coupled with a Thermo LCQ ion trap mass spectrometer (Thermo Fischer Scientific). Peptides were loaded on to a NANO HPLC CAPILLARY COLUMN  and eluted directly into a mass spectrometer  using a linear gradient  of acetonitrile at a flow rate of 300 nl/min . MS/MS data were acquired in a data-dependent mode. Collected data were analyzed using the SEQUEST program for peptide identification.The silver staining gel of the glycerol gradient sedimentation was excised and destained in 15 mM Potassium ferricyanide/ 50 mM Sodium thiosulfate for 10 min with shaking. The gel was washed three times with distilled water and once with 200 mM NHFlag-tagged EAChm was subcloned into a pET vector generating pET- EAChm. Flag-tagged EAChm was produced in E. coli strain Rosseta (DE3) pLysS (Invitrogen). Lysate of E. coli in which flag-tagged EAChm was expressed was immunopurified with flag M2 agarose (Sigma). After extensive washing of the agarose, flag-tagged EAChm was eluted with flag peptides (Sigma) and dialysed against 50 mM KCl. The purified recombinant EAChm was subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then visualized using Coomassie brilliant blue R staining.2, and then various active fractions from column purification were added as indicated. After 20 min, 6.67 mM Na(C3H7COO), 7.4 kBq [3H]acetyl-CoA, and 50 mM KCl HEG were added in a final volume of 7.5 μl. The reaction was incubated at 27°C for 2.5 h and analyzed on 13.5% SDS—PAGE with subsequent fluorography basically as described previously [Chromatin was made by mixing the pEcE4 DNA template containing five ecdysone response elements (EcRE) upstream of the adenovirus E4 promoter and core histone octamer in the presence of high salt concentration and subsequent salt dialysis. The resulting chromatin was centrifuged on a linear 15 to 40% glycerol gradient for further purification. For the acetylation assay, the incubation temperature of the whole interaction was performed at 27°C. At first, a 1 μl aliquot of chromatin containing 62.5 ng of plasmid DNA and 62.5 ng of histones was mixed with 3 mM ATP and 2 mM MgCleviously , 26.Drosophila nuclear extract from 0- to 12-h-old embryos (200 g) was applied to a 20 ml Q Sepharose FF column and the bound protein was eluted with a linear KCl gradient (10 column volumes) from 0.1 to 1 M KCl in HEG. General transcription factors were confirmed by Western blotting. TFIIH and TFIIA were eluted in the 0.25 M KCl fraction. TFFIID, TFIIE and TFIIF were eluted in the 0.4 M KCl fraction. RNA polymerase II was eluted in the 0.5 M KCl fraction. TFIIH was subjected to 2 ml POROS heparin column chromatography and eluted with a linear KCl gradient (10 column volumes) from 0.1 to 1 M KCl in HEG. TFIIH was eluted in the 0.5 M KCl fraction. TFFIID, TFIIE and TFIIF were subjected to 2 ml SP sepharose FF column chromatography and eluted with a linear KCl gradient (10 column volumes) from 0.1 to 1 M KCl in HEG. TFFIID, TFIIE and TFIIF were eluted in the 0.5 M KCl fraction. TFFIID, TFIIE and TFIIF were further purified with 1ml POROS heparin column chromatography. TFFIID, TFIIE and TFIIF were eluted in the 0.5 M KCl fraction. Bacterially expressed His-tagged TFIIB was purified by Ni-NTA affinity chromatography and Flag-tagged TFIIA was purified by flag M2 agarose. Purification of general transcription factor and recombinant proteins were basically performed as described previously .in vitro transcription assay in which partially purified Drosophila general transcription factors and recombinant TFIIB were used basically as described previously [Chromatin template assembled by salt dialysis, containing five ecdysone response elements (EcRE) upstream of the adenovirus E4 promoter, was employed for the eviously . The chr6 cells/ 3.5 cm dish in serum free Schneider’s Drosophila media for 6 h. The cells were subsequently had their medium changed to Schneider’s Drosophila media with 10% FBS and cultured at 26°C for 72 h.S2 cells were grown in Schneider’s Drosophila media (Gibco) with 10% FBS. Double-strand RNAs (dsRNA) corresponding to portions of the Chm, EAChm, and EGFP  were synthesized using the MEGAscript RNAi kit (Thermo Fischer Scientific). 25 μg of dsRNA was added to 8x 10The primer sequences for generation of dsRNA were as follows;Chm Forward,5’- TAATACGACTCACTATAGGGTACGCCTTGGAGTCCGGCAGC -3’,Chm Reverse,5’- TAATACGACTCACTATAGGGTACAGGGGAACCTTCCGCCCG -3’,EAChm Forward,5’- TAATACGACTCACTATAGGGTACGCAATCCATAGCATTTGCTC -3’,EAchm Reverse,5’- TAATACGACTCACTATAGGGTACCGATGTCATCTAGAACCTGG -3’,EGFP Forward,5’- TAATACGACTCACTATAGGGTACATGGTGAGCAAGGGCGAGGAG -3’,EGFP Reverse,5’- TAATACGACTCACTATAGGGTACTTACTTGTACAGCTCGTCCAT -3’For knockdown, S2 cells were treated with dsRNA for 3 days.Total RNA was extracted from S2 cells treated with dsRNA by ISOGEN II (NIPPON GENE). The amount of 0.5 μg of total RNA was used for RT reaction using random hexamers (TAKARA), oligo(dT) primer (Life Technologies), and M-MuLV reverse transcriptase (NEB) for 1 hour at 42°C. RNA quantification by real-time PCR was performed using the KAPA SYBR FAST qPCR Kit (Kapa Biosystems), and normalized with reference to GAPDH. The primer sequences for RT-qPCR were as follows;5’- ACCGACTTCTTCAGCGACACC -3’GAPDH Forward, 5’- GCTCTGCATATACTTGATCAG -3’GAPDH Reverse, 5’- AGCACCCTTCAAGCTCTCGG -3’Chm Forward, 5’- CTTAAGCAGGAGTCGTCGATC -3’Chm Reverse, 5’- CATAGTCTCAGTTGCACTCAG -3’EAChm Forward, 5’- CAAATTCATCTGGGGAGCTGC-3’.EAChm Reverse, melanogaster dm3 reference genome with the program of Miseq, allowing one mismatch. Reads that could be uniquely mapped to a gene were used to calculate the expression level. The level of gene expression was quantified by the number of uniquely mapped reads per kilobase of exon per million mapped reads (RPKM). Scatter plots were created using the Partek Genomics Suite to evaluate the correlation between the duplicate sample data sets. Gene ontology (GO) analysis was performed using a Partek Genomics Suite. Significantly enriched GO functional groups were defined as having an enrichment score equal to or greater than 3 , and each functional group was assigned with a GO enrichment score calculated using Fisher`s exact test. All RNA-seq data can be found online in the NCBI GEO SuperSeries GSE73098.Quality of total RNA was checked using the Agilent RNA 6000 Nano kit with an Agilent 2100 Bioanalyzer instrument (Agilent Technologies). A library was prepared using the TruSeq Stranded mRNA LT Sample Prep kit (Illumina) and sequenced with the MiSeq system (Illumina). Samples were sequenced to a depth of approximately 4 million uniquely mapped reads per sample. Sequences were aligned to the Drosophila Heat map and hierarchical clustering of the gene expression profiles were created using the Partek Genomics Suite. Heat maps of control KD, Chameau KD and EAChm KD were normalized by calculating the RPKM based on the sum of all reads found in the exon regions of that gene. To identify differentially expressed genes, one-way analysis of variance (ANOVA) was used. By setting P < 0.05 and fold-change (FC) settings FC > 2, we obtained lists of differentially expressed genes between control KD and Chameau KD or control KD and EAChm KD cells. Hierarchical clustering of control KD, Chameau KD, and EAChm KD cells was performed using 212 genes that varied significantly among each of the groups with a statistical P value < 0.05. We subtracted the mean of the gene expression levels in the six paired samples, normalized each row in the data table, calculated the distance using Pearson correlation, and then used a “pairwise average-linkage” hierarchical clustering method for clustering. The scale was the standardized RPKM value.t-test. The error bars indicate the standard deviation of four independent experiments.The statistical analysis was performed using Excel 2013 software (Microsoft). Comparison were performed with two-tailed Student’s S1 Fig(TIFF)Click here for additional data file.S2 FigAsterisk indicates the upregulated genes common to Chameau and EAChm KD.(TIFF)Click here for additional data file.S3 Fig(TIFF)Click here for additional data file.S1 File(XLS)Click here for additional data file.S2 File(XLS)Click here for additional data file.


G93A transgenic mice. In this study, we examined whether BVA’s effects depend on acupuncture point (ST36) in the organs, including the liver, spleen and kidney, of hSOD1G93A transgenic mice. We found that BV treatment at ST36 reduces inflammation in the liver, spleen, and kidney compared with saline-treatment at ST36 and BV injected intraperitoneally in symptomatic hSOD1G93A transgenic mice. Those findings suggest that BV treatment combined with acupuncture stimulation is more effective at reducing inflammation and increasing immune responses compared with only BV treatment, at least in an ALS animal model.Amyotrophic lateral sclerosis (ALS) includes progressively degenerated motor neurons in the brainstem, motor cortex, and spinal cord. Recent reports demonstrate the dysfunction of multiple organs, including the lungs, spleen, and liver, in ALS animals and patients. Bee venom acupuncture (BVA) has been used for treating inflammatory diseases in Oriental Medicine. In a previous study, we demonstrated that BV prevented motor neuron death and increased anti-inflammation in the spinal cord of symptomatic hSOD1 Amyotrophic lateral sclerosis (ALS) is characterized by progressive degeneration of motor neurons and muscle weakness. The death of ALS patients is caused by respiratory failure within 3–5 years of the diagnosis. ALS has two types: familial ALS (fALS) caused by genetic mutations, including superoxidase dismutase 1 (SOD1), alsin, senataxin, angiogenin, VAMP-associated protein B, dynactin, transactive response (TAR) DNA-binding protein 43 (TDP43), fused in sarcoma (FUS) and C9ORF72; and sporadic ALS (sALS), which includes ninety percent of all ALS cases and is induced by various environmental and genetic factors. The etiology of ALS is varied and there is no effective therapy for ALS patients. Riluzole, a glutamate release inhibitor approved by the FDA, is used only as a medical treatment for expansion of life by 3–5 months [G93A-overexpressed ALS-mimic transgenic mice [G93A transgenic mice. Bee venom (BV) is used for anti-inflammatory, anti-nociceptive, and anti-allergic effects in allergic rhinitis mice, complete Freund’s adjuvant (CFA)-induced arthritis models, and neuropathic pain models ,3,4. In nic mice ,6,7. HowG93A transgenic mice treated with saline at ST36 and those injected with BV intraperitoneally. Those findings suggest that BV treatment combined with acupuncture stimulation is more effective at reducing inflammation and increasing immune responses than is BV-only treatment, at least in an ALS animal model.In this study, we found that BV treatment at ST36 reduced inflammation in the liver, spleen, and kidney compared with that of symptomatic hSOD1G93A transgenic mice. As shown in G93A transgenic mice was increased by 3.4-fold compared with age-matched wild type (WT) (1 ± 0.24) mice. Furthermore, we found that BV treatment at ST36 significantly reduced Iba-1 by 2.8-fold compared with age-matched hSOD1G93A transgenic mice. In addition, the anti-inflammatory effects of BV treatment at ST36 increased by 2.3-fold compared with BV injected by i.p. in the hSOD1G93A mice. To confirm the anti-inflammatory effects of BV treatment at ST36, we studied the expression level of the cyclooxygenase 2 (COX2) protein in the liver of WT and hSOD1G93A transgenic mice. COX2 positive hepatocytes were increased in the liver of hSOD1G93A transgenic mice compared with WT (1 ± 0.55) mice, but BV treatment at ST36 significantly reduced them by 3.3-fold compared with saline-treated hSOD1G93A transgenic mice. Those findings suggest that BV treatment at ST36 is effective at reducing inflammation in the liver of hSOD1G93A transgenic mice. To investigate the effects of BV on inflammation specific to injection method, we conducted BV treatment two ways, namely at acupuncture point ST36 and intraperitoneally (i.p.), in 14-week-old hSOD1G93A transgenic mice. As shown in G93A transgenic mice compared with WT (1 ± 0.59) mice. BV treatment at ST36 significantly reduced Iba-1 by 5.2-fold compared with saline-treated hSOD1G93A transgenic mice. In addition, the expression level of Iba-1 in the white pulp of the spleen was decreased by 3.5-fold by BV treatment at ST36 compared with BV injected i.p. in the hSOD1G93A transgenic mice. BV treatment at ST36 in hSOD1G93A transgenic mice also significantly reduced COX2-immuno positive cells by 5.6-fold, which were increased by 11.2-fold compared with WT mice (1 ± 0.86). As a pro-inflammatory protein, TNF-α expression in the white pulp of the spleen of the hSOD1G93A transgenic mice also increased by 11.7-fold compared with WT mice (1 ± 0.25), but BV treatment at ST36 significantly reduced its expression by 5.3-fold compared with hSOD1G93A transgenic mice. BV injection by i.p. decreased the expression of TNF-α by 3.5-fold in the spleen of the hSOD1G93A transgenic mice compared with saline-treated hSOD1G93A transgenic mice. BV treatment at ST36 seems to be more effective at reducing the expression of the pro-inflammatory protein TNF-α in the spleen than BV-injected i.p., but it was not significantly different. These findings suggest that BV treatment at ST36 could augment immune responses by reducing the inflammatory proteins in the spleen of the hSOD1G93A transgenic mice. To study the anti-inflammatory effects of BV treatment at ST36 in the spleen, which is involved in the immune response, we immunostained with Iba-1, COX2, and tumor necrosis factor (TNF)-α antibodies for the tissues of WT or hSOD1G93A transgenic mice. The expression of Iba-1 was increased by 2.8-fold in the kidney, as was shown in the liver and spleen, in the hSOD1G93A transgenic mice compared with age-matched WT mice (1 ± 0.23)    A. BV trenic mice A. COX2 p ± 0.44) B. BV tre ± 0.93) C. HoweveG93A transgenic mice and that electroacupuncture and melittin treatment enhanced anti-inflammation proteins [G93A transgenic mice. We found that BV treatment at ST36 reduced inflammatory proteins, including Iba-1, COX2, and TNF-α, in the liver, spleen, and kidney of hSOD1G93A transgenic mice compared with BV injected by i.p. This suggests that BV’s effects may be more effective with treatment at an acupuncture point and it explains the synergistic effect of acupuncture combined with BV compared with BV treatment only.ALS causes the loss of motor neurons in the brainstem, cerebral cortex, and spinal cord and leads to irreversible paralysis of muscles and finally to respiratory impairment. There are several cellular and molecular pathological mechanisms involved, such as glutamate excitation, inflammatory events, oxidative stress, mitochondrial dysfunction, protein aggregation, and energy failure, as in other neurodegenerative diseases. However, it is insufficient to develop treatments and preventative measures for these biomarkers of ALS. Neuroinflammation in the central nervous system (CNS) from ALS contributes to the disease process and the immune system of sALS patients is altered by immune cells, including remarkable reductions in CD4+CD25+ T-regulatory (T-reg) cells as well as CD14+ monocytes . This suproteins ,12. Baseet al. reported liver abnormalities and atrophy, and an increase of cytokines and hepatic lymphocytes in hSOD1G93A transgenic mice [G93A transgenic mice, but BV treatment at ST36 significantly reduced the expression level of inflammatory proteins, including Iba-1 and COX2 compared with BV injected i.p. in symptomatic hSOD1G93A transgenic mice. This suggests that BV treatment at the acupuncture point ST36 may reduce hepatotoxicity from inflammation and affect liver metabolism in ALS patients.Finkelstein nic mice . In our G93A mice as an ALS animal model, the spleen is markedly reduced in size and weight compared with age-matched B6 wild type mice even though their spleen cell number is identical. In addition, splenic follicular architecture, T cell function, and the lymphoproliferative response are decreased in end-stage hSOD1G93A transgenic mice. Immune dysregulation affecting both the adaptive and innate immune systems is a consistent hallmark in ALS [G93A transgenic mice compared with age-matched non-transgenic mice. Furthermore, BV treatment at ST36 reduced the expression level of Iba-1, COX2, and TNF-α compared with saline-treatment at ST36 in symptomatic hSOD1G93A transgenic mice. Those findings suggest that BV treatment at ST36 improves the immune regulation of hSOD1G93A transgenic mice through a synergic effect of acupuncture combined with BV. In hSOD1k in ALS . In our et al. have reported that granular inclusion of mutant SOD1 protein is detected in the liver and kidney by immunohistochemical analysis of ALS patients [G93A transgenic mice. BV treatment at ST36 significantly increased anti-inflammation proteins in the kidney compared with saline-treatment at ST36 in hSOD1G93A transgenic mice. This suggests that BV treatment at this acupuncture point could improve kidney dysfunction in an ALS animal model. In addition, we suggest that the mechanism of BV treatment at ST36 may occur by combined effect of some components of BV suppressing inflammatory signaling and the activation of the endogenous modulatory systems by acupoint stimulation. Therefore, it should be required to confirm the activity of individual components of BV. Future studies should investigate whether BV’s effects at ST36 are specific or generalized to other acupuncture points. In addition, the relationship between acupuncture point stimulation and meridian, and the mechanism of BV’s synergistic effects when combined with acupuncture compared with BV alone need to be explored. Jonsson patients . We obserd codon of the cytosolic Cu/Zn superoxide dismutase (hSOD1G93A), were originally obtained from Jackson Laboratory . All mice were handled in accordance with the United States National Institutes of Health guidelines, and all procedures were approved by the Institutional Animal Care and Use Committees of the Korea Institute of Oriental Medicine (Protocol number: #13-109). Hemizygous transgenic B6SJL mice carrying the mutant human SOD1 gene, which has a glycine-to-alanine base pair mutation at the 93Transgenic mice were identified using polymerase chain reaction (PCR) as described previously . All of n = 7) or intraperitoneally  in 14 week-old hSOD1G93A transgenic mice. The mice were treated with BV once every other day for two weeks. According to the human acupuncture point landmark and a mouse anatomical reference [n = 3) and transgenic hSOD1G93A mice  were injected at the ST36 acupuncture point with normal saline of an equal volume. Bee venom (BV) was purchased from Sigma  and diluted with saline. At a dose of 0.1 µg/g, bee venom was injected bilaterally at the Joksamli (ST36) acupuncture point . The liver, spleen and kidney were removed and fixed in 4% paraformaldehyde for three days at 4 °C After three days, the liver, spleen and kidney were embedded in paraffin. The tissues were 5 µm-thick sections and were mounted on glass slides. The tissue sections were prepared for immunostaining through xylene treatment and gradual rehydration with 95%–75% ethanol. Following de-paraffinization, the slides were treated with 3% hydrogen peroxide (H2O2) for 15 min to inactivate endogenous peroxidases and then blocked in 5% bovine serum albumin (BSA) in 0.01% PBS-Triton X–100  for 1 h at room temperature. The sections were then incubated with various primary antibodies, including anti-Iba-1 , anti-TNF-α , and anti-COX2 , overnight. Next, the sections were incubated with the secondary antibody for 1 h. For visualizing, the ABC kit and 3,3'-diaminobenzidine (DAB)/H2O2 substrate were used with a hematoxylin counterstain. After rinsing, the sections were dehydrated in ethanol, cleared in xylene, and coverslipped. Immunostained tissues were observed with a light microscope  and analyzed by Image J 1.46j software (NIH) .hSOD1p < 0.05.All data were analyzed using GraphPad Prism 5.0  and are presented as the mean ± standard error of the mean (SEM) where indicated. The results of immunohistochemistry and Western blots were analyzed using one-way ANOVAs followed by Newman-Keuls tests. Statistical significance was set at G93A transgenic mice. We found that BV treatment at ST36 reduces inflammation-related proteins including Iba-1, COX2, TNF-α in the liver, spleen, and kidney compared with saline-treatment at ST36 and BV injected i.p. in symptomatic hSOD1G93A transgenic mice. Those findings suggest that BV treatment combined with acupuncture stimulation is more effective at reducing inflammation and increasing immune responses compared with only BV treatment, at least in an ALS animal model. In summary, we examined whether BVA’s effects depend on acupuncture point (ST36) in the organs, including the liver, spleen and kidney, of hSOD1


While the prevalence of X4-tropic viruses in subtype B is relatively well studied the distribution of tropism in CRF06_cpx (causing Estonian epidemic) is less known, especially in the treatment experienced patients with established multidrug-resistance and who are candidates for therapy with CCR5 antagonist.Aim: to describe HIV-1 drug resistance mutations (DRMs) and co-receptor tropism in antiretroviral (ARV) treatment failing patients infected with HIV-1 CRF06_cpx and to establish their suitability to treatment with CCR5 receptor antagonists.[coreceptor] 2.5. algorithm, respectively. All sequence prediction results with a false-positivity rate above 10% were considered R5-tropic.Altogether 12 patients were studied – all with ARV therapy failure and considered to start CCR5 antagonist therapy. Genomic viral DNA was directly sequenced in pol region and V3 loop of gp120 in triplicates. HIV-1 DRMs and tropism were detected using Stanford University Drug Resistance Database and Geno2phenoOf 12 viruses 11 were successfully sequenced and analyzed. Patients have been diagnosed and ARV therapy initiated in 2002-2013. 8/11 cases had failure of the second ARV regimen, 4/11 of the third and in 2/11 of the fourth ARV regimen. Median viral load at the time of tropism testing was 53,326  and median CD4+ count 184 (IQR 142-217) cells/cmm. The most commonly used regimens were EFV + 3TC plus AZT (5 cases) or plus ABC (4 cases). Of 11 viruses 10 had resistance against two ARV classes (NRTI + NNRTI or NNRTI + INI) and 1 had triple class resistance (NRTI + NNRTI + PI). In NRTI treated population the most common primary DRM was M184V (10/11), followed by L74V/IL/I and K70E/R/EKQ (both 4/11). In NNRTI treated population DRMs were as follows: K103N (5/11), P225H (4/411), G190A/S and K101H/E (both 3/11), others were seen in lower frequency. In PI treated patients two primary DRMs were represented – I54V, V82A (both in same case). In INI treated population one primary DRM was represented – Y143R. The majority of viruses (10/11) were R5-tropic and all determined as CRF06_cpx by phylogenetic analysis.The Estonian HIV epidemic has reached the stage where the first patients are considered to start treatment with CCR5 antagonists. Almost all highly resistant viruses were R5-tropic suggesting that CCR5 antagonists are an appropriate option for CRF06_cpx viruses resistant to other ARV agents.


Escherichia coli ST131 isolates among 116 carbapenemase-producing strains. Of isolates from 16 countries collected during 2008–2013, 35% belonged to ST131 and were associated with blaKPC, H30 lineage, and virotype C. This study documents worldwide incidents of resistance to “last resort” antimicrobial drugs among a common pathogen in a successful sequence type.We characterized  Escherichia coli sequence type 131 (ST131) was identified as pathogenic to humans in 2008; retrospective research suggests that its isolates have been present since at least 2003. The group has spread extensively and has been linked to the rapid global increase in the prevalence of antimicrobial resistance among E. coli strains  (http://www.merck.com/about/featured-stories/infectious_disease.html) started in 2002 and AstraZeneca's global surveillance study of antimicrobial resistance (unpublished data) began in 2012, to monitor global antimicrobial resistance trends among gram-negative bacteria  were positive for blaNDM, 38 (33%) for blaKPC, 30 (26%) for blaOXA-48-like, 2 (2%) for blaVIM-1 and 2 (2%) were positive for blaIMP  belonged to the sequence type ST131. Antimicrobial susceptibilities, types of β-lactamases, the presence of the blaKPC, 13 (32%) for blaOXA-48-like, 3 (7%) for blaNDM-1, and 1 (2%) for blaIMP-14. ST131 was present in 16 countries spanning 5 continents , of ST131strains were positive for ntinents . The disfimH alleles were identified among ST131 isolates: 24 H30 (58%), 3 H41 (7%), 3 H54 (7%), 2 H22 (5%), 2 H27 (5%), and 2 H191 (5%); and 1 each (2%) belonging to H24, H32, H65, and the new fimH alleles H434 and H435. Of the 24 H30 ST131 strains, 19 (79%) belonged to the H30-R sublineage and 5(21%) to the H30-Rx sublineage.Various E. coli strains from India and Vietnam  for NDMs and 13/30 (43%) for OXA-48-like strains. Our results suggest that ST131 is most likely responsible for the global distribution of E. coli with blaKPC.Because of the unprecedented global success of ST131, the presence of carbapenemases had been carefully monitored by molecular epidemiologists but has been limited to case reports from several countries ; it was associated with blaKPC and was especially prominent during 2012–2013. The increase of the ST131 H30 lineage with blaKPC during 2012–13 is cause for concern.The expansion of the E. coli ST131 has received comparatively less attention than other antimicrobial-resistant pathogens. Retrospective molecular surveillance studies have shown that ST131 with blaCTX-M-15 was rare during the early 2000s, but that an explosive global increase followed during the mid-to-late 2000s  in most regions of the world, in a common community and hospital pathogen  among a very successful sequence type . We urgently need well-designed epidemiologic and molecular studies to clarify the dynamics of transmission, risk factors, and reservoirs for ST131.E. coli ST131strains with carbapenemases. This sequence type poses a major threat to public health because of its worldwide distribution and association with the dominant H30 lineage. This sequence type among E. coli has the potential to cause widespread resistance to carbapenems.The medical community can ill afford to ignore A brief description is provided of antimicrobial resistance surveillance and molecular characterization programs by pharmaceutical companies Merck and AstraZeneca.