Emerin is an integral membrane protein of the inner nuclear membrane. Mutations in emerin cause X-linked Emery-Dreifuss muscular dystrophy (EDMD), a disease characterized by skeletal muscle wasting and dilated cardiomyopathy. Current evidence suggests the muscle wasting phenotype of EDMD is caused by defective myogenic progenitor cell differentiation and impaired muscle regeneration. We obtained genome-wide expression data for both mRNA and micro-RNA (miRNA) in wildtype and emerin-null mouse myogenic progenitor cells. We report here that emerin-null myogenic progenitors exhibit differential expression of multiple signaling pathway components required for normal muscle development and regeneration. Components of the Wnt, IGF-1, TGF-β, and Notch signaling pathways are misexpressed in emerin-null myogenic progenitors at both the mRNA and protein levels. We also report significant perturbations in the expression and activation of p38/Mapk14 in emerin-null myogenic progenitors, showing that perturbed expression of Wnt, IGF-1, TGF-β, and Notch signaling components disrupts normal downstream myogenic signaling in these cells. Collectively, these data support the hypothesis that emerin is essential for proper myogenic signaling in myogenic progenitors, which is necessary for myogenic differentiation and muscle regeneration. The nucleus is the primary site of nucleic acid regulation, including DNA replication, RNA transcription, and the organization of active and repressed chromatin domains. Proper regulation of these processes is essential for successful lineage specification and differentiation during embryonic development and for tissue repair after injury. Thus, these processes must be tightly controlled to ensure that the appropriate repertoire of genes is expressed during specification and differentiation into particular cell types.The nucleus is separated from the cytosol by the nuclear envelope, which is composed of two lipid bilayers: the outer nuclear membrane (ONM), which is contiguous with the endoplasmic reticulum, and the inner nuclear membrane (INM). Underlying the INM is a meshwork of type V intermediate filament proteins called lamins, which are the major scaffolding component of the nuclear lamina Lap2β, Emerin, and MAN1 Emerin was one of the first INM proteins to be discovered Skeletal muscle is composed of multi-nucleated, terminally differentiated myofibrils. Peripheral to these fibers is a niche populated by muscle stem cells called satellite cells or myogenic progenitors. Upon muscle damage quiescent satellite cells become activated. These activated satellite cells then asymmetrically divide to repopulate the niche and generate transient-amplifying myoblasts, which proliferate rapidly Several signaling pathways are important for muscle differentiation and regeneration, including the TGF-β TGF-β-family signaling inhibits muscle development and regeneration, with inhibitory effects on both satellite cell recruitment and myoblast differentiation Notch expression is upregulated in activated satellite cells and Notch signaling promotes satellite cell differentiation and increased numbers of proliferative myogenic progenitor cells. Addition of the constitutively active Notch intracellular domain was shown to repress initiation of the myogenic program by inactivating MyoD Notch and Wnt signaling are temporally coordinated to ensure proper differentiation, as these pathways promote proliferation and differentiation, respectively. A switch from Notch to Wnt signaling is necessary during adult myogenesis The importance of micro-RNAs (miRNAs), ∼22 nucleotide non-coding RNAs, as regulators of diverse cellular processes including development and differentiation has become evident in recent years To elucidate molecular pathways implicated in the EDMD disease mechanism, we performed mRNA and miRNA expression profiling in emerin-null myogenic progenitors. Emerin-null myogenic progenitors were used because the skeletal muscle phenotype of EDMD is predicted to be caused by the failure of myogenic progenitors to differentiate properly and regenerate damaged muscles We performed whole genome mRNA microarray analysis on proliferating wildtype and emerin-null H2K myogenic progenitors to determine how myogenic gene expression was affected by emerin loss. H2K myogenic progenitors were used for this analysis because myogenic progenitors derived from emerin-null H2K mice are superior to the C2C12 myoblast-derived cell line in many ways. For example, decades in cell culture have caused C2C12 myoblasts to diverge significantly from the myoblasts they were derived from and are aneuploid for multiple chromosomes. Emerin-null H2K myogenic progenitors conditionally express the SV40 large-T antigen, which allows primary cells derived from these mice to be passaged directly for multiple generations without losing their progenitor identity RNA was isolated from 500,000 wildtype or emerin-null myogenic progenitors and hybridized to Affymetrix Mouse Whole Genome 430 2.0 microarrays (n = 2). The microarray data is available through the NCBI Gene Expression Omnibus and accessible through GEO accession numbers GSM787509–GSM787512. This analysis showed extensive differences in gene expression between wildtype and emerin-null myogenic progenitors, as 13.3% of genes  showed perturbed expression in emerin-null H2K myogenic progenitors. QVALUE software developed by Alan Dabney and John Storey was then used to increase the confidence in these results. Using this approach we found that a p-value <0.035 was required to obtain a False Discovery Rate (FDR) of 5%. Applying this threshold to our data results in 2792 genes out of 21804 unique transcripts (12.5%) altering mRNA expression by greater than 1.5-fold in emerin-null H2K myogenic progenitors. mRNA with altered expression in emerin-null myogenic progenitors included components of the CREBBP/p300 transcriptional pathway, including CREBBP, Ep300 and Kat2b . StudiesDll1 (1.74-fold) and Gsk3b (1.45-fold) were upregulated in emerin-null myogenic progenitors, while Ckn1b (0.64-fold), Rbpj (0.63-fold), Mapkapk3 (0.59-fold), Numb (0.59-fold), Adam12 (0.52-fold), Ccnd1 (0.48-fold), Mapk14/p38 (0.32-fold), Adam10 (0.24-fold), and Adam17 (0.20-fold) were downregulated , Wisp1 (1.63-fold), Sfrp1 (1.59-fold), Wnt5b (1.5-fold), and Fzd3 (1.3-fold) were all upregulated. Wnt pathway transcripts Wnt4 (0.85-fold), Fzd4 (0.82-fold), Rhou (0.80-fold), Dvl2 (0.80-fold), Fzd7 (0.77-fold), Dvl1 (0.75-fold), Sfrp2 (0.66-fold), Fzd2 (0.61-fold), Fzd1 (0.54-fold), Fzd6 (0.41-fold), and Fbxw2 (0.33-fold) were downregulated. Decreased expression of several Wnt receptors suggests that Wnt signal transduction may be impaired in emerin-null myogenic progenitors.Expression of Wnt pathway components was also altered in emerin-null myogenic progenitors . Wnt patTgfb2 (2.89-fold), Foxo1 (1.84-fold), Tgfb1i1 (1.57-fold), Serpine1 (1.53-fold), Ep300/p300 (1.31-fold) and Tgfbr1 (1.28-fold) were upregulated. Tgfbr2 (0.85-fold), Tgfbr3 (0.82-fold), Col3a1 (0.76-fold), Kat2b/PCAF (0.73-fold), Stat1 (0.65-fold), Crebbp/CBP/p300 (0.62-fold), Col1a2 (0.55-fold), Pdgfb (0.48-fold), Bmp1 (0.37-fold), and Acvr1 (0.3-fold) were downregulated. These results suggest that TGF-βsignaling is attenuated in emerin-null myogenic progenitors, resulting in impaired activation of downstream target genes.Expression of TGF-β pathway components was altered in emerin-null myogenic progenitors . Tgfb2 (Igfbp2 (13.11-fold), Igf2bp3 (6.25-fold), Igf1 (4.42-fold), Igfbp3 (2.7-fold), Igfbp4 (2.5-fold), Igf2bp1 (2.04-fold), Igf2 (1.83-fold), Igfbp5 (1.61-fold), Igf2bp2 (1.59-fold), Igf1r (1.57-fold) and Igf2r (1.46-fold) were upregulated. Igfbp6 (0.77-fold) and Igfbp7 (0.39-fold) were downregulated. While Igf1 and Igf2 are upregulated, numerous IGF-binding proteins appear to be concomitantly upregulated, suggesting emerin-null progenitors are attempting to compensate for increased IGF signaling. From this expression data it cannot be predicted whether IGF signaling is disrupted in emerin-null myogenic progenitors.Emerin-null myogenic progenitors exhibit altered expression of IGF pathway genes . Igfbp2 Rbpj (1.9-fold) was upregulated and Notch, Gsk3b (0.78-fold), Mapk14/p38 (0.73-fold), Ccnd1 (0.69-fold), Mapkapk3 (0.46-fold) and Cdkn1b (0.29-fold) were downregulated (Wnt11 (5.45-fold), Wnt5b (2.56-fold), Frzb (1.98-fold), and Wisp1 (1.5-fold) were upregulated in emerin-null myogenic progenitors (Fzd1 (0.38-fold), Fzd5 (0.63-fold), Fzd6 (0.58-fold), Fzd7 (0.47-fold), Wnt2b (0.44-fold), Wnt4 (0.27-fold) and Wnt6 (0.19-fold). Components of the TGF-β and IGF pathways were also validated by qPCR, as Igfbp3 (5.27-fold), Igfbp5 (2.78-fold), Igf1 (3.3-fold), Tgfbr1 (1.96-fold), Foxo1 (2.01-fold) and Ep300/p300 (1.81-fold-fold) were upregulated and Tgfbr2 (0.6-fold), Crebbp/CBP/p300 (0.54-fold), Bmp1 (0.54-fold), Kat2b/PCAF (0.37-fold) and Tgfbr3 (0.02-fold) were downregulated (Quantitative RT-PCR (qPCR) was used to validate 49 components of the Notch, Wnt, TGF-β, and IGF signaling pathways. Supporting the microarray results, we found the Notch pathway component egulated . Wnt patgenitors . Emerin-egulated .Gsk3b, Mapk14 and Igfbp3 expression was measured to test if emerin was directly responsible for their altered gene expression. Emerin was overexpressed 601-fold, which is similar to the amount of decreased emerin expression seen in emerin-null myogenic progenitors (839-fold). mRNA levels were normalized to Gapdh expression. Changes in Gsk3b, Mapk14 and Igfbp3 mRNA expression were determined by comparing their expression in GFP-emerin expressing cells to GFP transfected cells. Importantly, expression of GFP-emerin increased the expression of Gsk3b (3.55±1.5 fold) and Mapk14 (1.56±0.35 fold) and decreased Igfbp3 expression (0.65±0.2 fold) in emerin-null cells , Ppp2r1a (0.81-fold), Dlv2 (0.74-fold), Fbxw2 (0.74-fold), Ctnnbip1 (0.73-fold), Lrp5 (0.73-fold), Ctbp1 (0.66-fold), Dvl1 (0.62-fold), Porcn (0.59-fold), Rhou (0.48-fold), and Sfrp2 , Inha (2.04-fold), Stat1 (0.42-fold), Col3a1 (0.28-fold), Col1a2 (0.26-fold), Gdf6 (0.26-fold), Pdgfb (0.26-fold), Itgb7 (0.13-fold), and Acvr1 , representing 7.7% of all known murine miRNAs. We used the MicroCosm Targets algorithm to identify potential mRNA targets. Interestingly, many of these miRNAs are predicted to target myogenic signaling components, some of which also showed aberrant mRNA expression .Of particular note are some of the most highly dysregulated miRNAs, including miR-183 (3.25-fold), miR-100 (1.97-fold), miR-713 (1.92-fold), miR-199a-5p (1.8-fold), miR-207 (1.74-fold), miR-132 (0.17-fold), miR-192 (0.39-fold), miR-195 (0.40-fold), miR-297a* (0.42-fold) and miR-298  at the Pax7 promoter nd or 3rd decade of life; Collectively, perturbation of these signaling pathways may help explain the relatively late onset . Several miRs that were shown to play roles in muscle differentiation, including miR-1 and miR-206, did not show disrupted expression in emerin-null myogenic progenitors. While myomiR expression is driven by myogenic regulatory factor expression, our studies do not show a direct relationship between perturbed myogenic regulatory signaling and disrupted expression of myomiRs in emerin-null myogenic precursors. Though it is now possible to acquire genome-wide miRNA expression data, the complexity of interactions between many miRs that target a single transcript, as well as the hundreds of transcripts that are targeted by a single miR make the annotation and validation of miR targeting an important goal for the future. Future studies will also analyze how changes in expression of miRs in emerin-null myogenic progenitors affect cell signaling and transcriptional pathways important for myogenic differentiation and skeletal muscle regeneration.Now that we have identified perturbations of the Notch, Wnt, TGF-β and IGF pathways in emerin-null myogenic progenitors, it will be important to test if impaired signaling persists during differentiation of emerin-null progenitors. It will also be important to determine the relative contributions of each signaling pathway to the impaired differentiation and muscle regeneration seen in emerin-null myogenic progenitors. Further studies also need to examine whether using pharmacological inhibitors or activators of these important myogenic signaling pathways are able to restore normal signaling pathway function in emerin-null myogenic progenitors. These compounds would then be candidates for treating the impaired muscle regeneration in emerin-null mice and EDMD patients. Using this approach we predict potential drugs will be identified to treat EDMD patients.btsA58 mice 2. Upon attachment of a single myogenic progenitor to the well, the fiber was removed and the myogenic progenitor was incubated in proliferation media for another 48 hours at 33°C, 10% CO2. Approximately 200 cells are expected after 48 hours and these were split and proliferated until enough cells were obtained for our analyses. H2K myogenic progenitors were maintained in proliferation media at 33°C and 10% CO2. Cells between passages 4–10 were used for these studies.Wildtype and Emerin-null H2K mouse myogenic progenitors were a generous gift from Tatiana Cohen and Terry Partridge . Emerin-null H2K mice were generated by Tatiana Cohen and Terry Partridge by breeding emerin-null and H-2KTotal RNA was isolated from 500,000 sub-confluent proliferative wildtype or emerin-null H2Ks using the miRNeasy Mini Kit  and processed according to manufacturer’s protocol. RNA was isolated from two independent cell culture plates for each replicate to determine significant changes in mRNA and miRNA expression. Each isolated RNA sample was then hybridized to GeneChip Mouse Genome 430 2.0 Arrays (Affymetrix) at the University of Chicago Functional Genomics Facility. The Affymetrix statistical software suite was used for statistical analysis using Robust Multichip Analysis at the Functional Genomics Facility to identify significant differences between both samples. To identify changes in miRNA expression 1 ug of total RNA was hybridized to GeneChip miRNA Arrays (Affymetrix) and processed at the University of Chicago Functional Genomics Facility. Principal component analysis was performed on the samples and the two conditions segregated into two independent and defined groups. Statistical analysis using Robust Multichip Analysis was performed by the Functional Genomics Facility to identify significant differences between both samples. The microarray data is MIAME compliant and is available through the NCBI Gene Expression Omnibus, accessible through GEO accession numbers GSM787505-GSM787512. Selected mRNA levels were confirmed by qPCR, as described below.2 First Strand Kit . qPCR was performed using a combination of individual primer sets  and TGF-β/BMP  pathways using RT2 Real-Time SYBR Green/fluorescein PCR master mix .Total RNA was prepared as above and cDNA was generated from RNA using MMLV Reverse Transcriptase  per manufacturer instructions for individual qPCRs. For qPCR arrays, cDNA was generated using the RT2. After 16–48 hours Fluorescence Activated Cell Sorting selected GFP or GFP-emerin expressing cells. On average 120,000 GFP-positive cells were received per electroporation. RNA was isolated and cDNA was generated as previously described. Gsk3b, Mapk14, Igfbp3 and Gapdh mRNA expression was monitored using qPCR. Gsk3b, Mapk14 and Igfbp3 levels in GFP or GFP-emerin expressing myogenic progenitors were normalized to Gapdh expression. Gsk3b, Mapk14 and Igfbp3 expression in GFP expressing progenitors is set to one and Gsk3b, Mapk14 and Igfbp3 expression is plotted as fold-change of GFP-emerin expressing progenitors as compared to GFP expressing progenitors.pEGFP (Clontech) or pEGFP-emerin was electroporated into 2 million emerin-null myogenic progenitors using the Neon transfection system (Invitrogen) with 2 pulses of 1225 volts at a width of 30 milliseconds. The electroporated progenitors were plated in separate 150 mm plates and incubated at 33°C, 10% COMisregulated miRNAs were entered into Microcosm Targets, an online repository of predicted and validated targets of all miRBase-annotated miRNAs. Targets of interest within the Wnt, TGF-β, IGF-1, and Notch pathways were selected based on their involvement in myogenesis or nuclear envelope structure and function.7 cells/ml. An equal number of wildtype and emerin-null cells was separated by electrophoresis on NuPAGE 4-12% Bis-Tris Gels , and transferred to nitrocellulose membranes. Membranes were blocked for 1 hour in 3% BSA in phosphate buffered saline (PBS) containing 0.1% Tween 20 (PBS-T) followed by incubation for 2 hours at room temperature or overnight with the indicated primary antibodies. Antibodies used were against p38 , phospho-p38 , Kat2b , GSK3β , Smad2 , Myf5 , and γ-tubulin  in 3% BSA in PBS-T. Membranes were washed 5 times in PBS-T and placed in appropriate secondary antibody  at a concentration of 1∶10,000 for 2 hours at room temperature. Antibody binding was assayed using ECL chemiluminescence detection reagent . Images were scanned and band intensities were determined using Quantity One software (Bio-Rad).H2K cells were grown under proliferative conditions as described. Wildtype and emerin-null cells were treated with 12.5 ng/ml IGF-1  for 10 minutes. Cells were collected and resuspended in NuPAGE LDS Sample Buffer  containing 14 µM β-mercaptoethanol  at a concentration of 10Figure S1Ingenuity Systems analysis to identify effect of emerin loss on IGF-1 signaling pathway. The network was generated through the use of IPA  on normalized mRNA values. Nodes represent molecules in a pathway, while the biological relationship between nodes is represented by a line (edge). Edges are supported by at least one reference in the Ingenuity Knowledge Base. The intensity of color in a node indicates the degree of up- (red) or down- (green) regulation. Nodes are displayed using shapes that represent the functional class of a gene product . Edges are marked with symbols to represent the relationship between nodes .(TIF)Click here for additional data file.Figure S2Ingenuity Systems analysis to identify effect of emerin loss on Notch signaling pathway. The network was generated through the use of IPA  on normalized mRNA values. Nodes represent molecules in a pathway, while the biological relationship between nodes is represented by a line (edge). Edges are supported by at least one reference in the Ingenuity Knowledge Base. The intensity of color in a node indicates the degree of up- (red) or down- (green) regulation. Nodes are displayed using shapes that represent the functional class of a gene product . Edges are marked with symbols to represent the relationship between nodes .(TIF)Click here for additional data file.Figure S3Ingenuity Systems analysis to identify effect of emerin loss on Wnt/β-catenin signaling pathway. The network was generated through the use of IPA  on normalized mRNA values. Nodes represent molecules in a pathway, while the biological relationship between nodes is represented by a line (edge). Edges are supported by at least one reference in the Ingenuity Knowledge Base. The intensity of color in a node indicates the degree of up- (red) or down- (green) regulation. Nodes are displayed using shapes that represent the functional class of a gene product . Edges are marked with symbols to represent the relationship between nodes .(TIF)Click here for additional data file.Figure S4Ingenuity Systems analysis to identify effect of emerin loss on TGF-β signaling pathway. The network was generated through the use of IPA  on normalized mRNA values. Nodes represent molecules in a pathway, while the biological relationship between nodes is represented by a line (edge). Edges are supported by at least one reference in the Ingenuity Knowledge Base. The intensity of color in a node indicates the degree of up- (red) or down- (green) regulation. Nodes are displayed using shapes that represent the functional class of a gene product . Edges are marked with symbols to represent the relationship between nodes .(TIF)Click here for additional data file.Figure S5Protein expression is similar between primary myoblasts and H2K myoblasts. A) Whole cell lysate from primary myoblasts and H2K myogenic progenitors were separated by SDS-PAGE and western-blotted for Kat2b, GSK3β, Myf5 and γ-tubulin. B) Densitometry was performed on blots in panel A to measure protein expression. Kat2b, GSK3β and Myf5 densitometry were normalized to γ-tubulin for each sample.(TIF)Click here for additional data file.


The development of human T- cell lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) may be related to genetic factors related to the presentation of viral antigens by human leukocyte antigen (HLA).To determine the HLA class I genotype of HTLV-1-infected patients.420 HTLV-1-infected patients  and 140 HAM/TSP) from the HTLV Reference Center in Salvador, Brazil were evaluated. HLA genotype was performed using automated DNA sequencers and analyzed using the software program Assign-SBT TM 3.2.The HLA types most frequently observed in all individuals combined were A*02 (26.1%), A*03 (10.5%); B*35 (13.1%), B*44 (10.4%), Cw*04 (21.9) and Cw* 07 (17.7%). The presence of HLA-A*02 reduced the odds of HAM-TSP . The frequency of homozygosity of HLA-A was 8%, 4% for HLA-B and 9% for HLA-C overall. Proviral load was significantly lower in HC among those heterozygous at all three HLA class I loci group relative to HC who were homozygous at one or more HLA class I loci group (p = 0.029). However, no difference in homozygosity between proviral load in the HAM/TSP patients was observed (p = 0.57).HLA-A*02 allele is more prevalent in the HC patients, which suggests a protective role of this allele against HAM/TSP. Furthermore, HC patients had a higher frequency of heterozygosity at all three HLA class than that in HAM/TSP patients.


The chemokine receptor CXCR4 regulates cell migration during ontogenesis and disease states including cancer and inflammation. Upon stimulation by the endogenous ligand CXCL12, CXCR4 becomes phosphorylated at multiple sites in its C-terminal domain. Mutations in the CXCR4 gene affecting C-terminal phosphorylation sites are a hallmark of WHIM syndrome, a genetic disorder characterized by a gain-of-CXCR4-function. To better understand how multi-site phosphorylation of CXCR4 is organized and how perturbed phosphorylation might affect CXCR4 function, we developed novel phosphosite-specific CXCR4 antibodies and studied the differential regulation and interaction of three C-terminal phosphorylation sites in human embryonic kidney cells (HEK293). CXCL12 promoted a robust phosphorylation at S346/347 which preceded phosphorylation at S324/325 and S338/339. After CXCL12 washout, the phosphosites S338/339 and S324/325 were rapidly dephosphorylated whereas phosphorylation at S346/347 was long-lasting. CXCL12-induced phosphorylation at S346/347 was staurosporine-insensitive and mediated by GRK2/3. WHIM syndrome-associated CXCR4 truncation mutants lacking the S346/347 phosphosite and the recently identified E343K WHIM mutant displayed strongly impaired phosphorylation at S324/325 and S338/339 as well as reduced CXCL12-induced receptor internalization. Relevance of the S346-S348 site was confirmed by a S346-348A mutant showing strongly impaired CXCL12-promoted phosphorylation at S324/325 and S338/339, defective internalization, gain of calcium mobilization, and reduced desensitization. Thus, the triple serine motif S346-S348 contains a major initial CXCR4 phosphorylation site and is required for efficient subsequent multi-site phosphorylation and receptor regulation. Hierarchical organization of CXCR4 phosphorylation explains why small deletions at the extreme CXCR4 C terminus typically associated with WHIM syndrome severely alter CXCR4 function. Stimulus-dependent phosphorylation of G protein-coupled receptors (GPCRs) represents a major mechanism regulating signal transduction and receptor trafficking G protein-coupled CXCR4, the receptor of the chemokine CXCL12, is essential for embryonic development of various organ systems, serves as HIV coreceptor, and plays a major role in diseases including cancer and inflammation warts, hypogammaglobulinemia, infections, and myelokathexis) and gain-of-CXCR4-function knockin mice carrying a heterozygous R334X truncation mutation, which corresponds to the most frequent WHIM syndrome-causing mutation in humans Early reports analyzing CXCR4 C-terminal truncation mutants described that deletions within the 12 C-terminal residues severely impair CXCL12-induced internalization – an effect expected to reduce CXCR4 desensitization Thus, while phosphorylation occurs at multiple dispersed CXCR4 C-terminal sites  125I-CXCL12 , AMD3100 , Lambda Protein Phosphatase , phorbol 12-myristate 13-acetate , forskolin , epidermal growth factor , staurosporine .CXCL12 ,Phosphosite-specific rabbit antibodies for the S338/339-phosphorylated and S346/347-phosphorylated forms of the CXCR4 receptor were generated against the following peptides: RGGH(pS)(pS)VSTE (S338/339) and VSTESE(pS)(pS)SFHSS (S346/347), which contained two phosphorylated serine residues (pS). The peptides were purified by HPLC, coupled to keyhole limpet haemocyanin and mixed 1∶1 with Freund’s adjuvant for immunization. Specificity of the antisera was tested using dot blot analysis. For subsequent analysis, antibodies were affinity purified against their immunizing peptide and against the non-phosphorylated peptide using the SulfoLink kit . The polyclonal phosphosite-specific antibody for the S324/325-phosphorylated form of the CXCR4 receptor (#5199) was a kind gift of Dr. Jeffrey L. Benovic TM3.1(+) vector . The cDNA for a CXCR4-ST/A mutant lacking C-terminal serines and threonines, a S346-348A mutant, an E343K mutant, and for CXCR4 mutants lacking the last 10 and 17 residues  were generated by gene synthesis  and inserted into CXCR4-WT using the endogenous Sph I site (bp 967) and the Not I extension. Sequence identity was verified by double strand sequencing.cDNA for wildtype CXCR4 (CXCR4-WT) was amplified by reverse transcription PCR from mouse spleen cDNA with Nhe I/Not I extensions. The construct received an N-terminal HA epitope tag or an N-terminal T7 epitope tag and was subcloned into pcDNAHuman embryonic kidney cells  were cultivated in DMEM  supplemented with 10% defined fetal bovine serum , 2 mM L-glutamine, and 100 units/ml penicillin/streptomycin (PAA). JetPEI reagent  was used for transient and stable transfections. Stable transfectants were selected in the presence of 400 µg/ml G418.125I-CXCL12. For homologous competitive radioligand binding, cells were incubated for 2 h at 4°C with 125I-CXCL12 containing increasing amounts of unlabeled CXCL12. Cells were washed twice with ice cold phosphate buffered saline (PBS) and then lysed in 10 mM Tris buffer (pH7.4). IC50 was calculated using the “homologous competitive binding curve” option of the GraphPad Prism 4.0a software. For pulse chase analyses of radioligand/receptor internalization, cells were loaded with 125I-CXCL12 for 2 h at 4°C. Cultures were washed with ice cold PBS and harvested either immediately  or transferred to 37°C to permit internalization. Residual surface-bound 125I-CXCL12 was stripped by a double wash with acidic citrate buffer. Then, cells were lysed in 10 mM Tris buffer. Counts of mock-transfected cultures were subtracted.Binding and internalization assays were performed as described Cells were processed as described Immunoblotting procedures were performed as described An established siRNA-mediated knockdown protocol for GRK2 and GRK3 was used i/o-coupled receptors to Ca2+ mobilization 2+]i measured using the FlexStation3 microplate reader as described Cells were co-transfected with CXCR4 constructs and a 16z44 G protein chimera engineered to link GStatistical tests were done by ANOVA including the indicated post-tests or by Student's t-test using GraphPadPrism software.To investigate multi-site phosphorylation properties of CXCR4, we generated two novel polyclonal phospho-selective antibodies for S338/339 and S346/347 and used them along with a polyclonal antibody for phosphorylated S324/325 The four antibodies were then characterized in immunoblots with lysates from HEK293 cells stably expressing CXCR4 with an N-terminal hemagglutinin (HA) epitope tag. The cultures received vehicle or CXCL12 for 15 min before they were harvested. Aliquots of CXCL12-stimulated lysates were dephosphorylated with Lambda-Protein Phosphatase (λ-PP) before loading. Anti-S341-S352 detected a broad band of the expected size at 47 kDa. The signal was strongly reduced after CXCL12-stimulation and was fully recovered after dephosphorylation . Thus, wThe phospho-selective antibodies produced strong CXCR4-specific signals in CXCL12-stimulated samples and weak signals in non-stimulated and dephosphorylated samples . CXCL12 We then used the four antibodies to examine kinetics of CXCL12-induced phosphorylation. Stable CXCR4 transfectants were harvested immediately before as well as 2, 5, and 15 min after CXCL12 stimulation. Aliquots of the lysates were loaded four times, immunoblotted, and detected with anti-S341-S352 and the three phospho-selective antibodies . Blots wDephosphorylation kinetics were studied after 30 min CXCL12 stimulation. The extended stimulation period was used to ensure maximum phosphorylation. The CXCL12 stimulus was ended by an acidic wash followed by 5, 15, 30, and 60 min ligand-free intervals in the presence of the CXCR4 antagonist AMD3100 . Thus, tIt has been reported that extended CXCL12 stimulation (≥180 min) causes CXCR4 degradation in HEK293 cells Stimulus-dependent phosphorylation of GPCRs is mediated by multiple kinases including protein kinase A (PKA), protein kinase C (PKC), and G protein coupled receptor kinases (GRKs) We then used the PKC inhibitor staurosporine to test the contribution of PKC to the CXCL12- and PMA-induced CXCR4 phosphorylation . This sh125I-labeled 12G5 anti-CXCR4 antibody We then studied the influence of the extreme CXCR4 C terminus on phosphorylation at S324/325 and S338/339. To this end, we used two CXCR4 mutants with an N-terminal T7-tag and deletions of the last 10 and 17 C-terminal residues , which correspond to WHIM syndrome-associated mutations in the human CXCR4 gene CXCL12-induced phosphorylation was studied in transient transfectants 15 min after vehicle and CXCL12 stimulation. Since the phospho-selective antibodies produced some constitutive signals under these experimental conditions, we subjected aliquots of the lysates to Lambda-PP dephosphorylation to demonstrate phosphoselectivity of the signal . CXCL12-Having shown that the CXCL12-promoted phosphorylation at S346/347 precedes that at S324/325 and S338/339 and that phosphorylation at S324/325 and S338/339 is severely reduced when the last ten CXCR4 residues are absent, we hypothesized that phosphorylation at residues S346-S348 is obligate for efficient phosphorylation to occur at the other sites. To test this hypothesis, we generated a HA-tagged S346-348A mutant . After pulse labeling of the extracellular HA-tag with anti-HA antibody in live cells at 4°C and several washes, cells were fixed and detected with a peroxidase-conjugated secondary antibody and a peroxidase-driven color reaction. The assay showed similar labeling intensities for wildtype CXCR4, E343K-CXCR4, and S346-8A-CXCR4 . Mock-trmutation . CXCL12-mutation  and quanmutation . After pmutation . Applicamutation  but littmutation . Consistmutation .i-coupled receptors to the calcium pathway G protein coupling of CXCR4 was examined in calcium mobilization experiments using FLIPR technology. The CXCR4 variants were co-transfected with a 16z44 G protein chimera that was engineered to link GSequential stimulation was applied to determine homologous CXCR4 desensitization. When CXCL12 (80 nM) was administered 180 s after a precedent 80 nM stimulus, it evoked only a small calcium response irrespective of the CXCR4 variant examined (not shown). To circumvent major CXCR4 desensitization, we reduced the CXCL12 concentration in the pre-stimulus to 5 nM. Normalization of the traces with the mean of the first maximum of wildtype CXCR4 showed that the calcium responses to both the first 5 nM stimulus and to the second 80 nM stimulus were larger with CXCR4-E343K and CXCR4-S346-8A than with wildtype CXCR4 , which cUsing novel and established phospho-selective antibodies for the CXCR4 C-terminal sites S324/325, S338/339, and S346/347, we provide evidence that S346/347 represents a major C-terminal phosphorylation site. Upon CXCL12 stimulation, S346/347 reached maximal phosphorylation faster than S338/339 and S324/325. Deletion of S346-S348 reduced CXCL12-promoted phosphorylation at S324/325 and S338/339, caused a gain-of-CXCR4-function and reduced receptor internalization and desensitization. Our findings indicate hierarchical organization of the multiple phosphorylation events taking place at the CXCR4 C-terminal domain and help explain why mutations in the CXCR4 gene which cause small alterations at the extreme C terminus can severely affect CXCR4 regulation and signaling.We established that the rabbit monoclonal anti-S341-S352 antibody UMB-2 is highly selective for CXCR4 and that the antibody recognizes only the non-phosphorylated C terminus. Immunoblots with lysates from CXCR4-overexpressing HEK293 cells showed that 15 minutes CXCL12 stimulation were sufficient to trigger an almost complete loss of UMB-2 signal. The finding that dephosphorylation fully restored the UMB-2 signal indicates that one or several of the potential phosphosites in the UMB-2 epitope  become quantitatively phosphorylated upon CXCL12 exposure. Thus, by detecting dephosphorylated and non-dephosphorylated aliquots of a sample with UMB-2 one can visualize total and non-phosphorylated CXCR4. The difference between the two signals reflects C-terminal CXCR4 phosphorylation and can serve as an indicator for CXCR4 activation. UMB-2 readily detects low endogenous CXCR4 levels in HEK293 cells and brain tissue Our dot blot analyses showed that phosphorylation at the S346/347 site is sufficient to prevent UMB-2 binding. Given that UMB-2 fails to recognize lysates from CXCL12-stimulated cells, we reasoned that the S346-S348 motif might contain a major CXCR4 phosphorylation site. To test this assumption, we developed a phospho-selective anti-pS346/347 antibody. Using this tool, we showed that this site is indeed rapidly phosphorylated upon CXCL12 stimulation. CXCL12-promoted phosphorylation at S346/347 was mediated by GRK2/3 and occurred in a PKC-independent manner. This property renders S346/347 distinct from previously identified phosphosites in the CXCR4 C terminus, which include S324/325, S330, and S338/339 Another distinguishing feature of S346/347 phosphorylation is its permanent nature: whereas S324/325 and S338/339 were rapidly dephosphorylated after CXCL12 washout, major dephosphorylation was not observed for S346/347 during the investigated 60 min ligand-free interval. Ligand-induced phosphorylation of GPCRs typically induces ß-arrestin/receptor interaction Most WHIM syndrome patients carry heterozygous mutations in the CXCR4 gene which eliminate the last 10 to 19 residues of the C terminus But how do WHIM patients lacking CXCR4 mutations Our studies in HEK293 cells show that serine residues 346 and 347 constitute a major CXCR4 phosphorylation site. Elimination of the S346-S348 cluster severely impairs phosphorylation at the intact C-terminal sites S324/325 and S338/339. We suggest that phosphorylation events at the CXCR4 C terminus occur hierarchically requiring initial phosphorylation at S346/347. These results identify a novel structural determinant of CXCR4 regulation and help decipher how mutations affecting the CXCR4 C terminus lead to signaling perturbances and disease.Figure S1Dot blot analysis of phospho-sensitive CXCR4 antibodies.A, Schematic representation of CXCR4 C-terminal residues 322–352. Sequences of peptides #1 - #3 used in the dot blot are underlined. Serine residues 324/325, 338/339, and 346/347 used for phosphorylation in the phosphopeptides are highlighted. B, Decreasing amounts (2–0.125 µg) of non-phospho and phosphopeptides #1 - #3 were blotted onto 4 PVDF membranes and detected with anti-S341-S352, anti-pS346/347, anti-pS338/339, and anti-pS324/325 as indicated. Anti-S341-S352 detects the S341-S352 epitope only when serine residues 346 and 347 are not phosphorylated. The phosphoantibodies recognize their phosphorylated epitopes with high selectivity showing little crossreactivity.(TIF)Click here for additional data file.


The expression of VH4 gene family during immunoglobulin heavy chain synthesis has been detected in autoimmune diseases. In particular, the rat monoclonal antibody 9G4 detects VH4-34-encoded immunoglobulins and B cells expressing these antibodies as surface receptor (autoreactive 9G4+ B cells).To determine whether autoantibodies commonly associated with rheumatoid arthritis (RA) express 9G4 idiotope (9G4-Id).Serum from 27 patients with established RA and 46 polyarthritis patients (<6 weeks duration) of whom 23/46 were subsequently diagnosed with RA  and 23/46 with other arthritis  was studied. 9G4-Id detection on anti-CCP, anti-tetanus toxoid (TT), Pneumococcal capsular polysaccharide (PCP) antibodies and total serum IgG and IgM was measured by ELISA.23/27 patients with established RA had anti-CCP antibodies, 8 of whom had 9G4+ anti-CCP. All were positive for both IgM and IgG anti-CCP. 9G4-Id detection levels correlated more closely with IgM than IgG-CCP. In ERA group, 15/23 patients had anti-CCP and 4/23 had 9G4+ anti-CCP. All 4 patients had both IgM and IgG anti-CCP. In ENRA, only one patient had 9G4+ IgM anti-CCP, albeit at low titer. 9G4-Id was not detected on TT or PCP antibodies.We describe for the first time the expression of VH4-34 heavy chain gene by autoantibodies to citrullinated peptides early after RA onset. In established RA, the expression of VH4-34 gene by anti-CCP antibodies is positively correlated with their titer, particularly with IgM-CCP. Therefore, we suggest that the expansion of CCP-specific B cell clones may be due to robust expansion of un-switched B cell clones, possibly including those in the splenic marginal zone or their analogous.


PCR-based testing has been widely utilized to assess the prevalence of Xenotropic Murine Leukemia Virus-related Virus (XMRV) in prostate cancer and chronic fatigue syndrome patients. An alternative approach, screening for antibodies elicited by XMRV infection, represents a more desirable option for large-scale epidemiologic studies. In this study, blood donor and retrovirus-infected populations were screened for serologic evidence of XMRV infection.Plasma from 1000 US blood donors, 100 HIV-1 infected Cameroonians, 486 HTLV-I infected Japanese, and 156 Japanese HTLV-uninfected controls were screened for antibodies to XMRV gp70 and p15E using recombinant-based chemiluminescence immunoassays (CMIAs). CMIA reactive samples were further evaluated by Western Blot (WB) and real-time RT-PCR for XMRV pol and env sequences.Of US donors, 0.8% (8/1000) were CMIA reactive: 1 p15E and 3 of 7 gp70 reactive samples WB confirmed yielding a 0.4% seroreactive rate. No HIV-1 infected specimens were reactive. Of the Japanese samples, 1/156 uninfected (0.6%) and 20/486 HTLV-infected samples (4.1%) had detectable p15E antibodies by CMIA and WB. Eight additional HTLV-infected samples were gp70 reactive; 4 of 486 (0.8%) were gp70 reactive in WB. No XMRV pol or env sequences were detected in the seroreactives.XMRV seroprevalence ranged from 0 - 0.6% in US blood donors, HIV-1 infected and HTLV uninfected subjects. Notably, 4.1% of Japanese HTLV-I infected individuals were p15E reactive. Inspection of sequence homology between HTLV and XMRV revealed a high level of conservation within the immunodominant region of HTLV gp21 suggesting increased seroreactivity is due to cross-reactive antibodies.


MicroRNA-21 (miR-21) is associated with the development of solid tumors progression including breast cancer. In this study we investigated matrix hyaluronan (HA)-CD44 (a primary HA receptor) interaction with c-Jun N-Terminal Kinase (JNK)/c-Jun signaling in MDA-MB-468 breast cancer cells [a triple-negative (estrogen receptor-negative/progesterone receptor-negative/HER2-negative) breast cancer cell line]. Our results indicated that HA binding to CD44 promotes c-Jun nuclear translocation and transcriptional activation. Further analyses revealed that miR-21 is regulated by an upstream promoter containing AP1 binding site(s), and chromatin immunoprecipitation (CHIP) assays demonstrated that stimulation of miR-21 expression by HA/CD44 interaction is c-Jun-dependent in these breast cancer cells. This process results in an increase of the anti-apoptosis protein Bcl-2 and upregulation of inhibitors of the apoptosis family of proteins (IAPs) as well as chemoresistance in MDA-MB-468 cells. Treatment with c-Jun specific small interfering RNAs effectively blocks HA-mediated c-Jun signaling and abrogates miR-21 production as well as causes downregulation of survival proteins (Bcl-2 and IAPs) and enhancement of chemosensitivity. In addition, our results demonstrated that anti-miR-21 inhibitor not only downregulates Bcl-2/IAP expression but also increases chemosensitivity in HA-treated breast cancer cells. Together, these findings suggest that the HA/CD44-induced c-Jun signaling plays a pivotal role in miR-21 production leading to survival protein (Bcl-2/IAP) upregulation and chemoresistance in triple negative breast cancer cells such as MDA-MB-468 cell line. This novel HA/CD44-mediated c-Jun signaling pathway and miR-21 production provide a new drug target for the future intervention strategies to treat breast cancer. Matrix Hyaluronan (HA) is an anionic, nonsulfated glycosaminoglycan distributed widely throughout connective, epithelial, and neural tissues . As a maCD44 is a family of cell-surface glycoproteins that are expressed in a variety of tissues, including breast cancer tissues ,3. RHAMMc-Jun and c-fos mRNA and AP-1 expression has been shown to be elevated in drug-resistant tumor cells (such as etoposide resistant human leukemia K562 cells) as compared to the c-Jun/c-fos mRNA/AP-1 levels found in drug-sensitive parental lines  leads to multidrug resistance in a variety of tumor cells  antibody, rabbit anti-c-Jun antibody, rabbit anti-JNK[pS63] antibody and rabbit anti-JNK antibody were from Cell Signaling Technology . JNK Inhibitor I, 420116 was purchased from EMD Millipore . Doxorubicin hydrochloride was from Sigma Chemicals . Healon HA polymers , purchased from Pharmacia & Upjohn Co. , were prepared as described previously ,31,38.MDA-MB-468 cells were transfected with anti-miR-21 inhibitor  (30 nmol/l) and its corresponding miRNA negative control  (30 nmol/l) using Lipofectamine 2000 reagent (Invitrogen) for 24 hours. Cells were then treated with HA or no HA in various experiments as described below.The NP-40 solubilized cell lysate materials from MDA-MB-468 cells  plus 50 μg/ml HA (or no HA) for various time intervals  at 37°C] were immunoblotted with rabbit anti-c-Jun antibody (2 μg/ml) or rabbit anti-phospho-c-Jun [pS63] antibody (2 μg/ml) or rabbit anti-c-JNK[pS183] antibody (2 μg/ml), respectively. In some cases, cell lysate of MDA-MB-468 cells  followed by HA (50 μg/ml) addition (or no HA or anti-CD44 antibody pretreatment plus HA) at 37°C were also immunoblotted using various immuno-reagents  or mouse anti-c-IAP-1 or mouse anti-c-IAP-2 antibody and anti-XIAP (2 μg/ml) or goat anti-actin (2 μg/ml) (as a loading control), respectively.To examine whether c-Jun or phospho-c-Jun directly interacts with the upstream promoter/enhancer region (containing AP-1 binding site) of miR-21, chromatin immunoprecipitation (ChIP) assays was performed in MDA-MB-468 cells  treated with HA (50 μg/ml) or without HA using a kit (EZ ChIP) from Millipore Corp according to the manufacturer’s instructions. Crosslinked chromatin lysates were sonicated and diluted with ChIP sonication buffer plus protease inhibitors, divided and incubated with normal rabbit IgG or rabbit anti-c-Jun antibody or rabbit phospho-c-Jun[pS63] antibody at 4°C overnight, then precipitated with protein G agarose. Crosslinking was reversed by overnight at 65°C incubation; DNA fragments were then extracted with PCR purification kit, analyzed by PCR and quantitated by PCR using primer pairs specific for the miR-21 upstream promoter/enhancer region containing the c-Jun binding sites: forward primer: 5′-TGGATAAGGATGACGCACAG-3′ and reverse primer: 5′-TGGTTTGAACCAATTAATAAGGAAA-3′ on an agarose gel as described previously ,29,38,60mirVana miRNA Isolation kit (Ambion). RNA concentrations were verified by measuring absorbance (A260) on the NanoDrop Spectrophotometer ND-1000 (NanoDrop). The mirVana miRNA probe construction kit (Ambion) was used to synthesize the 32P-labeled miR-21 antisense probe and miR-191 probe loading control as described previously  was enriched and purified using the eviously ,29,38.MDA-MB-468 cells (untreated or pretreated with anti-CD44 antibody) were incubated with HA (50 μg/ml) at 37°C for 30 minutes or with no HA. These cells were then fixed with 2% paraformaldehyde. Subsequently, these cells were rendered permeable by ethanol treatment followed by incubating with Texas Red-conjugated anti-phospho-c-Jun[pS63] antibody or fluorescein (FITC)-conjugated anti-c-Jun antibody followed by DAPI staining (a marker for nucleus). These fluorescence-labeled samples were then examined with a confocal laser scanning microscope.2/95% air. These cells (5 × 103 cells/well) were then incubated with various concentrations of Doxorubicin (4 × 10−9 M-1.75 × 10−5 M) with no HA or with HA (50 μg/ml). After 24h incubation at 37°C, MTT-based growth assays were analyzed as described previously  in the presence or absence of 50 μg/ml HA, as above. These cells were then plated in 96-well culture plates in 0.2 ml of Dulbecco’s modified Eagle’s medium/F12 medium supplement  containing no serum for 24 h at 37°C in 5% COeviously ,30. The All authors declare that they have no competing interest.LC performed the biochemical, molecular and immunofluorescence experiments and drafted the manuscript. LYWB conceived the idea, supervised the data collections/analysis and finalized the presentation of the manuscript. Both authors read and approved the final manuscript.


Human cytomegalovirus (HCMV) encodes microRNAs (miRNAs) that function as post-transcriptional regulators of gene expression during lytic infection in permissive cells. Some miRNAs have been shown to suppress virus replication, which could help HCMV to establish or maintain latent infection. However, HCMV miRNA expression has not been comprehensively examined and compared using cell culture systems representing permissive (lytic) and semi-permissive vs. non-permissive (latent-like) infection.Viral miRNAs levels and expression kinetics during HCMV infection were determined by miRNA-specific stem-loop RT-PCR. HCMV infected THP-1 (non-permissive), differentiated THP-1  and human embryo lung fibroblasts  were examined. The impact of selected miRNAs on HCMV infection  was determined by Western blotting, RT-PCR, qPCR, and plaque assay.US29 mRNA, confirming that US29 is a target of miR-US33.Abundant expression of 15 HCMV miRNAs was observed during lytic infection in HELs; highest peak inductions (11- to 1502-fold) occurred at 48 hpi. In d-THP-1s, fourteen mRNAs were detected with moderate induction (3- to 288-fold), but kinetics of expression was generally delayed for 24 h relative to HELs. In contrast, only three miRNAs were induced to low levels (3- to 4-fold) during quiescent infection in THP-1s. Interestingly, miR-UL70-3p was poorly induced in HEL (1.5-fold), moderately in THP-1s (4-fold), and strongly (58-fold) in d-THP-1s, suggesting a potentially specific role for miR-UL70-3p in THP-1s and d-THP-1s. MiR-US33, -UL22A and -UL70 were further evaluated for their impact on HCMV replication in HELs. Ectopic expression of miR-UL22A and miR-UL70 did not affect HCMV replication in HELs, whereas miR-US33 inhibited HCMV replication and reduced levels of HCMV Viral miRNA expression kinetics differs between permissive, semi-permissive and quiescent infections, and miR-US33 down-regulates HCMV replication. These results suggest that miR-US33 may function to impair entry into lytic replication and hence promote establishment of latency. Human cytomegalovirus (HCMV) is a ubiquitous pathogen infecting 50% to 90% of the population worldwide, with an extremely high prevalence (>90%) in China. HCMV infection is not believed to be deleterious to immunocompetent individuals. However, it can cause serious, often life-threatening complications in immunocompromised individuals, including solid organ and cell transplant recipients, AIDS patients, and patients suffering from late stage cancers  , UL112/113 and UL120/121, which are important for HCMV lytic infection In the present work expression and kinetics of 16 mature HCMV miRNAs were examined in different cell culture systems representing permissive, semi-permissive, and quiescent/latent-like infections. THP-1 acute monocytic leukemia cells The Wuhan Institute of Virology Institutional Review Board approved the isolation of primary human embryonic lung fibroblasts from postmortem fetal embryo tissue and waived the need for consent. The original source of the postmortem fetal embryo tissue was the Zhongnan Hospital.2.Primary HELs  and PCR-amplified using HCMV DNA (strain Towne) as the template. Primers and product sizes are listed in Lentivirus expression system, including plasmids pCDH-CMV-MCS-EF1-copGFP and pML-delta8.9 and pVSV-G , was used to ectopically express HCMV-encoded miRNAs. Sequences of HCMV-encoded pre-miRNAs were obtained from miRBase/Release 20 (HCMV Towne strain (ATCC-VR977) was propagated and titered as described previously HELs were transduced with lentiviruses lacking an insert (vector control) or expressing HCMV miRNAs at an MOI of 10. After confirmation of miRNA expression by RT-PCR at 48 h after transduction (described below), transduced HELs were infected with HCMV at MOIs of 0.01, 0.1, 1, or 5. Cells were harvested for RNA, DNA, and Western blotting studies at 24, 48 and 72 h post infection (hpi) and supernatants were collected and titered for infectious HCMV at 96 and 144 hpi.HCMV-encoded miRNAs were detected by stem-loop RT-PCR. Total RNA was isolated from HCMV-infected cells using Trizol Reagent (TaKaRa) and DNA was removed using Recombinant DNase I (TaKaRa). One µg of each RNA sample was reverse transcribed with RevertAid™ H Minus First Strand cDNA Synthesis Kit (Fermentas) using miRNA-specific stem-loop RT primers  as descrCAGCACCCGACAGAACTCAC 3′). Amplification of GAPDH sequences using primers F 5′ GTTTACATGTTCCAATATGA 3′ and R 5′ TCCTGGAAGATGGTGATGGG served as an internal reference. 20 µl qPCR reactions contained 2 µl (20 ng) DNA, 50 nM forward and reverse primers, and 10 µl 2× iTaqTMSYBR Green Supermix (BIO-RAD). Reactions were denatured at 95°C for 5 min, followed by 40 cycles of 95°C for 10 s and 54°C for 20 s using a CFX Connect™ Real-Time Sysem (BIO-RAD).DNA was extracted from cell pellets using TIANamp Genomic DNA Kit (TIANGEN) and adjusted to 10 ng/µl. HCMV DNA was detected using primers from the IE1 gene locus  to make plasmid pGL3-Luc-US29. β-galactosidase  expression from plasmid pCMV-SPORT-β-gal  was used as a transfection control. pGL3-Luc-US29 and pCMV SPORT-β-gal were cotransfected into 293T cells along with either pCDH-miR-US33 or the pCDH-copGFP empty vector control using calcium phosphate as described A luciferase reporter assay was constructed to assess miR-US33 regulation of All reactions were run in triplicate for each experiment. Three independent experiments were performed. Data were analyzed by One-way ANOVA. Results reported are means ± standard deviations (SD). Differences were considered to be significant when P values were less than 0.05.Twenty three mature miRNAs encoded by HCMV have been reported, however, only 16 of the 23 mature miRNAs are in the database of miRBase/Release 20 Based on patterns of expression, HCMV miRNAs could be classified into three groups. Group 1 miRNAs  increased gradually and reached high expression levels (>30-fold induction) at 48 hpi, then declined somewhat but remained high (>8-fold) at 72 hpi, when progeny virus production is vigorous . Group 2These results suggest that group 1 miRNAs may have functions that are important early in the HCMV replicative cycle, as significant levels are attained prior to the onset of DNA synthesis at ∼24 hpi. Group 2 and 3 miRNAs may have later functions as their levels do not rise until 48 hpi, when viral DNA synthesis is underway and progeny virus assembly has begun.i.e., 3-fold vs. 33-fold for miR-UL112, 4-fold vs. 1.5-fold for miR-UL70-3p, and 3-fold vs. 209-fold for miR-US5-1). Differences between the two cell types in kinetics and induction levels for four representative miRNAs are illustrated in To examine HCMV miRNA expression during quiescent infection, undifferentiated THP-1 monocytes were infected at an MOI of 10 and expression levels of the 16 HCMV miRNAs were determined at different hpi. Thirteen of the 16 miRNAs exhibited either little change or declined significantly after infection. This is in stark contrast to HELs in which these miRNAs increased 11- to 1502-fold. Three miRNAs  exhibited slight (3 to 4-fold) inductions, but only at 72 hpi . The relThese results suggest that, similar to lytic gene transcription, most HCMV miRNAs are repressed in THP-1 cells. MiR-UL70-3p appears to be uniquely expressed in THP-1s but its late time of induction would seem to preclude a role in establishing a quiescent state.To determine how differentiation to a semi-permissive state alters the expression of HCMV miRNAs, THP-1 cells were differentiated by culture for 24 h in the presence of PMA and hydrocortisone prior to HCMV infection. The resulting d-THP-1 cells were infected at an MOI of 10 and expression levels of the 16 HCMV miRNAs were determined at different hpi. Differentiation profoundly affected patterns and levels of miRNA expression in d-THP-1s relative to THP-1s. Thirteen of the 15 miRNAs (except miR-UL70-3p) that were strongly induced during HEL infection (but not induced in THP-1 cells) exhibited induction increases as a result of differentiation . Of thesDifferences between THP-1 and d-THP-1 cells in kinetics and induction levels for four representative miRNAs are illustrated in These results suggest that a subset of HCMV miRNAs are significantly expressed during lytic infection independent of cell type, while high expression levels of other miRNAs are unique to HELs or d-THP-1s. This suggests that differences in HCMV miRNA expression between THP-1, d-THP-1, and HEL cells cannot be simply explained by different percentages of cells entering quiescent vs. lytic infection in each cell type. The exclusive induction of miR-UL70-3p in d-THP-1s indicates that miR-UL70-3p induction may be THP-1 cell specific and cell differentiation associated.Three previously uncharacterized HCMV miRNAs, miR-US33, miR-UL22A and miR-UL70, were selected for further study based on changes in induction upon differentiation. HELs were transduced with lentiviruses expressing pre-miR-US33, pre-miR-UL22A, pre-miR-UL70, or empty vector control. Each miRNA was measured by stem-loop RT/qPCR at 48 h after transduction. The signal from miR-US33-transduced cells was 254-fold above that of empty vector-transduced cells, and those for miR-UL22A and miR-UL70 were 151-fold and 17-fold, respectively. However, transduced levels of miR-US33, miR-UL22A and miR-UL70 were 4375-, 106300- and 1267-fold lower, respectively, than levels induced 48 h post HCMV infection of untransduced HELs . Hence, 3 to 1.9×103 pfu/ml at 96 hpi (P = 0.0001) and from 8.6×105 to 3.1×105 pfu/ml at 144 hpi (P = 0.012) , early (UL44), and late  viral proteins was assessed by Western blotting, viral DNA replication was measured by qPCR, and virus yield was determined by titering infectious virus released into culture supernatants. Transduction of miR-UL22A-5p and miR-UL70-5p had no apparent effects on HCMV infection at all the MOIs tested, and miR-US33 had no effects at MOIs of 0.1, 1 or 5 . Viral D= 0.012) .US29 as a predicted target for miR-US33-3p US29 sequence functions as a target for miR-US33. The predicted US29 target sequences were inserted into the 3′ untranslated region of a mRNA encoding luciferase that was expressed from a plasmid by transient transfection. pCDH-miR-US33 or the pCDH-copGFP was cotransfected with the luciferease-US29 reporter plasmid and a control plasmid expressing β-gal into 293T/17 cells. Luciferase activities were normalized to β-gal activities measured in the same cells 48 h post transfection. Cells expressing miR-US33 had 2.6-fold lower luciferase activity compared to those transduced with empty vector (P = 0.002) (Most miRNAs contain short (2–8 nt) ‘seed’ regions that can base pair with target mRNAs to form exactly matched 6–7 nt regions of double-stranded RNA = 0.002) .US29 transcripts are affected by miR-US33 during HCMV infection, HELs transduced with miR-US33-expressing or vector control lentiviruses were infected with HCMV (MOI = 0.01) and US29 mRNA levels were quantitated by qRT-PCR. US29 transcript levels were reduced by nearly half at 24 hpi (P = 0.002), confirming that miR-US33 can down-regulate US29 mRNA in the context of HCMV infection  is crucial for driving cells forward in the lytic replication cycle. Conversely, repression of IE1/2 expression may promote establishment of latency in some cell types or abortive infection in others. During natural latency the MIEP is associated with markers of repressed chromatin and repressive chromatinization occurs rapidly after experimental infection in cell culture models of latency in vivo or in experimental cell culture latency models.The immunologically inert nature of miRNAs makes viral miRNA-mediated mechanisms for governing latency intellectually attractive, and indeed, control of viral and host genes by viral miRNAs during latency has been demonstrated for several viruses in the alpha- and gamma-herpesvirus subfamilies  as found to suppress HCMV lytic replication, adding to an emerging theme that a significant subset of HCMV miRNAs function to down-modulate lytic replication. The role(s) such miRNAs may play in latency awaits further studies to characterize miRNA expression during natural latency and to evaluate miRNA null mutants in cell culture latency models.Figure S1Expression kinetics of HCMV miRNAs during quiescent infection of THP-1 cells. Undifferentiated THP-1 monocytes were infected with HCMV strain Towne at an MOI of 10 and intracellular HCMV miRNAs were quantitated by stem-loop RT-PCR at the indicated times post infection. Results indicate fold-changes relative to levels measured at 3 hpi.(TIF)Click here for additional data file.Figure S2Expression kinetics of HCMV miRNAs during semi-permissive replication in d-THP-1 cells. THP-1 monocytes were differentiated into macrophages by culture for 24 h in medium containing PMA and hydrocortisone. Resulting d-THP-1 cells were infected with HCMV strain Towne at an MOI of 10 and intracellular HCMV miRNAs were quantitated by stem-loop RT-PCR at the indicated times post infection. Results indicate fold-changes relative to levels measured at 3 hpi.(TIF)Click here for additional data file.Table S1Comparison of Ct values of strongly induced miRNAs in d-THP-1 vs. HEL.(DOC)Click here for additional data file.


Tissue resident mesenchymal stem cells (MSCs) are multipotent, self-renewing cells known for their differentiation potential into cells of mesenchymal lineage. The ability of single cell clones isolated from adipose tissue resident MSCs (ASCs) to differentiate into cells of hematopoietic lineage has been previously demonstrated. In the present study, we investigated if the hematopoietic differentiated (HD) cells derived from ASCs could productively be infected with HIV-1.+, CD105+, CD45-, and CD34-). Transcriptome analysis revealed that HD cells acquire a number of elements that increase their susceptibility for HIV-1 infection, including HIV-1 receptor/co-receptor and other key cellular cofactors. HIV-1 infected HD cells (HD-HIV) showed elevated p24 protein and gag and tat gene expression, implying a high and productive infection. HD-HIV cells showed decreased CD4, but significant increase in the expression of CCR5, CXCR4, Nef-associated factor HCK, and Vpu-associated factor BTRC. HIV-1 restricting factors like APOBEC3F and TRIM5 also showed up regulation. HIV-1 infection increased apoptosis and cell cycle regulatory genes in HD cells. Although undifferentiated ASCs failed to show productive infection, HIV-1 exposure increased the expression of several hematopoietic lineage associated genes such as c-Kit, MMD2, and IL-10.HD cells were generated by differentiating clonally expanded cultures of adherent subsets of ASCs (CD90Considering the presence of profuse amounts of ASCs in different tissues, these findings suggest the possible role that could be played by HD cells derived from ASCs in HIV-1 infection. The undifferentiated ASCs were non-permissive to HIV-1 infection; however, HIV-1 exposure increased the expression of some hematopoietic lineage related genes. The findings relate the importance of ASCs in HIV-1 research and facilitate the understanding of the disease process and management strategies. Human immunodeficiency virus type 1 (HIV-1), the etiologic agent of acquired immune deficiency syndrome (AIDS), predominantly infects hematopoietic cells such as T-helper lymphocytes, monocytes and macrophages. Despite the development of highly active anti-retroviral therapy (HAART), the persistence of reservoirs of HIV-1 poses obstacles to the eradication of the disease. Although initial viral decay kinetics in plasma had indicated optimistic outcomes of HAART .+ HPCs and impaired clonogenic potential and apoptosis of these progenitor cells have been documented in HIV-1 infected individuals . Each viral stock was freshly prepared before exposure of ASCs or uninfected HUT78 cells. For controls, un-differentiated ASCs and HUT78 cells were exposed to the same number of viral particles. Following 24 hrs of virus exposure, cells were washed several times using fresh media to remove the unattached viral particles and cultured for 3 or 5 days post exposure. Prior to viral infection, ASCs were cultured in differentiation media for either 5 or 8 days, and subsequently exposed to HIV-1 in cell free viral media. Viral p24 levels were analyzed at each time point to monitor the viral replication in un-differentiated, HD, and HUT78 cells. A graphical representation of p24 level (pg/ml) vs time point (days) was carried out. Values with p < 0.0001 were considered significant.The uninfected T4-lymphocyte line HUT78, and HUT78 cells persistently infected with HTLV-IIIAlu-gag PCR technique described by Liszewski et al. [Alu-gag regions were amplified using following primers: 1. Alu (Forward): 5' GCC TCC CAA AGT GCT GGG ATT ACA G-3'; HIV gag (Reverse): nucleotides (nt) 1505-1486 5' GTT CCT GCT ATG TCA CTT CC-3'. On the second step, RU5 region in gag was detected using the following primers: RU5 (R Forward): nt 518-539 5'-TTA AGC CTC AAT AAA GCT TGC C-3'; RU5 (U5 Reverse): nt 647-628 5'-GTT CGG GCG CCA CTG CTA GA-3'; 5. RU5wildtype Probe: nt 584-559 5'-CCA GAG TCA CAC AAC AGA CGG GCA CA-3'; RU5degenerate1 Probe: nt 584-559 5'-CCA GAG TCA CAT AAC AGA CGG GCA CA-3'; and RU5degenerate2 Probe: nt 584-559 5'-CCA GAG TCA CAC AAC AGA TGG GCA CA-3'. The PCR products were analyzed by agarose gel electrophoresis.The genomic DNA from exposed HIV-1 ASCs was subjected to two step i et al. . In the http://www.sabiosciences.com. Differential gene expression was evaluated for statistical significance (p < 0.05). A cut off of 2 for fold change for up-regulated and 0.5 for down regulated genes was applied, so as to only consider genes whose expression was perturbed in magnitude as well as in a significant manner.Using total cellular RNA the gene expression was carried out on PCR array kits  which profiles the expression of 84 genes involved in susceptibility to HIV-1, infection and related immune response. The cellular RNAs from un-differentiated ASCs, HD, and HIV-1 infected HD cells (HD-HIV) (n = 3 donors) with HIV-1 infected HUT78 (HUT78-HIV) (serving as positive controls) were used. Data were analyzed using software provided by SABiosciences CD4: 5'-GTA GTA GCC CCT CAG TGC AA-3', 5'-AAA GCT AGC ACC ACG ATG TC-3'; CD14: 5'-ACA GGA CTT GCA CTT TCC AG-3', 5'-TCC AGG ATT GTC AGA CAG GT-3'; CD68: 5'-CAA CTG CCA CTC ACA GTC CT-3', 5'-CAA TGG TCT CCT TGG AGG TT-3'; IL10: 5'-AAG CCT GAC CAC GCT TTC TA-3', 5'-ATG AAG TGG TTG GGG AAT GA-3'; ITGAM: 5'-ACG GAT GGA GAA AAG TTT GG-3', 5'-CAA AGA TCT TCT CCC GAA GC-3'; c-KIT: 5'-CCG TGG TAG ACC ATT CTG TG-3', 5'-GTG CCC ACT ATC CTG GAG TT-3'; MMD2: 5'-GCA GAC CAA GGT GTC CAA AT-3', 5'-CTG GCT GTC ACC AGA AGT CA-3'; MRC1: 5'-GGC GGT GAC CTC ACA AGT AT-3', 5'-ACG AAG CCA TTT GGT AAA CG-3'; MSR1: 5'-TCC TCG TGT TTG CAG TTC TC-3', 5'-CAT GTT GCT CAT GTG TTC CA-3'; TNF: 5'-TCC TTC AGA CAC CCT CAA CC-3', 5'-AGG CCC CAG TTT GAA TTC TT-3'; gag: 5'-ATA ATC CAC CTA TCC CAG TAG GAG AAA T-3', 5'-TTT GGT CCT TGT CTT ATG TCC AGA ATG C-3'; Tat: 5'-GGA ATT CAC CAT GGA GCC AGT AGA TCC T-3', 5'-CGG GAT CCC TAT TCC TTC GGG CCT GT-3'; GAPDH 5'-CGA GAT CCC TCCA AAA TCA A-3' and 5'-GGT GCT AAG CAG TTG GTG GT-3'. The data were generated using an iCycler MyiQ (Biorad) and analyzed using the iQ5 V2.0 (Bio-Rad). The RT-PCR products were analyzed using agarose gel electrophoresis (1% agarose gel) and stained in 10 μg/ml ethidium bromid (Sigma) for visualization. For real-time RT-PCR, the equation 2-ΔΔCT was used for calculating fold changes. A threshold cycle of 35 was chosen as the cut-off for non-detectable genes, thus genes with CT values above 35 were considered not expressed.Real-Time RT-PCR was performed using SYBR Green Master mix  in a 2-step protocol (50 cycles of 10 sec at 90°C and 45 sec at Tm). The following primers were used to assess the gene expressions. HD cells were prepared and infected with HIV, the fixed, permeabilized, and incubated with human specific primary antibodies for CCR4, CCR5, CXCR4, and NOS2 at a final concentration of 0.02-0.04 mg/ml, then incubated with 0.002 mg/ml of the matching secondary antibody. The signal was detected with a Leica TCS SP-2 confocal microscope equipped with Argon  and HeNe lasers  at a magnification of HCX PL APO 63×/1.4 at 21°C. Data were processed with Leica confocal software.Adipogenic differentiation was determined in cultures of ASCs following HIV exposure using previously described methods . AdipogeAll data relating to this study were summarized using descriptive statistics such as mean, standard deviation and standard error. The analysis of variance method was used to compare the mean differences. Where meaningful, the results were presented graphically. The study hypotheses were tested at 5% level of significance throughout the analysis. Estimates of means and their 95% confidence intervals were calculated. R-computing software was used to plot the graphs.MSCs: mesenchymal stem cells; ASCs: adipose tissue derived mesenchymal stem cells; HD: hematopoietic differentiated cells; AIDS: acquired immune deficiency syndrome; HAART: highly active anti-retroviral therapy; HPCs: hematopoietic progenitor cells; LTBMC: long-term bone marrow cultures; HD-5: differentiate into HD cells for 5 days; HD-8: differentiate into HD cells for 8 days; MOI: multiplicity of infection; HD-HIV: infected HD cells with HIV-1; HUT78-HIV: infected HUT78 cells with HIV-1; ASCs-HIV: exposed ASCs to HIV-1; TRIM5: tripartite motif-containing 5; TAT-SF1: Tat specific factor 1; BTRC: b-transducin repeat containing.The authors declare that they have no competing interests.EF was responsible to cell cloning and cultures. EF, TN, and CS conducted differentiations. UR and CB were responsible for HIV-1 infection studies. AC and CD were responsible for tissue collections. RI, DM, and EA were responsible for experimental design. RI and EA were responsible for the overall experimental design and implementation of the project and contributed equally to this work.


Images, however, also showed significant peri-articular background activity. The folate receptor (FR)-β is a potential alternative target for imaging activated macrophages. Therefore, the PET tracer [18F]fluoro-PEG-folate was synthesized and evaluated in both in vitro and ex vivo studies using a methylated BSA induced arthritis model.Detection of  synovitis is relevant for both early diagnosis and monitoring of therapy of rheumatoid arthritis (RA). Previously, the potential of imaging (sub)clinical arthritis was demonstrated by targeting the translocator protein in activated macrophages using 18F]fluoro-PEG-folate was synthesized in a two-step procedure. Relative binding affinities of non-radioactive fluoro-PEG-folate, folic acid and naturally circulating 5-methyltetrahydrofolate (5-Me-THF) to FR were determined using KB cells with high expression of FR. Both in vivo [18F]fluoro-PEG-folate PET and ex vivo tissue distribution studies were performed in arthritic and normal rats and results were compared with those of the established macrophage tracer (R)-[11C]PK11195.[18F]fluoro-PEG-folate was synthesized with a purity >97%, a yield of 300 to 1,700 MBq and a specific activity between 40 and 70 GBq/µmol. Relative in vitro binding affinity for FR of F-PEG-folate was 1.8-fold lower than that of folic acid, but 3-fold higher than that of 5-Me-THF. In the rat model, [18F]fluoro-PEG-folate uptake in arthritic knees was increased compared with both contralateral knees and knees of normal rats. Uptake in arthritic knees could be blocked by an excess of glucosamine-folate, consistent with [18F]fluoro-PEG-folate being specifically bound to FR. Arthritic knee-to-bone and arthritic knee-to-blood ratios of [18F]fluoro-PEG-folate were increased compared with those of (R)-[11C]PK11195. Reduction of 5-Me-THF levels in rat plasma to those mimicking human levels increased absolute [18F]fluoro-PEG-folate uptake in arthritic joints, but without improving target-to-background ratios.[18F]fluoro-PEG-folate, designed to target FR on activated macrophages provided improved contrast in a rat model of arthritis compared with the accepted macrophage tracer (R)-[11C]PK11195. These results warrant further exploration of [18F]fluoro-PEG-folate as a putative PET tracer for imaging (sub)clinical arthritis in RA patients.The novel PET tracer [ Activated macrophages infiltrate inflamed synovium of rheumatoid arthritis (RA) patients in early stages of the disease and play a pivotal role in sustaining the chronic phase of RA ,2. AssesIn a large subset of RA patients, clinical manifestations of disease are preceded by a preclinical phase characterized by the presence of auto-immune antibodies and presumably subclinical synovitis ,4. Visua(R)-[11C]PK11195 (1-[2-chlorophenyl]-N-methyl-N-[1-methyl-propyl]-3-isoquinoline carboxamide), that binds to the translocator protein that is up-regulated in activated macrophages fluoro-polyethylene glycol (PEG)-folate was developed. Several 18F-based folate tracers have been synthesized thus far [18F]fluoro-PEG-folate was anticipated to show improved kinetics because of the introduction of the PEG moiety fluoro-PEG-folate (1) was synthesized in a two-step procedure  was obtained by fluorination of the SFB precursor (2) . The precursor (5 mg) was dissolved in 1 mL acetonitrile and, after standard work-up of the [18F]fluoride solution, the precursor was added and the reaction was allowed to proceed for 10 minutes at 90°C. The labeled precursor (3) was then deprotected with 40 µL of tetrapropylammonium hydroxide, and the reaction mixture was evaporated and treated with tetramethyl-o-(N-succinimidyl)uranium tetrafluoroborate to generate [18F]SFB (4). This [18F]SFB solution was passed over a preconditioned (10 mL sterile ethanol 96% and then 10 mL water for injection) Waters Sep-Pak C18 cartridge . After washing with 10 mL 15% acetonitrile in water for injection and subsequent elution of the product from the cartridge with 1.5 mL acetonitrile, the [18F]SFB solution was evaporated to a volume of approximately 100 µL. A solution of 2 mg PEG-folate precursor (5) in 1 mL borate buffer  was added to the 100 µL solution of [18F]SFB (4) in acetonitrile and reacted for 30 minutes at ambient temperature. The reaction mixture was injected onto a Gemini 5µ C18 HPLC column , which was eluted with an 80/20 mixture of acetonitrile/0.1% trifluoroacetic acid in water at 3.0 mL/minute and UV at 280 nm. The product, [18F]fluoro-PEG-folate, eluted at 31 to 33 minutes. This fraction was collected in 30 mL water and passed over a preconditioned (10 mL sterile ethanol 96% and then 10 mL water for injection) Waters Sep-Pak tC18 . After washing with 20 mL water for injection, the product was eluted from the cartridge with 1.0 mL sterile ethanol (96%) and 14 mL of a sterile and pyrogen free sodium phosphate solution  in saline. The final mixture was passed over a Millex GV 0.22 µm filter , yielding a sterile, isotonic and pyrogen free solution of 0.5-1.5 GBq of [18F]fluoro-PEG-folate. The product was analyzed using HPLC on a Varian ChromSpher 5 C18 column , which was eluted with a 20/80 mixture of acetonitrile/0.1% trifluoroacetic acid in water at 1.2 mL/minute and UV at 280 nm.8Ffluoro-(R)-[11C]PK11195 was synthesized routinely with a radiochemical purity >98% and mean specific activity of 95.7 ± 28.4 GBq/μmol, as described previously fluoro-PEG-folate via a jugular vein canula, an emission scan was acquired for one hour. PET data were normalized and corrected for scatter, randoms, attenuation, decay and dead time. Data were converted into 16 sinograms . Images were reconstructed using an iterative 3D ordinary Poisson ordered-subsets expectation-maximization algorithm with 8 iterations and 16 subsets. Resulting images had a matrix size of 256 × 256 × 207 voxels, each with a dimension of 1.21 × 1.21 × 1.21 mm3.Scans were performed using a double LSO/LYSO layer ECAT High Resolution Research Tomograph , a small animal and human brain 3-dimensional (3D) scanner with high spatial resolution  and high sensitivity . Arthrit3) were manually drawn over left and right knees. ROIs were projected onto the dynamic image sequence, thereby generating time-activity curves, expressed as standardized uptake values (SUV), where SUV represents mean ROI radioactivity concentration normalized for injected dose and body weight. For determining [18F]fluoro-PEG-folate uptake in both knees, SUV was averaged over the period from 50 to 60 minutes post-injection. Blood concentrations, also expressed as SUV, were obtained from manually drawn ROIs  in the heart. The image derived injected dose (IDID) was obtained from a ROI covering the whole rat.PET images were processed using AMIDE software  . Ellipso18F]fluoro-PEG-folate or 10.5 ± 2.9 MBq (R)-[11C]PK11195, blood was collected from anesthetized rats by heart puncture. Subsequently, rats were sacrificed by cervical dislocation. Both knees (in toto), bone obtained from the right hind leg, and blood and tissue from various internal organs were excised and weighed. Radioactivity in each tissue  was determined using an LKB 1282 Compugamma CS gamma counter . Tissue distribution studies were performed in four arthritic rats and eight normal rats for [18F]fluoro-PEG-folate and in five arthritic rats for (R)-[11C]PK11195.Sixty minutes after intravenous injection of 15.1 ± 3.2 MBq fluoro-PEG-folate binds specifically to FR, blocking studies were performed in rats (n = 6) by pre-administration of a ≥100-fold molar excess of the folate conjugate glucosamine-folate five minutes prior to the injection of [18F]fluoro-PEG-folate. Glucosamine-folate was preferred over folic acid because of its increased solubility at low pH, preventing precipitation in the kidneys [ex vivo tissue distribution studies were performed as described above.In order to investigate whether [ kidneys . PET and18F]fluoro-PEG-folate for binding to FR. To investigate this, one subgroup of rats (n = 4) was fed with folate deficient chow  for 14 days. Plasma 5-Me-THF levels were determined by positive electrospray liquid chromatography tandem mass spectrometry (LC-MS/MS)  with a plasma intra-assay CV <4% and inter-assay <2% fluoro-PEG-folate in the arthritic versus contralateral knee. Mann-Whitney (exact) tests were performed to analyze differences in (a) [18F]fluoro-PEG-folate uptake in arthritic versus normal knee, (b) [18F]fluoro-PEG-folate versus (R)-[11C]PK11195 uptake, (c) [18F]fluoro-PEG-folate uptake in the arthritic knee under normal versus folate deficient conditions, and (d) tissue uptake ratios. A P-value <0.05 was considered as statistically significant. Results are presented as mean ± standard deviation (SD), unless stated otherwise.Statistical tests were performed using SPSS version 15.0 for Windows . A Wilcoxon signed rank (exact) test was used to determine differences in paired observations, for example, uptake of [18F]fluoro-PEG-folate was synthesized via a two-step radiochemical reaction sequence  .PK11195. [18F]fluoro-PEG-folate cleared more rapidly from blood than (R)-[11C]PK11195, resulting in significantly lower blood levels of [18F]fluoro-PEG-folate than of (R)-[11C]PK11195 . Consequently, mean arthritic knee-to-blood ratios of [18F]fluoro-PEG-folate were higher than those of (R)-[11C]PK11195  -[11C]PK11195 in various tissues including arthritic knees , contralateral knees  and other internal organs, such as heart, lungs, spleen and gastrointestinal organs (data not shown).Tissue distribution of [) Figure . Mean ab18F]fluoro-PEG-folate (1.42 ± 0.15) and (R)-[11C]PK11195 (1.39 ± 0.10) were similar. Nevertheless, with peri-articular bone as the most relevant background tissue, mean arthritic-to-ipsilateral bone ratios of [18F]fluoro-PEG-folate (1.64 ± 0.15) were significantly higher than those of (R)-[11C]PK11195   in the arthritic knee (0.03 ± 0.02) was found in ex vivo tissue distribution studies  and spleen (0.02 ± 0.01), while uptake in kidneys (1.52 ± 0.81) and intestines (0.24 ± 0.31) was reduced but still appreciable.After s Figure , indicatMean plasma serum levels in rats fed with folate deficient chow for 14 days decreased from 110 ± 21 to 11.0 ± 4.3 nM .P <0.05). Mean arthritic knee-to-bone ratios (1.38 ± 0.17) did not improve under folate deficient conditions, since uptake of the tracer in ipsilateral bone tissue increased in parallel.Mean tissue radioactivity (%ID/gram) found in arthritic knees of rats on folate deficient chow (0.52 ± 0.10) was increased compared with that of rats on normal chow  with comparable affinity [in vivo PET scans and by ex vivo tissue distribution studies. Specific binding of [18F]fluoro-PEG-folate to FR in the macrophage-rich synovium was demonstrated by in vivo blocking studies.The results of the affinity . Accumul18F]fluoro-PEG-folate shows promise, as the lower peri-articular uptake in bone (marrow) and the faster clearance from blood compared with (R)-[11C]PK11195 provide improved contrast when imaging arthritic joints. This will especially be important for the imaging of subclinical disease activity.As a tracer, fluoro-PEG-folate may, therefore, have been underestimated. Statistical significance of the differences between uptake (ratios) of [18F]fluoro-PEG-folate and (R)-[11C]PK11195, and between uptake in arthritic versus control joints was further reduced due to lack of statistical power related to the relatively small numbers of animals (n = 4 to 8) per group.The mBSA induced rat arthritis model is an established model of RA, which resembles human pathology of RA at the joint level . A limit18F]fluoro-PEG-folate uptake was observed in the liver and spleen of rats with mBSA induced arthritis, which was largely abolished by FR receptor blockade with glucosamine-folate, suggesting specific receptor binding of the tracer. Conceivably, this observation may be explained by an increase of macrophage-like cells in the liver and spleen due to systemic immune activation as part of locally antigen induced arthritis [18F]fluoro-PEG-folate in the liver and spleen were respectively 3.8 and 2.2 times higher in arthritic than in normal rats (results not shown). Part of the liver and intestinal uptake of the novel folate tracer was, however, not blocked by glucosamine-folate, which could be due to the hepatobiliary secretion of the tracer. Likewise, [18F]fluoro-PEG-folate kidney uptake could be partly abolished under FR-blockade conditions, which was anticipated from folate receptor expression in proximal tubule cells, but leaving retention of the tracer in the kidney, possibly due to renal clearance. Additionally, non-specific tissue uptake of [18F]fluoro-PEG-folate or radiometabolite formation could also have influenced tissue distribution studies.Relatively high fluoro-PEG-folate for imaging of RA has to be corroborated in human studies, which is readily achievable since the current method for the synthesis of [18F]fluoro-PEG-folate can easily be translated into a Good Manufacturing Practice compliant method yielding a clinical grade [18F]fluoro-PEG-folate radiotracer. Since human 5-Me-THF plasma and tissue levels are substantially lower compared to rat it is conceivable that absolute uptake of [18F]fluoro-PEG-folate in inflamed joints may be higher in humans. Nevertheless, simultaneously increased background uptake of [18F]fluoro-PEG-folate was shown in rats fed with folate deficient chow. Furthermore, it should be acknowledged that humans exhibit generally higher FR expression levels in the kidneys compared to rats [18F]fluoro-PEG-folate in human patients than was found in our experiments performed in rats. Thus, it remains to be seen whether lower circulating 5-Me-THF plasma levels and generally higher FR expression tissue levels in humans set favorable conditions for [18F]fluoro-PEG-folate PET imaging of RA.The ultimate value of fluoro-PEG-folate tracer was demonstrated in an mBSA induced arthritis model in rats. [18F]fluoro-PEG-folate shows higher contrast due to a lower background signal than the established, clinically available macrophage tracer (R)-[11C]PK11195. These promising results set the stage for proof of concept studies in RA patients.In conclusion, highly sensitive arthritis imaging by PET and macrophage targeting using the novel fluoro-PEG-folate. (A) Semi prep HPLC chromatogram of the purification of [18F]fluoro-PEG-folate; top: UV detection, bottom: radioactivity detection. The product elutes at 31 to 33 minutes, radiochemical yield, calculated from this chromatogram is 73%. The non-radioactive reference compound eluted at the same retention time as the radioactive compound (result not shown) (B) Analysis  of the final solution of [18F]fluoro-PEG-folate; top: UV detection, bottom: radioactivity detection. Radiochemical purity >99%. (C) Analysis  of the final solution of [18F]fluoro-PEG-folate, with the addition of reference compound; top: UV detection, bottom: radioactivity detection. The reference compound elutes at the same time as [18F]fluoro-PEG-folate and confirms the identity of [18F]fluoro-PEG-folate.Representative HPLC chromatograms of the purification and analysis of [Click here for file18F]fluoro-PEG-folate in arthritic knee and blood. (A) Time-activity curves of [18F]fluoro-PEG-folate uptake in arthritic knee and blood. (B) Arthritic knee-to-blood ratio as function of time. Results are presented as mean ± SD of four arthritic rats.Uptake [Click here for file18F]fluoro-PEG-folate imaging of arthritic rats. [18F]fluoro-PEG-folate uptake in the right arthritic knee at (A) baseline (arrow) and (B) after blocking the FR with an excess dose of glucosamine-folate. In the latter case abolishment of the signal in the arthritic knee is evident.[Click here for file


The bacteriophage P22 protein cage can be bioengineered to contain a high-relaxivity gadolinium (Gd) payload internally and targeting ligands externally. It also enables phage-library-based identification of novel targets. Thus, P22 may have advantages for molecular/cellular imaging by MRI.-1s-1, superior to Gd-DTPA for the equivalent Gd concentration.1) P22: The P22 protein cage (60 nm) is bioengineered with an internal polymer network with amine functional groups allowing incorporation of ~9100 Gd-DTPA molecules per cage via the amine groups  and FVB mice were used. ApoE-/- mice develop atherosclerosis enhanced by high-fat diet. FVB mice develop macrophage-rich carotid lesions with carotid ligation in combination with high-fat diet and diabetes induction [2) Atherosclerosis Models: Both ApoE-deficient  or Magnevist . Vascular MRA at 1T was performed  using 3D-SPGR . Vessel wall MRI at 3T was performed  with a phased-array mouse coil , using a double inversion recovery fast spin echo sequence  up to 24 hours after injection.3) P22-polymer-Gd ex vivo fluorescence imaging: Molecular targeting of P22 was evaluated by attaching RGD peptides externally, which targets the αVβ3 integrin, upregulated on activated macrophages. ApoE-/- mice (N=4) were injected intravenously with RGD+P22 or RGD-P22 . Forty-eight hours later, ex vivo fluorescence imaging was performed using MaestroTM imaging system . Maximum plaque signal intensities were measured and compared.4) RGD-targeted P22 -/-) and ligated carotid (FVB) at 3T .Low dose P22-polymer-Gd showed strong enhancement for 1T vascular MRA (Figure s Figure . RDG tarin vivo MRI as well as external RGD-targeting for enhanced uptake in vascular inflammation. Thus, P22 is a novel, multi-functional nanoparticle platform for targeted-imaging of atherosclerosis.The P22 protein cage nanoparticle demonstrates both internal high-relaxivity Gd-loading for GE healthcare, Kowa, Inc.


Panax ginseng C.A.Meyer, Pueraria lobata, Dioscorea batatas Decaisne, Rehmannia glutinosa, Amomum cadamomum Linné, Poncirus fructus and Evodia officinalis was investigated for its anti-diabetic effects in cell and animal models.An aqueous extract of multi-hypoglycemic herbs of db/db mice .Activities of PPARγ agonist, anti-inflammation, AMPK activator and anti-ER stress were measured in cell models and in db/db mice treated with the extract showed reduced fasting blood glucose and HbA1c levels, improved postprandial glucose levels, enhanced insulin sensitivity and significantly decreased plasma free fatty acid, triglyceride and total cholesterol.While the extract stimulated PPARγ-dependent luciferase activity and activated AMPK in C2C12 cells, it inhibited TNF-α-stimulated IKKβ/NFkB signaling and attenuated ER stress in HepG2 cells. The The aqueous extract of these seven hypoglycemic herbs demonstrated many therapeutic effects for the treatment of type 2 diabetes in cell and animal models. All OADs, however, have adverse effects, eg weight gain with sulfonylurea, non-sulfonylurea secretagogues or TZD, edema and anemia with TZD [Caused by complex interactions of multiple factors, diabetes mellitus type 2 (type 2 diabetes) is characterized by decreased secretion of insulin by the pancreas and resistance to the action of insulin in various tissues , Pueraria lobata (Pea family), Dioscorea batatas DECAISNE (Dioscoreaceae), Rehmannia glutinosa (Phrymaceae), Amomum cadamomum Linné (Zingiberaceae), Poncirus fructus(Rhamnaceae)) and Evodia officinalis DODE(Rutaceae) were purchased from Kwangmyungdang Natural Pharmaceutical (Korea) and identified morphologically, histologically and authenticated by Professor Su-In Cho  according to standard protocol in National Standard of Traditional Medicinal Materials of The Korean Pharmacopeia [The dried herbs of macopeia . VoucherMyristica fragrans Houtt (Myristicaceae), was prepared for positive control [Powders of the herbs were mixed in equal amount (200 g each) and extracted in hot-water. The extract was freeze dried to powder and melt by dimethylsulfoxide (DMSO) when used. Macelignan, an active compound of  control .2C12 skeletal myoblast cells were grown in DMEM supplemented with 2% horse serum to induce differentiation into myotubes.Cell lines of human embryonic kidney (HEK) 293 (CRL-1573), 3T3-L1 pre-adipocytes (CL-173), HepG2 hepatocytes (HB-8065) and C2C12 skeletal myoblast cells (CRL-1772) were obtained from the American Type Culture Collection . HEK293 and HepG2 were cultured in Dulbecco's modified Eagle's medium (DMEM) containing glucose  supplemented with 10% (v/v) fetal bovine serum . The 3T3-L1 pre-adipocytes were differentiated as described previously . C2C12 s5 cells/well) were transfected with PPRE-tk-Luc and incubated with the extract, rosiglitazone or macelignan for 24 hours. The luciferase activities were then determined with a luciferase assay system kit .The PPAR ligand-binding activity was measured with a GAL4/PPAR chimera assay and PPRE-tk-Luc reporter assay as described previously . HEK293 5 cells/well) with NFkB-Luc reporter or ERSE-Luc reporter using a Cignal™ Reporter Assay kit . The cells were then incubated with the extract, rosiglitazone or macelignan for 24 hours. The luciferase activities were determined with a Dual-Glo Luciferase assay system kit .To determine the anti-inflammatory activities and anti-endoplasmic recticulum (ER) stress, we transfected HepG2 cells , acyl-CoA synthetase (ACS) and carnitine palmitoyltransferase-1 (CPT-1). The total RNA was extracted with TRIzol reagent  and subjected to reverse transcription with M-MLV Reverse Transcriptase . The total RNA was then amplified (with gene-specific primers) and quantified with a fluorescence thermocycler .Total proteins were extracted with PRO-PREP reagent  and immuno-blotted with the antibodies of p-AMPK, IkBα, GRP78 or p-elf2α  . The immdb/db mice aged 8 weeks were purchased from Jackson Laboratory (USA) and individually housed in polycarbonate cages under a 12-hour light-dark cycle at 21-23°C and 40-60% humidity. After a 2-week adaptation period, the body weight and fasting blood glucose level of the 10-week-old mice were measured. Then, the mice were equally divided into four groups (n = 7): (1) diabetic control, (2) rosiglitazone, (3) macelignan and (4) treatment (with the extract). All groups were fed a standard AIN-76 semi-synthetic diet (American Institute of Nutrition) and three experimental groups  were orally administered with rosiglitazone (10 mg/kg body weight), macelignan (15 mg/kg body weight) or the extract (150 mg/kg body weight) for three weeks. After starved for 12 hours, the mice were anesthetized with ether and their blood samples were collected from the inferior vena cava for the measurement of the blood and plasma biomarkers such as HbA1c and insulin. All animal handlings during the experiments were in accordance with the Pusan National University guidelines for the care and use of laboratory animals.Twenty-eight (28) male C57BL/KsJ-1c) collected from sacrificed mice was measured with a MicroMat™ II Hemoglobin A1c Test . All blood samples obtained were centrifuged at 1000 × g for 15 min at 4°C for biochemical analysis. The plasma insulin, glucagon and C-peptide levels were measured with the enzyme-linked immunosorbent assay (ELISA) kits .During the experiments, the fasting blood glucose concentration was monitored by a Glucometer  with venous blood drawn from the mouse tail vein after a 12-hour fast. Moreover, the blood glycosylated hemoglobin  while the plasma free fatty acid (FFA) concentration was determined with an ACS (acyl-CoA synthetase)-ACOD(ascorbate oxidase) method .db/db mice after a 12-hour overnight fast. To determine the glucose and insulin tolerance, we injected the mice intraperitoneally with glucose (0.5 g/kg body weight) or insulin (2 unit/kg body weight). The glucose concentrations of blood drawn from the tail vein were determined immediately upon collection at 30, 60 and 120 min after glucose injection or at 30, 60 and 120 min after insulin injection.On the third week of treatment, an intraperitoneal glucose and insulin tolerance test (IPGTT and IPITT) were performed on all All statistical tests were two-sided, and the level of significance was set at 0.05. All data are presented as mean ± standard deviation (SD) for all groups. Statistical analyses were performed with the SPSS, version 18. One-way ANOVA with post-hoc test by Duncan's multiple-range test was used to examine differences among groups. The data were analyzed by Student's t-test for two group comparison.P = 0.035 vs non-treatment) similar to that of rosiglitazone (P = 0.001 vs non-treatment), a well-known PPARγ agonist, and macelignan (P = 0.005 vs non-treatment), a PPARα/γ dual agonist used as positive control throughout the experiments. To further explore the PPARγ agonist potential of the extract, transient transfections were performed in differentiated 3T3-L1 adipocytes with the tk-luciferase vector containing PPAR-responsive elements (PPREs) and then treated with the extract. The treatment stimulated PPRE-dependent luciferase activities in transfected cells (P = 0.005 vs non-treatment)   Figure . To prov) Figure . Moreove) Figure . Taken t2C12 cells incubated with the extract. As with the AMPK activator, aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) (P = 0.001 vs non-treatment), the treatment activated AMPK in C2C12 cells (P = 0.007 vs non-treatment), similar to when samples were treated with macelignan (P = 0.042 vs non-treatment)  and carnitine palmitoyltransferase-1(CPT-1) (P = 0.041 vs non-treatment)  activation, we measured the AMPK phosphorylation and expression of fatty acid oxidation genes in C) Figure . Consist) Figure , suggestP = 0.001 vs non-treatment), The extract effectively prevented this increase (P = 0.034 vs TNF-α treatment)  Figure . These r2-terminal kinase (JNK) activation [P = 0.001 vs non-treatment), the extract effectively blocked the thapsigargin-mediated stimulation (P = 0.039 vs thapsigargin treatment)   Figure . When ER) Figure . Taken tin vivo anti-diabetic effects of the extract on diabetes, we orally administered rosiglitazone (10 mg/kg), macelignan (15 mg/kg) and the extract (150 mg/kg) to C57BL/KsJ-db/db mice every day for three weeks and the extract's effects were compared with rosiglitazone and macelignan. Treatment with the extract did not have a significant effect on the body weights in the db/db mice; however, mice treated with rosiglitazone had final body weights significantly higher than those of the others (P = 0.001 vs control)  and so did the other groups treated with rosiglitazone (P = 0.001 vs control) and macelignan (P = 0.002 vs control). The blood glucose levels of the extract-treated mice were significantly reduced by about 15% compared to the control  Figure . The basl Figure .P = 0.001 vs control) similar to rosiglitazone (P = 0.003 vs control) and macelignan (P = 0.004 vs control) used as positive controls compared with the diabetic control groups. The insulin tolerance test also showed that reduction in blood glucose levels in response to insulin was much greater in mice treated with the extract than in untreated db/db mice (P = 0.002 vs control)  Figure . These fP = 0.021 vs control), triglyceride (P = 0.012 vs control) and total cholesterol (P = 0.003 vs control) concentrations of the diabetic control db/db mice compared with untreated db/db mice when the experiment ended . Both the plasma insulin (P = 0.042 vs control) and C-peptide levels (P = 0.038 vs control) were significantly higher in the extract-treated db/db mice than in the diabetic control db/db mice; however, the glucagon levels were significantly lower than those of the diabetic control db/db mice (P = 0.018 vs control). Therefore, treatment with the extract significantly improved the ratio of insulin/glucagon (I/G) when compared to the diabetic control db/db mice -ptivities . TherefoActivation of AMPK enhances insulin sensitivity through increased glucose uptake and lipid oxidation in skeletal muscle and inhibition of glucose and lipid synthesis in the liver . MetformInflammatory cytokines and IKK attenuate insulin signaling through serine phosphorylation of IRS-1 . High doBy activating c-Jun amino-terminal kinase (JNK), which induces insulin resistance in liver and skeletal muscle and inhibits beta cell function, ER stress induces the development of type 2 diabetes . Thus, aThe aqueous extract of these seven hypoglycemic herbs demonstrated anti-diabetic effects on type 2 diabetes.1c: blood glycosylated hemoglobin; HDL-cholesterol: high density lipoprotein-cholesterol; HEK293: human embryonic kidney293; IKK: IκB kinase; IPGTT: intraperitoneal glucose tolerance test; IPITT: intraperitoneal insulin tolerance test; JNK: c-Jun N-terminal kinases; LPL: lipoprotein lipase; OAD: oral antidiabetic drug; PBA: phenyl butyric acid; PPAR: peroxisome proliferator-activated receptor; PPREs: PPAR-responsive elements; SD: standard deviation; TZD: thiazolidinedioneACS: acyl-CoA synthetase; AICAR: aminoimidazole carboxamide ribonucleotide; AMPK: AMP-activated protein kinase; aP2: adipose fatty acid-binding protein 2; CPT-1: carnitine palmitoyltransferase-1; DMSO: Dimethylsulfoxide; ER: endoplasmic reticulum; ERSE: ER stress response element; FFAs: free fatty acids; eIF: elongation initiation factor; GLP-1: glucagon-like peptide-1; HbAThe authors declare that they have no competing interests.in vivo experiments. YMK conducted the experiments in cultured cells. All authors read and approved the final version of the manuscript.MHJ designed the study and wrote the manuscript. SIC prepared the aqueous extract from the herbs. JY conducted the