Expression of either CA-Lyn or DN-Lyn had no effect on the survival of U87 GBM cells grown under nutrient-rich conditions. In contrast, under nutrient-deprived conditions  CA-Lyn expression enhanced survival and promoted autophagy as well as inhibiting cell death and promoting proliferation. Expression of DN-Lyn promoted cell death. In the nutrient-deprived GBM cells, CA-Lyn expression enhanced AMPK activity and reduced the levels of pS6 kinase whereas DN-Lyn enhanced the levels of pS6 kinase. Similar results were obtained in vitro using another cultured GBM cell line and primary glioma stem cells. On propagation of the transduced GBM cells in the brains of nude mice, the CA-Lyn xenografts formed larger tumors than control cells and autophagosomes were detectable in the tumor cells. The DN-Lyn xenografts formed smaller tumors and contained more apoptotic cells. Our findings suggest that on nutrient deprivation in vitro Lyn acts to enhance the survival of GBM cells by promoting autophagy and proliferation as well as inhibiting cell death, and Lyn promotes the same effects in vivo in xenograft tumors. As the levels of Lyn protein or its activity are elevated in several cancers these findings may be of broad relevance to cancer biology.Members of the Src family kinases (SFK) can modulate diverse cellular processes, including division, death and survival, but their role in autophagy has been minimally explored. Here, we investigated the roles of Lyn, a SFK, in promoting the survival of human glioblastoma tumor (GBM) cells  Lyn is one of eight members of the Src family of kinases (SFK) expressed in human cells Lyn activity is elevated in GBM, the highest grade of glioma tumors, as well as in breast cancer, acute myelocytic leukemia (AML), B-cell chronic lymphocytic leukemia (CLL) and Ewing’s sarcoma Here, we investigated the role of Lyn in promoting survival of GBM cells under nutrient-rich conditions and conditions of nutrient deprivation focusing on its role in autophagy. During autophagy, cytoplasmic proteins and organelles are sequestered in autophagasomes, which allows the cell to generate energy and nutrients We found that the survival of GBM cells transduced with either a lentiviral vector carrying constitutively-active (CA) Lyn or a dominant-negative (DN) Lyn construct grown under nutrient-rich conditions did not differ from control cells. In contrast, the results of similar analyses carried out under conditions of nutrient deprivation indicated that Lyn promotes survival of nutrient-deprived GBM cells through both promotion of autophagy and inhibition of apoptosis. When grown as xenografts, the CA-Lyn tumor cells formed larger tumors that contained autophagosomes and DN-Lyn cells formed smaller tumors with increased evidence of apoptosis. These studies suggest that Lyn can play a role in promoting survival of GBM cells by facilitating autophagy and underscores the importance of gaining an improved understanding of SFK-associated mechanisms in stressed cells.The animal experiments were performed in accordance with an approved protocol from the Cleveland Clinic Institutional Animal Care and Use Committee (IACUC) (#2011–0554).i.e., L-glutamine-free DMEM supplemented with 1% BSA  and without FBS. GBM cells have an increased demand for L-glutamine U87 human GBM cells were obtained recently from the American Type Culture Collection and maintained in L-glutamine-free DMEM  supplemented with 10% FBS and 1 mM L-glutamine. SNB19 human GBM cells The primary human glioma stem cell (GSC) line (3832) i.e., 4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolopyrimidine (PP2) (EMD Millipore), perifosine and rapamycin , 3-methylamine (3-MA), and chloroquine (Invitrogen).In some experiments, inhibitors were added to the culture medium, 6 infectious particles/ml) with vectors expressing CA-Lyn, DN-Lyn, or empty vector. Stable populations of U87 and SNB19 cells were selected with 5 µg/ml puromycin (2 weeks), monitored for GFP fluorescence on a regular basis, and sorted for GFP-expressing cells as necessary. After transduction, GSCs were sorted by FACS based on GFP positivity.CA-Lyn (Y508F) and DN-Lyn (Y397F) cDNAs were kind gifts from Dr. Evan Ingley at the Western Australian Institute for Medical Research Antibodies were purchased as follows: rabbit anti-pY397FAK and anti-total FAK , rabbit anti-Fyn, anti-Lyn and anti-cSrc ; monoclonal antibody (mAb) anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and mAb anti-actin ; rabbit anti-LC3B, anti-phospho-Akt and anti-total Akt, and antibodies to pS6 kinase and total S6 kinase ; rabbit anti-LC3B (Nanotools); rabbit polyclonal IgG (sc-25575) (Santa Cruz Biotechnology); anti-phospho-AMPK (Thr172) and anti-total AMPK  and anti-phospho-a1-AMPK (Thr172) (Millipore).Cells were lysed using lysis buffer containing NP-40 and protease inhibitors and electrophoresed or immunoprecipitated followed by western blotting and densitometry Viability was assessed by trypan blue exclusion and apoptosis determined by FACS analysis of Annexin V-APC-stained cells, blotting for cleaved caspase-7, or TUNEL assays Autophagosomes were detected by analysis of microtubule-associated protein light chain 3 (LC3) Xenografting was performed as described previously This was performed as described Statistical analyses were performed by the biostatistician and author (ASN). The statistical test utilized is stated in each figure legend.th panel, The levels of Lyn activity were manipulated by transducing U87 GBM cells with lentiviral vectors expressing a CA-Lyn construct or a DN-Lyn construct. The SFKs, including Lyn, are maintained in an inactive state by the C-terminal Src kinase (Csk), which phosphorylates the C-terminal negative regulatory peptide (Y508 in Lyn). The phosphorylated tyrosine binds to the SH2 domain folding SFK into an inactive configuration Analysis of cell viability indicated that neither the expression of CA-Lyn nor DN-Lyn affected viability under nutrient-rich conditions . In contTo confirm that the kinase activity of CA-Lyn is required for Lyn promotion of GBM cell survival under nutrient deprivation, we utilized PP2 (a broad SFK inhibitor). We first selected the dosage of PP2 that inhibited SFK activity of the U87 cells when added to the media 24 h after initiation of nutrient deprivation. Blotting for pSrcY418 on day 5 confirmed inhibition of Lyn activity in the U87-CA-Lyn cells . In the We then transduced the cells with lentiviral vector expressing RFP-tagged LC3 Similar results were obtained using another human GBM cell line, SNB19. We found that lentiviral-mediated expression of CA-Lyn resulted in a broad band migrating at 53–56-kDa on blotting for active Src(pY418)  that wasTo confirm the effects of Lyn on autophagy in the nutrient-deprived cells we used several different approaches. Overnight treatment of nutrient-deprived cells with chloroquine, an inhibitor of lysosomal fusion, resulted in an increase in the levels of LC3B-II protein in the U87-CA-Lyn, U87-LV and U87-DN-Lyn cells , consistAs autophagy is negatively regulated by the mTORC1 complex, we estimated mTORC1 activity by assessing phosphorylation of S6 kinase, a downstream effector of mTORC1 Akt activation of mTORC1 can inhibit autophagy GSCs are thought to be the tumor-initiating cell in GBM in vivo, we injected U87-CA-Lyn, U87-DN-Lyn and U87-LV cells into the brains of nude mice. After 35 days, the U87-CA-Lyn cells had formed significantly larger tumors than the U87-LV cells whereas the U87-DN-Lyn cells had formed significantly smaller tumors  in tumor cells and the results of quantitation are shown in in vitro correlated with increased activation of AMPK and inhibition of the mTORC1 complex. The decrease in phosphorylation of S6 kinase in the GBM cells expressing CA-Lyn and the increase in the cells expressing DN-Lyn was consistent with the effects of these constructs on autophagy. Glucose, growth factor and amino acid deprivation activate AMPK thereby relieving mTORC1 inhibition of autophagy Here, we show that Lyn promotes the survival of nutrient-stressed human GBM cell lines and primary human GSCs by promoting autophagy and proliferation as well as inhibiting cell death. The mechanism underlying the pro-autophagy effect of Lyn in the nutrient-deprived U87 GBM tumor cells and in the growth factor-deprived GSCs SFK activity was necessary for the prosurvival effect of Lyn in nutrient-deprivation conditions, which suggests that Lyn kinase activity is necessary. However, we have not yet identified its substrate. Although multiple SFK substrates have been identified, none are known to positively regulate autophagy. The tyrosine phosphorylation of the known SFK substrates, FAK(Y397 and Y925) , cortactThe manipulation of Lyn activity also affected apoptosis and proliferation of nutrient-deprived cells. Expression of CA-Lyn resulted in a significant reduction in cell death and significant increase in EdU incorporation whereas expression of DN-Lyn resulted in a significant increase in cell death but no change in EdU incorporation. Although miRNA-205 has been shown to inhibit mRNA and protein expression of Lyn, c-Src, and c-Yes in A498 cells resulting in G0/G1 cell cycle arrest and apoptosis in vivo studies in which GBM cells expressing DN-Lyn were propagated in the nude mouse brain showed a significant reduction in tumor size that was associated with increased tumor cell death, suggesting Lyn is important for tumor survival in vivo. This is consistent with a prior report that dasatinib inhibits malignant glioma growth in a mouse model e.g., in addition to blocking the function of all SFKs it also targets the cytoplasmic non-receptor tyrosine kinase c-Abl, which is necessary for apoptosis of brain microvessel endothelial cells induced by an integrin αvβ3/αvβ5 inhibitor and by latrunculin in vivo, but a focal lack of nutrients does occur in GBM Our Elevation of Lyn is associated with resistance to therapy. In CML, it is associated with the development of resistance to imatinib mesylate, a Bcr-Abl tyrosine kinase inhibitor in vivo using a mouse model. Lyn promotes the malignant phenotype of GBM and multiple other cancers, including promotion of the epithelial-mesenchymal transition in breast cancer in vivoIn summary, we demonstrate the novel findings that Lyn promotes autophagy and proliferation, as well as inhibits cell death, in nutrient-deprived GBM cells in culture and Figure S1(TIF)Click here for additional data file.Figure S2(TIF)Click here for additional data file.Figure S3(TIF)Click here for additional data file.Figure S4(TIF)Click here for additional data file.


Archaea had been absent from the caspase inheritance discussion due to a lack of gene homologues. We recently demonstrated extremely high, basal caspase-like catalytic activity in the model haloarcheon, Haloferax volcanii, which was linked to the cellular stress response and was widespread among diverse Archaea. Here, we rigorously tested the catalytic specificity of the observed archaeal caspase-like activities using hydrolytic assays with a diverse suite of protease substrates and inhibitors compared with known model serine and cysteine proteases . Our experiments demonstrate that exponentially growing H. volcanii possesses a highly specific caspase-like activity that most closely resembles caspase-4, is preferentially inhibited by the pan-caspase inhibitor, zVAD-FMK, and has no cross-reactivity with other known protease families. Our findings firmly root the extremely high levels of caspase-like activity as the dominant proteolytic activity in this extreme haloarcheaon, thereby providing further support for housekeeping functions in Haloarchaea. Given the deep archaeal roots of eukaryotes, we suggest that this activity served as a foundation for stress pathways in higher organisms.Caspase-like proteases are key initiators and executioners of programmed cell death (PCD), which is initiated by environmental stimuli and manifests in organisms ranging from unicellular microbes to higher eukaryotes.  Hydra to humans  activity and expression of immunoreactive proteins to human caspase-8 antisera, both of which were induced by salt stress and death and were abolished by in vivo addition of a broad-spectrum caspase inhibitor  and in diverse members of Euryarchaeota (the methanogen Methanosarcina acetivorans and the hyperthermophile Pyrococcus furiosus) and Crenarchaeota (the acidophile Sulfolobus solfataricus) argue for broad representation within the archaeal domain , VDVAD- (caspase-2), DEVD- (caspase-3), LEVD- (caspase-4), WEHD- (caspase-5), VEID- (caspase-6), IETD- (caspase-8), and LEHD-AMC (caspase-9)] to expand our coverage from previously documented caspase-8-like activity  h−1 mg protein−1] and VEID-AMC (36 001 RFU h−1mg protein−1) substrates, respectively, compared with IETD-AMC (6909 RFU h−1mg protein−1). AMC fluorescence calibration curves placed the corresponding substrate hydrolysis rates at 4124, 2000, and 383 nmol h−1mg protein−1  activities in diverse unicellular marine phytoplankton, including cyanobacteria, diatoms, coccolithophores, and chlorophytes, are comparatively low for exponentially growing cells [10 s to 100 s of RFUs hH. volcanii cell extracts to four model proteases, trypsin, cathepsin, papain, and recombinant human caspase-8, helped to further place it in the caspase catalytic class  and aspartate-AMC (Asp-AMC), respectively, given the H. volcanii genome  has 13 annotated aminopeptidases . Leu-AMC is a commonly used aminopeptidase substrate for diverse environmental microbes .We also tested for general leucine and aspartate aminopeptidase activity in H. volcanii cells and refined the activity as more caspase-4-like, and demonstrate that the previous findings using IETD actually underestimated H. volcanii caspase activity. In order to address the possibility that the absence of some enzyme activities  in our extracts may have been due to competing small molecules, we also independently assayed for these activities in cell extracts heated to 60°C and in partially purified proteins at 42°C obtained using standard ammonium sulfate (AmSO4) precipitation techniques  and successful LLVY-AMC cleavage has only been reported at 60°C ; likewise, LLVYase activity was also observed in the 80% and 100% AmSO4 precipitate fractions, albeit much lower  and in 80% and 100% AmSO4 precipitate fractions . Boiling of cell extracts at 100°C for 30 min completely abolished all hydrolysis activities, indicative of protein dependence. Our results confirm prior observations  of the maximum observed caspase activity (i.e. LEVDase) and was not detectable at physiological temperatures.Our results confirmed the consistent and extremely high basal activity in exponentially growing ractions . Neitherractions ; they weH. volcanii was further verified by challenging cell extracts with a panel of protease inhibitors that target a diverse array of proteases including serine proteases , cysteine proteases , metalloproteases (EDTA), and a cocktail of these inhibitors  or caspases (z-VAD-FMK). The aforementioned four model proteases, trypsin (serine protease), papain and cathepsin (cysteine proteases), and caspase-8 (caspase), were used to diagnose the specificity and efficacy of the chosen inhibitors when incubating with their preferred substrate. As expected, each specific inhibitor exhibited between 84 and 100% inhibition of protease activity for its given substrate  and caspase-6-like (VEIDase) activities further supporting that this activity is produced by a caspase-like enzyme(s) despite possessing distinct sequence differences to classic caspase superfamily proteins  of caspase-8-like activity (IETDase) when zVAD-FMK , corrobo et al., . Likewisactivity . These rHaloferax volcanii has a very high tolerance for MgCl2 , indicating a possible requirement for magnesium ions  stress . Our results highlight the need to identify the protein(s) that confer caspase activity and function. Given the high specificity of H. volcanii to caspase catalytic activity our findings strongly suggest that recently developed in situ inhibitor trapping techniques (Tu et al., Our findings firmly root extremely high levels of specific caspase activity, particularly resembling caspase-4, as the dominant proteolytic activity in this extreme halophilic archeaon. These findings, along with our previous work that linked very high basal caspase activity to the cellular salt stress response (Bidle


Although a majority of detected polymorphisms were due to point mutations, two consecutive nucleotide substitutions  were also identified in nine miRNAs. We determined that miR-SNPs are frequently located within the quantitative trait loci (QTL), chromosome fragile sites, and cancer susceptibility loci, indicating their potential role in the genetic control of various complex traits. To test this further, we performed an association analysis between the mmu-miR-717 seed SNP rs30372501, which is polymorphic in a large number of standard inbred strains, and all phenotypic traits in these strains deposited in the Mouse Phenome Database. Analysis showed a significant association between the mmu-miR-717 seed SNP and a diverse array of traits including behavior, blood-clinical chemistry, body weight size and growth, and immune system suggesting that seed SNPs can indeed have major pleiotropic effects. The bioinformatics analyses, data and tools developed in the present study can serve researchers as a starting point in testing more targeted hypotheses and designing experiments using optimal species or strains for further mechanistic studies.MicroRNAs (miRNAs) are a class of non-coding RNA that plays an important role in posttranscriptional regulation of mRNA. Evidence has shown that miRNA gene variability might interfere with its function resulting in phenotypic variation and disease susceptibility. A major role in miRNA target recognition is ascribed to complementarity with the miRNA seed region that can be affected by polymorphisms. In the present study, we developed an online tool for the detection of miRNA polymorphisms (miRNA SNiPer) in vertebrates ( MicroRNAs (miRNA) are non-coding RNA molecules with approximately 21 nucleotides in length, which play an important role in posttranscriptional regulation of mRNA. By binding to the target gene's complementary sequence of the 3′ untranslated region (3′UTR) they repress translation Gpc3 gene, miRNA gene (mmu-miR-717), and miR-seed-SNP identified in the lean mouse strain 129/Sv The key binding location for translational suppression resides in the mature miRNA sequence, more accurately nucleotides 2–7 or 2–8 from the 5′ end of the miRNA, also called the seed region Polymorphisms within miRNA genes have been reported to be rare, with only approximately 10% (65/474 reported miRNAs at that time) of human pre-miRNAs having documented SNPs, and <1% (3/474) of miRNAs having SNPs in the functional seed region http://www.mirbase.org/) http://www.targetscan.org/) http://www.ensembl.org/) http://integratomics-time.com/miRNA-SNiPer/.A web based tool named “miRNA SNiPer” was developed for the detection of polymorphisms residing within miRNA genes in vertebrates. It accepts a list of miRNA genes and returns a table of variations within different regions of miRNA genes: pre-miRNA, mature, and seed region. The mature sequences are designated as “miR” and the precursor hairpins as “mir” http://www.integratomics-time.com/miR-seed-SNPs/catalog/). The assembled list was supplemented with information relevant for further analysis from the literature and from other sources. Validation status and allele frequencies were retrieved from the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/). Overlaps with host genes were retrieved from miRBase, release 18 http://diana.cslab.ece.ntua.gr/tarbase/) http://mirecords.biolead.org) databases The developed tool miRNA SNiPer was used to generate an assembly of miR-seed polymorphisms in vertebrates (http://www.integratomics-time.com/miR-seed-SNPs/genomic_view/) using Flash GViewer web tool (http://gmod.org/wiki/Flashgviewer/). Overlap analysis of miRNAs comprising seed-SNPs with genomic fragile sites was performed using data retrieved from Ensembl via BioMart. Human and mouse QTL were retrieved from the Rat Genome Database (RGD) (http://rgd.mcw.edu/) http://www.animalgenome.org/cgi-bin/QTLdb/index/) Genomic distribution of miR-seed polymorphisms was presented on a genomic view (http://www.targetscan.org/) was used to analyze whether the miR-seed-SNP cause the formation of seed regions annotated to different miRNA http://watson.compbio.iupui.edu:8080/miR2Disease/) TargetScan Custom feature (mmu-miR-717 (rs30372501) and phenotypes was analyzed. Data for the genotype-phenotype association analysis was downloaded from the Mouse Phenome Database  mmu-miR-717  consisting of a various number of measurements, ranging from one to 311 for each strain.Association between the mouse seed SNP in ijkl represents the observation for the traits, μ trait average, Gi fixed effect of genotype mmu-miR-717 seed SNP rs30372501 , Lij nested effect of strain (j = 1−14), Sk fixed effect of sex , and eijkl random error.Statistical package SAS/STAT mmu-miR-717 showed association with a diverse array of traits.We developed a web-based tool for the detection of genetic variations in miRNA genes in vertebrates and generated an open access catalog of polymorphisms that overlap with miRNA seed regions Fig. 1A.http://www.integratomics-time.com/miRNA-SNiPer/) was developed using data assembled from miRBase, TargetScan and Ensembl Variation database . These polymorphisms included SNPs, double nucleotide polymorphisms (DNPs), and insertion/deletions (indels). An example of miR-SNPs located within the pre-miRNA, mature and seed regions of miRNA gene is demonstrated in Display settings enable the miR-SNPs to be arranged according to their location in pre-miRNAs, mature or seed regions. In six vertebrate species  149 polymorphisms overlapped with miRNA seed regions . The miRNA SNiPer tool is going to be updated with each new release of compatible databases.http://www.integratomics-time.com/miR-seed-SNPs/catalog/). From the total of 149 identified miR-seed polymorphisms only 29 have previously been described in the miRNA context Table S1). Genomic distribution of the assembled miR-seed-SNPs was presented on the genomic view (http://www.integratomics-time.com/miR-seed-SNPs/genomic_view)  was supplemented with data retrieved from literature and databases, and presented as an open access online catalog (ic_view) Fig. 3.et al.gga-mir-1658 had one SNP within the mature sequence (gga-miR-1658) and two SNPs within the minor miR sequence (gga-miR-1658*). Our study also revealed four human miRNAs  that had two validated consecutive nucleotides altered, so called DNPs, which have been found to occur with a frequency of ∼1% of the total number of SNPs in the genome The frequency estimations of miRNAs having polymorphic seed regions at this point should be treated with caution because all miRNAs have not yet been systematically sequenced and screened for polymorphisms, and some SNPs in the databases still have unvalidated status. Our preliminary data showed that in human 88 of 1527 (5.7%) currently annotated miRNAs had polymorphic seed regions . Similarly, in mouse 13 of 741 (1.7%) miRNAs had miR-seed-SNPs , whereas in chicken six of 499 (1.2%) miRNAs had miR-seed-SNPs . Nevertheless, our analysis of currently available data revealed a much higher frequency (5.9%) of miR-seed polymorphisms in human than the frequency of >1% reported by Didiano and Hobert hsa-miR-96 and mmu-miR-96 which have been previously linked with hearing loss in both human and mouse Allele frequency was available for 90 SNPs in human, 12 in mouse, and eight SNPs in chicken (NCBI). Population-based differences were observed for 41 human SNPs; among them rs12975333 described as polymorphic in three studies The data from the catalog were further analyzed to prioritize promising SNPs for further functional analysis. We examined each miR-seed SNP for their potential to generate novel seed regions that also match to a different miRNA, for their genomic distribution, overlaps with host genes, QTL, and fragile regions, as well as their association with diseases and phenotypes.et al., hsa-miR-96 caused a change in the seed region to perfectly match another annotated miRNA hsa-miR-514, implying a possibility of targets shared by both miRNAs. To determine whether miR-seed-SNPs cause the formation of seed regions annotated to different miRNAs we screened the catalog using TargetScan Custom. SNPs in hsa-miR-3117-3p and -4467 matched two different seed regions of hsa-miR-499-5p and -885-3p, respectively . A change of annotated seed regions may lead to altered recognition and selection of miRNA targets, which could possibly be a part of a different biological pathway.As shown in Mencia Figures S2 and S3 for human, mouse, and chicken, respectively. The highest number of miR-seed polymorphisms was present on human chromosomes 1, 15, 19, and 20. Several miRNAs with polymorphic seed regions overlapped with host genes, QTL, fragile sites, or cancer susceptibility sites.Genomic locations of miRNAs comprising seed polymorphisms are shown in Table S1). MiRNA genes and their host genes in antisense orientation have been shown to have independent transcription mechanisms Table S1). Intronic miRNAs have previously been found to be co-expressed and regulated by co-activation of both miRNA and its host gene We observed 75 miRNAs with polymorphic seed regions residing within protein coding host genes; 16 in antisense, 55 in sense orientation and four miRNAs that overlapped with host genes both orientations (hsa-miR-4737 and hsa-miR-4756 each overlapping 43 QTL. In mouse mmu-miR-628 overlapped with 23 QTL and in chicken gga-miR-1658 with seven QTL (data not shown). MiRNA genes have also been observed to be frequently located near the mouse cancer susceptibility loci, which is in concordance with a previous study of Sevignani et al.By comparing locations of polymorphic seed regions with QTL and fragile sites, several overlaps were found. MiRNAs with validated seed SNPs overlapped with 830 QTL in human, 118 in mouse and 20 in chicken. Highest number of overlapped QTL in human was observed for Hsa-miR-1302-1 overlapped with a common fragile site located at 12q24.1 and hsa-miR-122-3p overlapped with a fragile site located at 18q21.3  genotypes and all 2586 phenotypes within the Mouse Phenome Database  and also groups of traits can be highly correlated . Therefore, one should not interpret these associations as causal but rather as a list of potential groups of traits that a particular miR-SNP could affect. In this sense a visual presentation  of associations shown in Table S2 can be informative as one can observe immediately which groups have the highest number of significant associations and hence string support and which trait groups can be further joined into related “super” groups . Such examination can help researchers to prioritize further causation experiments by providing them only a small number of different traits likely to be controlled by a miR-SNP.We reviewed published associations between miRNAs with polymorphic seed regions and diseases/phenotypes. Additionally, we performed a statistical genotype-phenotype association analysis using the data from the Mouse Phenome Database. Four human and two mouse miRNAs comprising seed-SNPs have already been associated with diseases and phenotypes Table 1 base see . The SNPet al.Associations between SNP rs30372501 and obesity traits reported by Kunej The following open questions could be addressed in future studies: 1) To examine the effects of SNPs that cause a formation of seed regions already annotated to different miRNAs. 2) To experimentally validate DNPs found in seed regions for their effect on miRNA target selection. 3) To study effects of miR-seed-SNPs identified herein on shared transcriptional regulation, expression and function of polymorphic miRNAs and their host genes. 4) Our statistical association analysis of seed-SNP with mouse phenotypes showed a diverse array for associated phenotypes. Further studies should be designed to examine the molecular mechanism for such differential miR-SNP pleiotropic effects.In conclusion, miR-seed polymorphisms may have a profound effect on a wide range of phenotypes. Using the database integration we assembled all known and identified novel miR-seed-SNPs, and performed a first systematic case-study in this field. The project is ongoing, as novel miRNAs and SNPs are yet to be discovered in human as well as in other animal species. However, results and tools developed in this study can be immediately used by interested scientific community to help retrieve valuable information and design efficient experimental plans in the field of miR-SNP research.Figure S1miR-seed-SNP causing formation of novel seed regions. Three examples of miRNAs (green) with seed-SNPs which cause a formation of a seed region annotated to another miRNA are indicated (red).(TIF)Click here for additional data file.Figure S2Genomic location of miRNAs with polymorphic seed regions in mouse.(TIF)Click here for additional data file.Figure S3Genomic location of miRNAs comprising seed polymorphisms in chicken.(TIF)Click here for additional data file.Figure S4mmu-miR-717 seed SNP rs30372501 and 363 traits clustered into 25 groups: behavior, blood-clinical chemistry, blood-hematology, blood-lipids, blood-miscellaneous, body composition, body weight size and growth, bone, brain, breathing pattern, cancer, cardiovascular, cell and tissue damage, ear, endocrine, gallbladder, immune system, ingestive preference, kidney, liver, local experiment parameter, muscle, respiratory, sensory gating and spleen.Graphical representation of (TIF)Click here for additional data file.Table S1Catalog of miRNAs with polymorphic seed regions in human, mouse, chicken, chimpanzee, rat, and zebra finch: genomic location, host gene orientation, nucleotide substitution and validation status of the SNP.(DOC)Click here for additional data file.Table S2Estimated differences between miR-seed- SNP (rs30372501) alleles (C>T), associated standard errors and P_values for 363 traits.(DOC)Click here for additional data file.


Archived tissues from previously completed prospective trials represent invaluable resource for biomarker development. However, such specimens are often stored as sections on glass slides, in which RNA is severely degraded due to prolonged air exposure. We evaluated whether a proportion of archived sectioned formalin-fixed paraffin-embedded (AS-FFPE) tissues yield transcriptome profiles comparable to freshly cut (FC) FFPE tissues, which can be used for retrospective class prediction analysis.Genome-wide transcriptome profiles of 6 to 7-year-old AS-FFPE tissue sections (generated from 5 to 16-year-old blocks) of 83 hepatocellular carcinoma (HCC) and 47 liver cirrhosis samples were generated by using whole-genome DASL assay (Illumina) and digital transcript counting (nCounter) assay (NanoString), and gene signature-based prediction of HCC subclasses and prognosis was compared with previously generated FC-FFPE profiles from the same tissue blocks.RNA quality and assay reproducibility of AS-FFPE RNA were comparable to intermediate to poor quality FC-FFPE samples . Analyzable transcriptome profiles were obtained in 64 (77%) HCC and 36 (77%) cirrhosis samples. Statistically more confident predictions based on random resampling-based method (nearest template prediction) were obtained in 37 (58%) HCC and 13 (36%) cirrhosis samples. Predictions made in FC-FFPE profiles were reproduced in 36 (97%) HCC and 11 (85%) cirrhosis AS-FFPE profiles. nCounter assay was tested in 24 cirrhosis samples, which yielded confident prediction in 15 samples (63%), of which 10 samples (67%) showed concordant predictions with FC-FFPE profiles.AS-FFPE tissues yielded poorer quality RNA and transcriptome profiles compared to FC-FFPE tissues. Statistically more confident class prediction was feasible in 37 of 83 HCC samples and 13 of 47 cirrhosis samples. These results suggest that AS-FFPE tissues can be regarded as a resource for retrospective transcriptome-based class prediction analysis when they are the only available materials. Clinical deployment of transcriptome-based biomarker has been a challenging task due to multiple reasons We have successfully utilized the cDNA-mediated annealing, selection, extension and ligation (DASL) assay We analyzed AS-FFPE tissue sections (one to three 10 micron-thick slices for each sample) sectioned from 5 to 16-year-old FFPE tissue blocks and archived for additional 6 to 7 years on glass slides at room temperature with drying reagent in sealed bags. These are subsets of samples analyzed in our previous studies, in which RNA was isolated from FC-FFPE tissues: 83 out of 118 hepatocellular carcinoma (HCC) tissues used to determine molecular subclasses Total RNA was isolated as previously described Whole-transcriptome profiling was performed by using the HumanHT-12 whole-genome DASL beadarray ver. 4.0 (Illumina). Total RNA was converted to cDNA using biotinylated oligo-dT18 and random nonamer primers, followed by immobilization to a streptavidin-coated solid support. The biotinylated cDNAs were then simultaneously annealed to a set of assay-specific oligonucleotides based on content derived from the National Center for Biotechnology Information (NCBI) Reference Sequence Database (release 98). The extension and ligation of the annealed oligonucleotides generate PCR templates that are then amplified using fluorescently-labeled (P1) and biotinylated (P2) universal primers. The labeled PCR products are captured on streptavidin paramagnetic beads, to yield single-stranded fluorescent molecules which are then hybridized, via gene-specific complementarity, to the HumanHT-12 BeadChip, whereafter fluorescence intensity is measured for each bead. Hybridized chips were scanned by using iScan (Illumina) and raw measurements were extracted by Genome Studio software ver. 3.0 (Illumina).www.broadinstitute.org/genepattern) As a quality measure of the expression profiles, we calculated proportion of gene probes with a “present” signal computed based on built-in negative control probes by the Genome Studio software . We also computed inter-sample correlation to detect outlier poor quality profiles as previously described The prognostic 186-gene signature was implemented in the digital transcript counting (nCounter) assay (NanoString) www.ncbi.nlm.nih.gov/geo) with accession numbers GSE10186 and GSE10140 for the HCC and liver datasets, respectively. AS-FFPE profiles newly generated in the current study are available at GEO with accession number GSE46444.Prediction of HCC subclasses  The quality of RNA extracted from AS-FFPE tissues was compared with FC-FFPE tissues. 2 values of 0.91–0.93 suggest that the amount of assayable RNA molecule was reduced to approximately 1/40 in AS-FFPE compared to FC-FFPE tissues. We next assessed how microarray signal detection is affected by archiving FFPE sections by comparing the proportion of probes with detected signal namely % present call . It is expected that %P-call is similar and inter-sample correlation is high within adult tissues sharing the same tissue lineage Consistent with the Bioanalyzer result, whole-transcriptome profiling of technical replicates of AS-FFPE RNA showed reproducibility comparable to the intermediate to poor quality FC-FFPE RNA samples Figure 2We next evaluated gene signature-based class prediction in the AS-FFPE profiles in comparison to the FC-FFPE profiles. In the 64 HCC samples, a meta-analysis-based signature of three HCC subclasses , which has been well-validated in multiple independent cohorts and assay platforms In the profiles of 36 liver cirrhosis samples, we performed class prediction using a prognostic 186-gene-expression signature, which has been extensively validated in multiple independent cohorts and assay platforms  certification Recently developed genomic profiling technologies such as DASL have enabled analysis of archived FFPE tissue specimens with rich clinical annotations and follow-up necessary for validation of predictive/prognostic molecular signatures. However, these assays are optimized for FC-FFPE materials, and AS-FFPE is often the only available archived materials in many pathology labs and clinical trial study coordination centers. In addition, FC-FFPE tissue processing requires scheduling of sectioning and RNA isolation within a certain timeframe, which is limited by sample processing capability of each pathology lab. Therefore, analysis of AS-FFPE tissues will substantially increase the opportunities of retrospective assessment of predictive/prognostic molecular signatures.Our results clearly demonstrated that the quality of AS-FFPE RNA is distinctly inferior to that of FC-FFPE RNA. Approximately three-fourths of AS-FFPE tissues yield analyzable transcriptome profiles for the purpose of retrospective class prediction analysis, and statistically more confident prediction was feasible in approximately one-third of the samples. The relatively good prediction confidence p-values and comparable prediction consistency in the nCounter assay suggest that the assay is another option to retrospectively analyze AS-FFPE tissues. The lack of target amplification may contribute to the higher prediction confidence. This may also indicate that high-throughput sequencing-based transcript counting could be an option once the assay is optimized for AS-FFPE specimens. A recent study showed minimal lab-to-lab and day-to-day experimental variations in the gene expression measurements in nCounter assay, further supporting its utility in the analysis of FFPE tissues The sites of degradation in FFPE RNA was observed roughly in random fashion in our previous study In summary, RNA isolated form AS-FFPE tissues was more degraded than that from FC-FFPE tissues, and yielded poorer quality transcriptome profiles. However, our results suggest that transcriptome signature-based disease classification is still feasible in a subset of AS-FFPE tissues, which will help increase the opportunities of retrospective validation and facilitate eventual clinical development of predictive/prognostic molecular signatures.


HIV-1 neutralizing antibodies (Abs) bind to the envelope glycoprotein (Env) spike, which functions as a trimer of gp120-gp41 heterodimers that is anchored in the viral membrane. However, Env trimers are low in copy number and coexist with irrelevant forms of Env and its byproducts, which typically elicit non-neutralizing Abs.Here, we have attempted to generate immunogens by fixing trimeric spikes of HIV-1JR-FL using a defined chemical crosslinker, purifying the crosslinked spikes from virions, and immunodepleting them of irrelevant Env contaminants using non-neutralizing Abs.Purified, crosslinked spikes were bound by a panel of neutralizing Abs, but not by non-neutralizing Abs, and are virtually devoid of non-trimeric Env. However, at least one neutralizing epitope on gp120, the crown of V3, appears to be occluded by crosslinking. An immunization was performed using the purified, crosslinked Env trimers as a boosting agent following a DNA prime using full-length env, either as soluble protein or captured onto small proteoliposome nanoparticles (PLNs). The binding titers of the Ab response to crosslinked Env spikes were quite weak, possibly reflecting an overall weaker immune response. Boosting animals with trimer-PLNs however elicited a qualitatively different neutralizing Ab response than uncrosslinked Env on virions, with sporadic activity against neutralization-resistant HIV-1 isolate JR-CSF, reduced neutralization of sensitive Tier 1 isolates, and reduced antibody responses against host protein.Immunizing animals using purified, crosslinked Env spikes captured on PLNs elicited a qualitatively different and broader neutralizing Ab response than uncrosslinked, heterogeneous Env. We conclude that with further changes to the crosslinking and immunization strategy crosslinked and purified Env spikes hold promise as a vaccine candidate.


Simvastatin (SV), a competitive inhibitor of 3-hydroxy-3-methylglutaryl CoA reductase and a widely prescribed treatment for hypercholesterolemia, exerts numerous positive pleiotropic effects that are thought to occur independent of its cholesterol-lowering properties. In previously published work, we have shown that chronic SV treatment rescues cognitive function in a transgenic mouse model of Alzheimer’s Disease, and enhances learning and memory in non-transgenic mice without affecting total brain cholesterol and amyloid-beta levels. More recently, we demonstrated the ability of SV to enhance long-term potentiation (LTP) in the CA1 region of the hippocampus in slices from wild type C57BL/6 mice via a mechanism dependent upon phosphatidylinositol 3-kinase (PI3-K)/Akt activation during LTP induction. The present study was conducted to better understand the molecular mechanisms underlying SV-induced enhancement of LTP. Specifically, it was found that inhibiting production of isoprenoid intermediates in the biosynthetic pathway for cholesterol triggers the downstream events leading to enhanced LTP. Interestingly, two major isoprenoid intermediates exhibit differential effects. Replenishment of farnesyl pyrophosphate, but not geranylgeranyl pyrophosphate, abolished the LTP-enhancing ability of SV. Parallel to this finding, inhibiting farnesylation, but not geranylgeranylation replicated the enhancement of LTP caused by SV. Finally, inhibiting farnesylation promotes the activation of Akt during the induction phase. Together, these results suggest that SV enhances LTP in CA1 by modulating isoprenylation-dependent molecular pathways downstream of farnesyl transferase. These findings will aid in the identification of novel therapeutic targets that modulate synaptic and cognitive function.Grant support: Supported by grants of NIH AG031846 and an Anonymous Philanthropic Foundation to L Li.This study was supported by Program for Excellent talent in University of Chongqing (2010).


Four of these CpG sites  and an additional hypomethylated CpG site (BIRC4BP) were implemented in a model to predict donor age. This Epigenetic-Aging-Signature was tested on a validation group of eight independent datasets corresponding to several cell types from different tissues. Overall, the five CpG sites revealed age-associated DNA-methylation changes in all tissues. The average absolute difference between predicted and real chronological age was about 11 years. This method can be used to predict donor age in various cell preparations - for example in forensic analysis.All tissues of the organism are affected by aging. This process is associated with epigenetic modifications such as methylation changes at specific cytosine residues in the DNA (CpG sites). Here, we have identified an Epigenetic-Aging-Signature which is applicable for many tissues to predict donor age. DNA-methylation profiles of various cell types were retrieved from public data depositories - all using the HumanMethylation27 BeadChip platform which represents 27,578 CpG sites. Five datasets from dermis, epidermis, cervical smear, T-cells and monocytes were used for Pavlidis Template Matching to identify 19 CpG sites that are continuously hypermethylated upon aging (R > 0.6; p-value <10 Aging has different consequences in different tissues - it results for example in wrinkle formation of dermis, graying of epidermally-derived hair, loss of bone formation, myeloid bias of blood, and compromised function of the immune system . Despitein vitro culture for several weeks these DNA-methylation profiles still reflected age-associated changes that relate to their donors, but this regulation differed markedly between MSC and fibroblasts indicating cell type specificity. Many other authors have used this platform to determine age-associated changes in primary tissues including dermis [Among epigenetic modifications, DNA methylation is best characterized. CpG dinucleotides in the mammalian genome can be enzymatically methylated at cytosines – and many studies demonstrated the occurrence of age-associated modifications in the DNA- methylation pattern -12. Receg dermis , epidermg dermis , blood [g dermis , 18, corg dermis , 20 and g dermis . Recentlg dermis . Based og dermis . Overallg dermis .On the other hand, some age-associated DNA-methylation changes do not appear to be tissue specific: Teschendorff and co-workers have identified a specific subset of 69 CpGs which are associated with polycomb group protein target genes and which revealed age-associated changes – notably, they described similar modifications in seven independent data sets including normal and cancerous tissues as well as cultured MSC . FurtherFor this study, we have combined several datasets which were retrieved from public data repositories. We have only considered datasets that 1) used the same Infinium HumanMethylation27 BeadChip platform, 2) were generated with freshly isolated cells to exclude effects by culture expansion, 3) used non-cancerous material since malignant transformation might influence age-related changes, and 4) provided reliable information about donor age. DNA-methylation datasets of 13 different cell types or tissues were used: 5 datasets were implemented as a training-set for identification of the Epigenetic-Aging-Signature and 8 datasets were reserved for subsequent validation table . Several−13). 19 CpG sites passed this criterion - notably, all of them revealed hypermethylation upon aging  resulting in age-associated hypermethylation at 431 CpG sites whereas 25 CpG sites were hypomethylated. This is in line with previous reports that demonstrated predominant hypermethylation at specific sites upon aging whereas hypomethylation might be less tightly regulated [Subsequently, we used Pavlidis Template Matching (PTM)  to identg Figure . These mg Figure . Subsequegulated , 21, 22.−8). Selection of CpG sites was irrespective of gene function as it has been shown, that site-specific methylation changes are hardly associated with differential gene expression [Next, we selected a subset of CpG sites to be integrated into the Epigenetic-Aging-Signature. Therefore, we have chosen CpGs which correlated with donor age across the whole training-set as well as in individual datasets. Another criterion was the variation in DNA-methylation level between young and elderly donors as larger changes are less prone to technical noise. Comparison of age-predictions in the training set led us to four hypermethylated CpG sites corresponding to tripartite motif-containing 58 , KCNQ1 downstream neighbor , neuronal pentraxin II  and glutamate receptor ionotropic AMPA 2 . We reasoned that predictions might be more robust by additional consideration of a hypomethylated CpG site. Therefore, we have also included XIAP associated factor-1  despite a lower correlation coefficient  and Array Express . After literature search we decided to include the following 13 datasets for subsequent analysis which were divided in a training-group for identification of the Epigenetic-Aging-Signature and a validation-group.In this study we have considered all at the time publically available datasets with the Infinium HumanMethylation27 BeadChip platform in the public repositories Gene Omnibus (+ T-cells and CD14+ monocytes were isolated from fresh venous whole blood as described by Rakyan and co-workers (GSE20242 and GSE20236) [+ hematopoietic progenitor cells (HPC) were isolated from cord blood and from G-CSF mobilized peripheral blood as described by Bocker and colleagues [The authors of these important primary studies have to be acknowledged: Grönniger and co-workers isolated keratinocytes from epidermal suction blisters and dermal fibroblasts from punch biopsies E-MTAB-202) . EpithelSE20236) . Saliva SE20236) . CD34+ hlleagues  . These beta-values represent the percentage of methylation at each of the 27,578 CpG sites – they are continuous variables between 0 and 1 and represent the intensity ratio of the methylated bead to the combined locus intensity. Background normalized raw data of these beta-values were determined with the BeadStudio software (Illumina) and retrieved from the public data repositories Gene Onmibus and Array Express. Initially we considered various normalization regimen including quantile normalization to minimize chip effects . On the To identify CpG sites which reveal continuous age-associated hypermethylation or hypomethylation we performed Pavlidis Template Matching (PTM)  with theversus donor age with EXCEL 2007 (Microsoft). These linear regression models were then used for age-predictions in the datasets of the training-group as well as for the validation-group: the five CpG sites (i) were inversely used to predict the age (N) by inserting the specific DNA-methylation levels of the corresponding CpG site (β).For each of these CpG sites we performed a linear regression analysis of beta-values A is the Y-axis intercept and B is the slope of the corresponding CpG site in the training group (Figure Where p Figure . The mea


Since publication of our article , we have"Therefore, we plan to repeat this study in a larger sample size including well defined endometrial samples obtained during menstrual, follicular and secretory phase, to validate the reproducibility of SELDI-TOF MS technology in these samples and to identify the protein peaks observed after proteomic analysis, which are expensive and labour intense requiring High-performance liquid chromatography or high-pressure liquid chromatography (HPLC) and matrix assisted laser desorption ionization Time-of-Flight-Mass Spectrometry (MALDI-TOF/TOF MS)."


BRCA1 or 2 genes. Results from such studies may define a new therapeutic paradigm, wherein simultaneous loss of the capacity to repair DNA damage may have antitumor activity in itself, as well as enhance the antineoplastic potential of cytotoxic chemotherapeutic agents.The poly (ADP-ribose) polymerase (PARP) family of enzymes plays a critical role in the maintenance of DNA integrity as part of the base excision pathway of DNA repair. PARP1 is overexpressed in a variety of cancers, and its expression has been associated with overall prognosis in cancer, especially breast cancer. A series of new therapeutic agents that are potent inhibitors of the PARP1 and PARP2 isoforms have demonstrated important clinical activity in patients with breast or ovarian cancers that are caused by mutations in either the  Environmental exposures and cell replication result in DNA damage that is repaired by a variety of mechanisms, including base excision repair (BER), mismatch repair (MMR), nucleotide excision repair (NER), single strand annealing (SSA), homologous recombination (HR), and nonhomologous end joining (NHEJ) . Poly , iniparib (BSI-201), olaparib , veliparib , MK-4827, BMN-673, CEP-9722  and E7016 GPI 21016, oral). The loss of BER capacity produced by PARP inhibition has prompted the evaluation of these drugs as potential enhancers of DNA damaging cytotoxic chemotherapeutic agents such as alkylating agents  and topoisomerase 1 inhibitors  [16, oral.BRCA1/2 defects. This could also be extended to include treatment of tumors with defects in other HR pathway proteins. For instance, PTEN-deficient cells have been shown to be sensitive to PARP inhibition, due to the role of PTEN in the expression of RAD51 [PARP inhibition enhances the therapeutic index of cytotoxic chemotherapy only if DNA damage is selectively increased in tumor compared to normal tissues, such as the gastrointestinal mucosa or bone marrow. The opportunity to achieve selectivity in tumor cell killing with these agents would, therefore, be improved in tumors that already harbor DNA repair defects. Simultaneous dysfunction of two DNA damage repair (DDR) pathways, termed 'synthetic lethality', decreases the ability of tumor cells to withstand the DNA damage produced during normal cellular replication . Duplicaof RAD51 . One queNew data are emerging on the myriad of effects of PARP on DNA repair and other pathways . However, a major concern with any On the other hand, measurement of PARylated substances produced by PARP1/2 activity reflects the balance between degradation of those PARylated molecules by an enzyme called poly (ADP-ribose) glycohydrolase (PARG), and production by PARP1/2. A sandwich immunoassay (IA) was developed and validated at the US National Cancer Institute to quantify the level of poly-ADP-ribosylated macromolecules from a calibration curve of poly-ADP-ribose standard ('PAR antigen') . This PAin vitro. However, iniparib and its two major metabolites failed to cause any change in the level of PARylated substances in the model systems, as measured by the PAR-IA [The PAR IA has been used to define a reproducible response of PARP1/2 to veliparib in tumor biopsies and mononuclear cell samples from treated patients. In addition, the PAR IA has been used to confirm pharmacodynamic effects, similar to those observed with veliparib, produced by olaparib and MK-4827 in human tumor xenografts and human tumor cell lines e PAR-IA . The appe PAR-IA .BRCA mutations, or in combination with DNA damaging therapies  glycohydrolase; PARP: poly (ADP-ribose) polymerase; TNBC: triple negative breast cancerThe authors declare that they have no competing interests.SK, AC, REP, RK, and JHD jointly wrote the manuscript. All authors were involved in revising the manuscript, and gave final approval. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1741-7015/10/25/prepubEarly phase clinical trials with PARP inhibitors. A table listing clinical trials of PARP inhibitors currently in development in early phase clinical trials.Click here for fileClinical trials with PARP inhibitors in defined diseases. A table listing clinical trials of PARP inhibitors currently in development in specified cancers.Click here for fileStructural and functional characteristics of PARP1. A: Poly(ADP-ribose) polymerase 1 (PARP1) is shown with its DNA-binding (DBD), automodification (AD) and catalytic domains. The PARP signature sequence  comprises the sequence most conserved among PARPs. Crucial residues for nicotinamide adenine dinucleotide (NAD+) binding  and for polymerase activity  are indicated. B: Consequences of PARP1 activation by DNA damage. Although not shown to simplify the scheme, PARP1 is active in a homodimeric form. PARP1 detects DNA damage through its DBD. This activates PARP1 to synthesize poly(ADP) ribose  on acceptor proteins, including histones and PARP1. Owing to the dense negative charge of pADPr, PARP1 loses affinity for DNA, allowing the recruitment of repair proteins by pADPr to the damaged DNA (blue and purple circles). Poly(ADP-ribose) glycohydrolase (PARG) and possibly ADP-ribose hydrolase 3 (ARH3) hydrolyse pADPr into ADP-ribose molecules and free pADPr. ADP-ribose is further metabolized by the pyrophosphohydrolase NUDiX enzymes into AMP, raising AMP:ATP ratios, which in turn activate the metabolic sensor AMP-activated protein kinase (AMPK). NAD+ is replenished by the enzymatic conversion of nicotinamide into NAD+ at the expense of phosphoribosylpyrophosphate (PRPP) and ATP. Examples of proteins non-covalently (pADPr-binding proteins) or covalently poly(ADP-ribosyl)ated are shown with the functional consequences of modification. It is important to note that many potential protein acceptors of pADPr remain to be identified owing to the difficulty of purifying pADPr-binding proteins in vivo. PARP inhibitors prevent the synthesis of pADPr and hinder subsequent downstream repair processes, lengthening the lifetime of DNA lesions. ATM, ataxia telangiectasia-mutated; BER, base excision repair; BRCT, BRCA1 carboxy-terminal repeat motif; DNA-PKcs, DNA-protein kinase catalytic subunit; DSB, double-strand break; HR, homologous recombination; NHEJ, non-homologous end joining; NLS, nuclear localization signal; PPi, inorganic pyrophosphate; SSB, single-strand break; Zn, zinc finger. Reprinted by permission from Macmillan Publishers Ltd: Nature Reviews Cancer [s Cancer , copyrigClick here for file


During limb development, chondrocytes and osteoblasts emerge fromcondensations of limb bud mesenchyme. These cells then proliferate anddifferentiate in separate but adjacent compartments and functioncooperatively to promote bone growth through the process of endochondralossification. While many aspects of limb skeletal formation are understood,little is known about the mechanisms that link the development ofundifferentiated limb bud mesenchyme with formation of the precartilaginouscondensation and subsequent proliferative expansion of chondrocyte andosteoblast lineages. The aim of this study was to gain insight into theseprocesses by examining the roles of c-Myc and N-Myc in morphogenesis of thelimb skeleton.Prx1-Cre, in chondro-osteoprogenitors withSox9-Cre and in osteoblasts withOsx1-Cre. We show that c-Myc promotes the proliferativeexpansion of both chondrocytes and osteoblasts and as a consequence controlsthe process of endochondral growth and ossification and determines bonesize. The control of proliferation by c-Myc was related to its effects onglobal gene transcription, as phosphorylation of the C-Terminal Domain(pCTD) of RNA Polymerase II, a marker of general transcription initiation,was tightly coupled to cell proliferation of growth plate chondrocytes wherec-Myc is expressed and severely downregulated in the absence of c-Myc.Finally, we show that combined deletion of N-Myc andc-Myc in early limb bud mesenchyme gives rise to aseverely hypoplastic limb skeleton that exhibits features characteristic ofindividual c-Myc and N-Myc mutants.To investigate c-Myc function in skeletal development, we characterized micein which floxed c-Myc alleles were deleted in undifferentiated limb budmesenchyme with Our results show that N-Myc and c-Myc act sequentially during limbdevelopment to coordinate the expansion of key progenitor populationsresponsible for forming the limb skeleton. The limb skeleton develops from limb buds that are initially composed of rapidlyproliferating multipotent mesenchymal stem cells encased in ectoderm. These cellsare maintained in a proliferative and undifferentiated state primarily by thesecretion of Fgf and Wnt ligands from ectoderm Sox9, deletion of Runx2 completely blocksdevelopment of the osteoblast lineage and therefore bone ossification Sox9 deletion in the early limb bud also blocksRunx2 expression and osteogenesis because the osteogeniclineage is derived from condensing mesenchyme of the limb bud that fails to properlyform in the absence of Sox9Runx2 transcriptional activity and its ability to promoteosteogenesis Chondrogenic and osteogenic lineages arising from condensing mesenchyme in the limbbud are defined by the expression and activity of the transcription factors Sox9 andRunx2/Cbfa1 respectively Runx2,Indian Hedgehog (Ihh) expression is alsoinitiated in prehypertrophic chondrocytes and Ihh appears to have a positiveinfluence on blood vessel development or function Runx2 is not only a cell autonomous regulator of the osteogenic lineage, but also hascell non-autonomous effects on osteoblasts by promoting chondrocyte maturation andexpression of Vegfa in prehypertrophic chondrocytes N-Myc andc-Myc are sequentially expressed in developing limbs, withN-Myc expressed primarily in undifferentiated limb budmesenchyme and c-Myc expressed in proliferating chondrocytes of thegrowth plate and osteoblasts in the perichondrium (see below). N-Myc is known topromote the proliferative expansion of undifferentiated limb bud mesenchyme, whichin turn influences the formation of condensing mesenchyme and production ofosteochondro-progenitors N-Myc and c-Myc in the early limb budgives rise to a severe limb miniaturization phenotype that exhibits featurescharacteristic of individual c-Myc and N-Mycmutants. Together, our results indicate that N-Myc and c-Myc function to maintainand expand distinct and sequentially formed progenitor populations responsible forforming the limb skeleton.Despite major advances in the understanding of chondrocyte and osteoblast lineagedetermination and endochondral growth, little is known about the mechanisms thatlink the development of undifferentiated limb bud mesenchyme with formation of theprecartilaginous condensation and subsequent development and expansion ofchondrocyte and osteoblast lineages. Potential candidates for coordinating theseactivities are Myc family transcription factors, particularly c-Myc and N-Myc, whichare known to strongly influence the development and maintenance of pluripotent andmultipotent in a number of homeostatic tissues  mice and in chondrocyctes and perichondrialcells at E13.5 (c-Myc expression observed in c-Myc ckolimb buds at E12.5 is likely in non-targeted muscle cells streaming into thelimb buds since c-Myc expression is apparent in muscle cells atlater stages (not shown).To investigate the role of limb bud  in a reglimb bud ', but nolimb bud  that applimb bud  that ovelimb bud ,c-Myc iautopod . At E13.s reside . Consistat E13.5 . The resc-Myc playing a role in development of theinitial cartilage template of the limb skeleton, Alcian blue staining at E13.5revealed a reduction in the size of all limb skeletal elements , adevelopmental stage roughly equivalent to E10.5 in forelimb buds. In contrast,mutant forelimb buds at E11.5 consistently exhibited reduced intensity and areaof Sox9 expression compared to Prx1-Crecontrols . In addition to decreased proliferation in the proliferativezone of the growth plate, there appeared to be lower levels of proliferation inthe epiphyseal articulating surface  in the extracellular matrix. An even greater decreasein cell density was observed in the hypertrophic region marked by Collagen TypeX (ColX)  . Incontc-Myc mutants, we examined the relationship between ColXprotein expression in hypertrophic chondrocytes of the growth plate andCollagen type I (Col1) mRNA expression inosteoblasts. In E18.5 tibias, the area of hypertrophic ColX expression wasreduced, but what was more striking was a marked decrease inCol1 expression in perichondrium and bone collar regions aswell as in the primary ossification center . In addition, Runx2 expressionin perichondrium was reduced at E18.5  was markedlydecreased in the perichondrium and ossification front of c-Mycmutant tibias , but theirexpression levels appeared reduced.The reduced number of chondrocytes in proliferative and hypertrophic zones ofof c-Myc '. In addc-Myc expression domain  of RNA Polymerase II, anevent required for transcription to be initiated n domain . Moreoven  at E10.5, Prx1-Credeletion of c-Myc had little or no effect on mesenchymeproliferation at this stage , like deletion ofN-Myc alone, resulted in strongly reduced cellproliferation and produced small limb buds at E10.5  in the zone ofpolarizing activity  and the demonstrated role of N-Myc (but notc-Myc) in proliferation of limb bud mesenchyme ,indicating that any differences observed were not due to differences in ablationefficiency. Taken together with our analysis of Prx1-Cre andSox9-Cre c-Myc mutants, these results are consistent withN-Myc and c-Myc playing distinct and complementary roles during development ofthe limb skeleton.While all size . Theforall size . Thehinsenchyme . Further missing , they weorelimbs , a resuldcko limb skeletons at E13.5 arereinforced by examination of the skeleton at later stages. At E15.5, very littlecan be deciphered from forlimbs of dcko embryos because they are so poorlydeveloped  were formed, and likeN-Myc mutants The basic observations of eveloped .Howevera tissue and the a tissue . In the he tibia . The hinomponent . In addidcko mice remainedextremely small throughout development and exhibited very small or no primaryossification centers, suggesting persistent disruption of endochondral growthand ossification. Because forelimb skeletal elements that formed indcko mice were extremely small and misshapen, we examinedendochondral growth and ossification on less severely affected hindlimb.Histological analysis of E18.5 proximal tibia sections fromdcko mice revealed a combination of architectural featuresfound in individual c-Myc and N-Myc cko mice,Prx1-Cre deletion of N-Myc in the hindlimb (latelimb bud deletion) results in a less hypoplastic skeleton.Both forelimb and hindlimb elements of cko mice. First,  mutants '. While 2, Vegfa.Moreovemutants. . As in c mutants , these ehondrium . SuppresN-Myc deficiency alone decreased the size ofthe epiphyseal head of the tibia (and other limb elements – not shown),and resulted in small declines in expression of Ihh,Runx2, Vegfa in the growth plate, it had arelatively strong effect on Col1 and Runx2expression in the perichondrium, and on the thickness of perichondrium . Thus, given the well-established role of Myc in promoting andmaintaining the pluripotent or multipotent character of stem and progenitorpopulations and the ability of forced Myc expression to suppress cell fatetransitions and cell differentiation N-Myc orc-Myc are expressed in most if not all proliferating celltypes Myc gene to another appears to be a common event associatedwith various developmental transitions during organogenesis and in homeostatictissues N-Myc andc-Myc may have evolved to facilitate and coordinatedevelopmental transitions that incorporate, and presumably require, a transientexit from the cell cycle. Understanding these codes may provide insight not onlyinto the underlying mechanisms that drive various critical developmentaltransitions, but also into how to manipulate the expression of Myc proteins invivo to facilitate the therapeutic proliferative expansion of specificprogenitor populations in injury and disease settings.Genetic swap experiments indicate that surround . Therefoc-Myc-floxed mice Prx1-Cre mice , Osx1-Cre mice Sox9-Crec-Myc-floxed mice weremated with N-Myc-floxed mice Prx1-Cre mice to generate double conditional knockout mice.All mice were maintained and used in accordance with animal protocols approvedby the Oregon Health & Science University Institutional Animal Care and UseCommittee.Alcian blue, Alizarin red staining and von Kossa staining were performed usingstandard procedures. To examine detailed bone architecture , limbs wEmbryos were fixed in 4% paraformaldehyde at 4°C overnight andprocessed for whole mount in situ hybridization using digoxigenin-UTP-labeledprobes or embedded in OTC and sectioned before hybridization as previouslydescribed . For immunohistochemistry, limb sections wereincubated with the following primary antibodies: anti-phospho-Rpb1 CTD - CellSignaling, Danvers, MA, anti-CD31(PECAM) - BD Bioscience, San Jose, CA, Type Xcollagen - Dr. William Horton), followed by incubations with AlexaFlour-conjugated secondary antibodies  or DAB stainingusing the Vectastain ABC Kit  as previouslydescribed t-test.BrdU (Sigma) labeling (1.5 hr) was performed using BD Biosciences BrdU In Situ Detection Kit as previously described t-test.Cell density measurements were made from within equivalent areas of proliferativeand hypertrophic zones. The specific regions selected were identified by theircharacteristic cell morphology, as well as by BrdU labeling of serial sectionsand ColX staining. DAPI stained sections from at least five different tibiaswere used in these calculations. Data are presented as mean ± SEM andstatistical significance of differences were calculated using Student'sFigure S1Limb skeletal phenotype of mice withSox9-Cre deletion ofc-Myc. (A) Alcianblue/Alizarin red staining of forelimb skeletal elements at E15.5 and (B)Alcian blue and H&E staining of E15.5 proximal tibia sections.Comparison of Alcian blue/Alizarin red staining of forelimb skeletalelements at E18.5 of the indicated mouse strains is shown.(TIFF)Click here for additional data file.Figure S2Osx1-Cre deletion ofc-Myc has little or noeffect on endochondral ossification and growth. (A) Alcian blueand Alizarin red skeletal preparations of the indicated mice at E18.5 mice.(B) Isolated hindlimbs E18.5 mice. (C) Alcian blue and H&E staining ofproximal tibia sections.(TIFF)Click here for additional data file.Figure S3Immunohistochemical analysis of RNA Polymerase CTD phosphorylation inE18.5 tibias of the indicated mouse strains. Approximatelocations of the Resting (RZ), Proliferative (PZ), Prehypertrophic (PHZ) andHypertrophic Zones (HZ) are shown. Higher magnification images of the boxedregions are shown on the right.(TIFF)Click here for additional data file.


MicroRNAs (miRNAs) have important roles in various biological processes. miRNA cross-mapping is a prevalent phenomenon where miRNA sequence originating from one genomic region is mapped to another location. To have a better understanding of this phenomenon in the human genome, we performed a detailed analysis in this paper using public miRNA high-throughput sequencing data and all known human miRNAs. We observed widespread cross-mapping events between miRNA precursors (pre-miRNAs), other non-coding RNAs (ncRNAs) and the opposite strands of pre-miRNAs by analyzing the high-throughput sequencing data. Computational analysis on all known human miRNAs also confirmed that many of them could be involved in cross-mapping events. The processing or decay of both ncRNAs and pre-miRNA opposite strand transcripts may contribute to miRNA enrichment, although some might be miRNA-mimics due to miRNA mis-annotation. Comparing to canonical miRNAs, miRNAs involved in cross-mapping events between pre-miRNAs and other ncRNAs normally had shorter lengths (17–19 nt), lower prediction scores and were classified as pseudo miRNA precursors. Notably, 4.9% of all human miRNAs could be accurately mapped to the opposite strands of pre-miRNAs, which showed that both strands of the same genomic region had the potential to produce mature miRNAs and simultaneously implied some potential miRNA precursors. We proposed that the cross-mapping events are more complex than we previously thought. Sequence similarity between other ncRNAs and pre-miRNAs and the specific stem-loop structures of pre-miRNAs may provide evolutionary implications. MicroRNAs (miRNAs) are small non-coding RNAs with a length of approximately 22 nucleotides (nt), which play important roles in various biological processes, including various pathophysiological conditions To elucidate the potential role of miRNAs in biological processes, it is pivotal to accurately profile the entire miRNA repertoire and compare miRNAomics between different samples. Recently, high-throughput sequencing technologies have been widely applied in identifying and profiling miRNAs at an unprecedented scale with high sensitivity and accuracy. For example, lightly expressed miRNAs can be well profiled in high-throughput miRNA sequencing. IsomiRs, multiple miRNA variants with end sequence variations, have been observed in miRNA deep sequencing datasets from various species Although high-throughput sequencing has accelerated our understanding of miRNA biogenesis and function, there are still some issues to be addressed in the analysis of high-throughput miRNA sequencing data. Recent analyses have found a widespread phenomenon of multi-mapping or cross-mapping in deep sequencing datasets of small RNAs To analyze high-throughput miRNA sequencing data, we first filtered the sequencing reads that could be mapped to other ncRNAs. The remaining reads were then aligned to known human miRNA precursors for identification. Theoretically, filtered reads (those mapped to other ncRNAs) could not be simultaneously mapped to known pre-miRNAs. However, we found that 0.26% of filtered reads were mapped to human pre-miRNAs. This indicated that cross-mapping events could occur between some pre-miRNAs and other ncRNAs due to their high sequence similarity. We also detected cross-mapping events among different pre-miRNAs. 36.88% of sequencing reads could be mapped to more than one (even more than 10 in some cases) human pre-miRNA . The croBased on the widespread occurrence of cross-mapping events between miRNA precursors and other ncRNAs in high-throughput sequencing data, we further performed a systematic analysis on all known human miRNAs to identify their potential relationships. Seven human miRNAs  could be accurately mapped to other ncRNAs, which included snoRNAs, snRNAs, tRNAs and rRNAs . HoweverOther than multiple mapping events between miRNA precursors and other ncRNAs, we also found many miRNAs could be accurately mapped to different miRNA precursors including opposite strands of known pre-miRNAs . 13.49% Interestingly, cross-mapping of miRNAs also could be detected between miRNA precursors and opposite strands of different pre-miRNAs. For example, both hsa-miR-338-5p and hsa-miR-338-3p could be accurately mapped to the opposite strand of hsa-mir-3065 . These tIn analyzing high-throughput sequencing dataset, we found that some short reads could be simultaneously mapped to miRNA precursor sequences and other ncRNAs. Cross-mapping indicated common sequences or high sequence similarities between miRNA precursors and some ncRNAs. This cross-mapping phenomenon was also widely observed among miRNA precursors, especially for the multi-copy precursors and homologous miRNA genes. Notably, many opposite strands of pre-miRNAs could also be involved in cross-mapping, as found in 9.91% of sequencing reads detected. Some miRNA could be mapped to the opposite strand of its own or other unrelated pre-miRNAs . All of We then asked what this cross-mapping phenomenon would reveal when all human miRNAs were taken into consideration. We performed a comprehensive analysis based on all known human miRNAs, pre-miRNAs and other ncRNAs. As we expected, some miRNAs could be simultaneously mapped to other ncRNAs including rRNAs, tRNAs, snoRNAs and snRNAs . The crohttp://www.mirbase.org/cgi-bin/mirna_entry.pl?acc=MI0000110) Analysis all known human miRNAs found that 4.9% could be accurately mapped to opposite strands of known miRNA precursors, which could be their own pre-miRNAs or some other pre-miRNAs . ProductMoreover, we found some miRNAs could be accurately mapped to opposite strands of their own pre-miRNAs . This inWhen miRNA cross-mapping occurs, the target reference sequences, pre-miRNAs and other ncRNAs, showed different levels of similarity in consensus sequences or large sequence divergences except in common regions . If mismhttp://SOLiDsoftwaretools.com/gf/project/srna/) and analyzed using the SOLiD miRNA analysis pipeline. The pipeline includes three major steps: 1) Filter known human non-coding RNAs, such as tRNAs, rRNAs, snoRNA, snRNAs, etc. All ncRNAs were collected from GenBank (http://www.ncbi.nlm.nih.gov/genbank/). 2) Detect known miRNAs by aligning them to human miRNA precursor sequences in the miRBase database . 3) Discover novel miRNAs by mapping unaligned reads using the human genome sequence as a reference. To detect known miRNAs, the short reads were first aligned to miRNA precursors using a seed 16-mer with allowing only 2 mismatches. For those reads matching the seed sequence, full-length (35 nt for SOLiD reads) alignments were performed with 4 mismatches allowed. Short reads were removed from the analysis if their lengths are less than 16 nt after 3′ adaptor sequences were discarded. Only short RNAs that could be mapped to known miRNA precursors were used for further analysis of multiple isomiRs. Due to shorter sizes and tolerant mismatches, the phenomenon of multiple mapping or cross-mapping could be detected with an equal number of mismatches.Public high-throughput sequencing dataset of small RNAs from human placental sample generated from the SOLiD™ System  were obtained  Figure S1Human miRNA precursors' stem-loop structures from the miRBase database. Sequences with a pink background indicate miRNA and miRNA*. (A) Generally, miRNA precursors can form stem-loop structures with some incomplete complementary miRNA regions. (B) If miRNAs can simultaneously map to sense and antisense strands of their own pre-miRNAs, these special precursors may form complete complementary miRNA regions.(TIF)Click here for additional data file.Figure S2Different miRNAs from sense and antisense strands in the same genomic region can complementarily bind to each other. The binding events maybe provide potential regulation among different miRNAs.(TIF)Click here for additional data file.Figure S3Inconsistent expression levels of miRNAs (sense/antisense miRNAs) from the same genomic region. Pairwise miRNAs are generated from the same genomic region; however, their expression levels show significant differences. Generally, one miRNA shows a higher percentage (more than 90%).(TIF)Click here for additional data file.Table S1Accurate cross-mapping of miRNAs with pre-miRNAs and the opposite strands of pre-miRNAs.(DOC)Click here for additional data file.


Several alphaherpesviruses breach the basement membrane during mucosal invasion. In the present study, the role of proteases in this process was examined. The serine protease-specific inhibitor AEBSF inhibited penetration of the basement membrane by the porcine alphaherpesvirus pseudorabies virus (PRV) by 88.1% without affecting lateral spread. Inhibitors of aspartic-, cysteine-, and metalloproteases did not inhibit viral penetration of the basement membrane. Further analysis using the Soybean Type I-S trypsin inhibitor for the serine protease subcategory of trypsin-like serine proteases resulted in a 96.9% reduction in plaque depth underneath the basement membrane. These data reveal a role of a trypsin-like serine protease in PRV penetration of the basement membrane. The basement membrane (BM) constitutes part of the extracellular matrix and creates a formidable barrier to invading pathogens . Crossin7 TCID50 of PRV field strain 89V87 [Breach of the BM in disease states is best characterized in metastatic progression in oncology following neoplastic establishment and often involves disruption of the BM by proteolytic enzymes ,14. Therin 89V87 . After i® (Sigma) and frozen at -70°C. The time point of sampling was specified at 20 h pi because PRV was found to cross the BM between 12 and 16 h pi (data not shown). Cryosections were made, fixed in methanol, stained for collagen IV (BM component) and PRV and analyzed by confocal microscopy. Viral invasion across and lateral spread above the BM were analyzed using ImageJ, as reported previously [Proteases are classified according to their catalytic activity: serine-, cysteine-, metallo- and aspartic peptidases ,18. The eviously . For eacFigure To further delineate which of the serine-, cysteine- and/or metalloproteases are involved in PRV invasion through the BM, type-specific inhibitors were used: AEBSF (4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride) inhibits serine proteases, E-64 (trans-Epoxysuccinyl-l-leucylamido-(4-guanidino)butane) inhibits cysteine proteases and phosphoramidon inhibits metalloproteases (Sigma). PRV-inoculated explants were treated with 100 or 250 μM AEBSF, 1 or 10 μM E-64 or 10 μM phosphoramidon and the effect on the plaque depth underneath the BM and the plaque latitude above the BM was evaluated. Figure Glycine max, Soybean Type I-S, was first isolated by Kunitz [p-Tosyl-L-phenylalanine chloromethyl keton) inhibits chymotrypsin-like serine proteases and elastatinal is an inhibitor of elastase-like serine proteases (Sigma) [Based on their substrate specificity, serine proteases are generally classified into three main categories: trypsin-like, chymotrypsin-like and elastase-like serine proteases . The tryy Kunitz  and inhi (Sigma) . A time  (Sigma) . TreatmeInteractions of herpesviruses with respiratory mucosa remain poorly understood. Invasion of different alphaherpesviruses towards the lamina propria involves passage of the BM barrier. This facilitates viral access to blood capillaries and nerve endings which enables further dissemination throughout the body -12. To dThe current study shows that serine protease activity is involved in PRV invasion through the BM. Over one third of all known proteolytic enzymes are serine proteases which mediate diverse biological processes requiring timely and specifically regulated proteolytic activity, where insufficient or excessive proteolysis can cause pathological processes ,23. To dMore specifically, in the current study, we demonstrate that a trypsin-like serine protease is involved in viral penetration of the BM. Since serine protease activity involvement has been described in different stages of the herpesvirus life cycle , the effWe can conclude that trypsin-like serine protease activity is involved in PRV invasion through the BM towards the lamina propria. We hypothesize that this trypsin-like serine protease activity is involved in local degradation of BM barrier components, thereby enhancing BM penetration. An important question that remains is whether the proteases involved are of cellular and/or viral origin. Interestingly, transcriptome studies have indicated that several cellular components involved in trypsin-like serine protease activity are differentially regulated upon infection with alphaherpesviruses like PRV and HSV, including factor IX, tissue-type plasminogen activator (t-PA) and urinary plasminogen activator receptor 1 (uPAR1) ,31. FurtThe authors declare that they have no competing interests.SG set up the study design, carried out the experiments, processed all samples, performed the statistical analysis and drafted the manuscript. HWF participated in the design of the study. LS and APV assisted in sampling. HJN took part in the design and coordination of the study. All authors read and approved the final manuscript.


Osx-Cre mouse line  expresses GFP::Cre fusion protein from a BAC transgene containing the Osx regulatory sequence. The mouse strain was initially characterized during embryogenesis, and found to target mainly osteoblast-lineage cells. Because the strain has been increasingly used in postnatal studies, it is important to evaluate its targeting specificity in mice after birth. By crossing the Osx-Cre mouse with the R26-mT/mG reporter line and analyzing the progenies at two months of age, we find that Osx-Cre targets not only osteoblasts, osteocytes and hypertrophic chondrocytes as expected, but also stromal cells, adipocytes and perivascular cells in the bone marrow. The targeting of adipocytes and perivascular cells appears to be specific to those residing within the bone marrow, as the same cell types elsewhere are not targeted. Beyond the skeleton, Osx-Cre also targets the olfactory glomerular cells, and a subset of the gastric and intestinal epithelium. Thus, potential contributions from the non-osteoblast-lineage cells should be considered when Osx-Cre is used to study gene functions in postnatal mice.Osterix (Osx or Sp7) is a zinc-finger-family transcriptional factor essential for osteoblast differentiation in mammals. The  Runx2 expression is relatively normal, but other osteoblast markers including Col1a1, Bsp, and osteocalcin are either absent or severely suppressed Osx is abolished in Runx2-null embryosOsterix (Osx or Sp7) is a zinc finger family transcriptional factor critical for osteoblast differentiationOsx-Cre (Osx1-GFP::Cre), a BAC transgenic mouse line expressing a GFP::Cre fusion protein from the regulatory sequence of Osx, was generated to direct gene deletion in the osteoblast lineage Osx-Cre mouse line has been increasingly used to study the osteoblast lineage in postnatal mice Osx-Cre in postnatal bones is yet to be formally evaluated.The Cre/loxP technology enables gene deletion in specific cell types and has significantly advanced our understanding of gene functions in both physiological and pathological conditions. In this system, specificity is achieved by expression of the Cre recombinase under the control of cell type-specific regulatory sequences. R26-mT/mG reporter allele. The R26-mT/mG allele ubiquitously expresses a membrane-targeted red fluorescent protein (mTomato) but switches to expressing a membrane-targeted green fluorescent protein (mGFP) upon Cre recombination. We find that within the skeleton, Osx-Cre targets not only osteoblast lineage cells and a subset of chondrocytes, but also stromal cells, adipocytes and perivascular cells specifically within the bone marrow. Moreover, Osx-Cre also targets cells within the olfactory bulb, the intestine and the stomach.Here we assess the cell types targeted by Osx-Cre in two-month-old mice by monitoring GFP expression from the Osx-Cre (Osx1-GFP::Cre) and R26-mT/mG mouse lines are as previously described Two-month-old mice were perfused with 4% PFA as described previously2 Fragment of Goat Anti-Rabbit IgG (for perilipin), and Alexa Fluor® 647 Goat Anti-Mouse IgG2a (for αSMA) . Sections were mounted with VECTASHIELD Mounting Medium containing DAPI .For detection of GFP, perilipin or αSMA, immunostaining was performed on cryostat sections using a chicken polyclonal GFP antibody , or rabbit monoclonal perilipin antibody , or mouse monoclonal αSMA antibody . The secondary antibodies are as follows: Alexa Fluor® 488 Goat Anti-Chicken IgG (for GFP); Alexa Fluor® 647 F(ab')All quantitative data were obtained from three independent animals. Statistical analyses were performed with student's t-test.Osx-Cre in postnatal mouse bones, we generated Osx-Cre; R26-mT/mG mice (one copy each of Osx-Cre and R26-mT/mG) and analyzed GFP expression on sections of long bones at two months of age. As expected, bone sections from the control R26-mT/mG mice did not exhibit any GFP . In either case, the progenitors do not appear to continuously express Osx or Osx-Cre throughout postnatal life, as cells actively expressing Osx postnatally, when marked with Osx-CreER in response to tamoxifen, failed to sustain the turnover of either osteoblasts or stromal cells Osx-expressing cells give rise to the bone marrow mesenchymal progenitors in postnatal mice. Additional experiments are necessary to test this hypothesis formally.Evidence suggests that mesenchymal stem cells (MSCs) residing within the adult bone marrow produce osteoblasts, adipocytes and bone marrow stromal cells necessary for postnatal tissue homeostasis Osx-expressing progenitors. While this work was in review, others using the Ai9 Cre reporter mouse reported similar findings about the targeting of bone marrow adipocytes and perivascular smooth muscle cells by Osx-Cre, thus allaying the concern over limitations of a single Cre reporter Bone marrow adipose tissue is believed to be metabolically different from non-marrow peripheral fat depots Osx-Cre, when activated in the embryo, targets more than osteoblast-lineage cells in postnatal mice. These findings echo the increasing concern that many Cre strains exhibit some degree of unintended recombination activity Osx-Cre targets olfactory glomerular cells and the GI tract epithelia. Targeting of the olfactory bulb is consistent with endogenous Osx expression as previously reported in this organ Osx is normally expressed in the GI tract, or the mosaic Cre activity there simply reflects a phenomenon specific to the Osx-Cre transgene. Furthermore, it is not known whether Osx-Cre-mediated gene deletion in the olfactory bulb or the GI track affects bone physiology. Nonetheless, caution needs to be taken when one interprets postnatal bone phenotypes caused by gene deletion with Osx-Cre beginning in the embryo.Our data clearly indicate that


Various foot orthoses have been shown to alter tibialis anterior and peroneus longus electromyographic (EMG) muscle activity during walking. However, it is unclear whether these changes represent optimisation in muscle function. Therefore, two studies were conducted to determine; (i) whether people with flat-arched feet display altered muscle activity compared to those with normal-arched feet during gait and (ii) whether foot orthoses change muscle activity in people with flat-arched feet towards a pattern observed in those with normal-arched feet.A screening protocol that included clinical and radiographic measures was used to recruit participants with normal-and flat-arched feet. Sixty adults aged 18 to 47 years qualified for this study. Of these, 30 had normal-arched feet  and 30 had flat-arched feet . Two different foot orthoses were dispensed to the participants with flat-arched feet: (i) a heat-moulded (modified) foam prefabricated foot orthosis and (ii) a 20-degree inverted-style customised foot orthosis. EMG activity was recorded while walking from tibialis posterior and peroneus longus via in-dwelling wire electrodes, and from tibialis anterior and medial gastrocnemius via surface electrodes.During the contact phase of gait, the flat-arched group exhibited increased activity of tibialis anterior (19% increase) and decreased activity of peroneus longus (13% decrease). During midstance/propulsion, the flat-arched group exhibited increased activity of tibialis posterior (26% increase) and decreased activity of peroneus longus (14% decrease), compared with those with normal-arched feet. During the contact phase of gait, tibialis posterior EMG amplitude decreased significantly with the prefabricated orthosis (19% decrease) and the customised orthosis (12% decrease) compared to the shoe only condition (p < 0.008). In contrast, during the midstance/propulsive phase, peroneus longus EMG amplitude increased significantly with the prefabricated orthosis compared to the shoe-only and customised orthosis conditions .Both foot orthoses had a significant effect on EMG amplitude of tibialis posterior and peroneus longus, however only the modified prefabricated orthosis changed peroneus longus EMG amplitude towards a pattern observed with normal-arched feet. Otherwise, there were few differences found between the modified prefabricated and customised orthoses.


To the Editor: The effect of influenza on young children is substantial, but most infections are clinically unrecognized  diagnostic codes specific to influenza  are easily retrieved from hospital discharge records. However, researchers and public health officials have rarely used them for influenza hospitalization surveillance, presumably because they lack sensitivity for identifying true influenza infections, although this assumption has never been tested.To determine the sensitivity and positive predictive value of influenza-specific ICD-9 admission or discharge codes , we conducted a retrospective cohort study of all patients <21 years of age hospitalized at the Children's Hospital of Philadelphia with laboratory-confirmed influenza during 3 consecutive influenza seasons (July 2001 through June 2004) , H. parainfluenzae (n = 1 wound infection), adenovirus (n = 1), and RSV (n = 2) infections. For 5 patients, influenza infection was documented in their charts, but they had either negative influenza test results or no influenza test performed. Seven patients had the expression "follow-up" written as "f/u" in the assessment section of their admission note, which may have been interpreted by medical coders as flu. We could not determine the reason for miscoding in 2 patients.Of 715 cases of laboratory-confirmed influenza identified , 617 86. Of 66 pThe sensitivity of influenza-specific diagnosis codes was related to the method of laboratory confirmation. Seventy-three percent (452/617) of patients (95% CI 70%–77%) who had positive rapid test results had influenza-specific admission or discharge diagnosis codes, whereas only 11% (11/98) (95% CI 6%–19%) who had positive influenza viral cultures (and negative rapid test results) had influenza-specific diagnosis codes.Our results have a few policy implications. First, they suggest that in hospitals where routine influenza viral testing is performed, use of admission and discharge ICD-9 codes from hospital billing data for surveillance purposes will systematically underestimate actual influenza-related hospitalizations by 35%. The higher sensitivity of influenza-specific ICD-9 codes in patients with positive rapid test results compared with positive culture results suggests that unlike viral culture results, which generally are not available before discharge, rapid test results are often used to assign influenza-specific ICD-9 codes. Thus, rapid diagnostic tests that are more sensitive  may increase the sensitivity of influenza-specific ICD-9 codes in hospitals that routinely evaluate children admitted with respiratory symptoms of unclear cause. However, the imperfect specificity (94%–98%) of rapid influenza tests will produce a small but not negligible number of false-positive results. In hospitals where influenza testing is not commonly performed, the sensitivity of influenza-specific ICD-9 codes is likely to be lower.H. influenzae infections as patients with influenza demonstrates the potential for systematic coding errors even when influenza testing is routine.Second, the high positive predictive value of influenza-specific ICD-9 codes observed in this study suggests that in hospitals where influenza testing is routinely performed, most patients whose hospitalization summary includes an influenza-specific ICD-9 code actually have influenza. However, misclassification of patients with parainfluenza and Epidemiologists and public health officials should be aware that influenza-specific ICD-9 codes assigned in a setting of routine rapid diagnostic testing may be useful for following trends. However, these codes will substantially underestimate the actual number of influenza-related hospitalizations.


Thrombin and activated protein C (aPC) are two pleiotropic proteases whose opposing functions in hemostasis and endothelial function are dysregulated during sepsis. Exogenous supplementation of aPC, the ligand for endothelial protein C receptor (EPCR), is the only known therapeutic shown to reduce mortality in severe septic patients. Paradoxically, both thrombin and aPC signal the endothelium via the same receptor, protease-activated receptor-1 (PAR-1), by cleaving its N-terminus to produce an identical tethered ligand, yet result in opposing signaling networks. Once activated, PAR-1 triggers at least three separate signaling pathways  and it is the relative contribution of each pathway that determines the endothelial response. Thrombin is a potent proinflammatory, endothelial barrier disruptive agonist, while aPC induces an anti-inflammatory and barrier protective phenotype, thought to be important to its therapeutic mechanism. We hypothesized that when bound to its ligand, aPC, EPCR functionally dimerizes with activated PAR-1, thereby altering its specificity for Gq, an important mediator of proinflammatory pathways in endothelial cells.We used bioluminescent resonance energy transfer to dynamically monitor the interaction of recombinant PAR-1 and EPCR in HEK cells. The effect of EPCR on PAR-1 G-protein selectivity was determined by EPCR siRNA knock down in cultured endothelial cells. Relative activation of Gq was determined by assaying agonist-induced intracellular calcium mobilization. G13 activation was determined by monitoring agonist-induced changes transendothelial electrical resistance across monolayers.We found that in the absence of protease ligands, unactivated PAR-1 dimerizes with EPCR. However, proteolytically activated PAR-1/EPCR interaction was maintained with aPC but not thrombin. Both aPC and thrombin induced G13 signaling; however, aPC failed to activate Gq compared with thrombin. aPC-induced PAR-1/Gq signaling appears to be impaired by aPC-bound EPCR and is relieved when EPCR is depleted using siRNA.aPC-bound EPCR neutralizes the proinflammatory function of PAR-1 signaling by maintaining interaction with activated PAR-1, thereby abrogating Gq signaling. Thus it is not the difference in protease activation between thrombin and aPC, but rather the ability of aPC to direct PAR-1/EPCR dimerization that controls PAR-1 signaling, and thereby provides the therapeutic barrier protective/anti-inflammatory effects associated with aPC treatment.