Privacy protection is a core principle of genomic but not proteomic research. We identified independent single nucleotide polymorphism (SNP) quantitative trait loci (pQTL) from COPDGene and Jackson Heart Study (JHS), calculated continuous protein level genotype probabilities, and then applied a naïve Bayesian approach to link SomaScan 1.3K proteomes to genomes for 2812 independent subjects from COPDGene, JHS, SubPopulations and InteRmediate Outcome Measures In COPD Study (SPIROMICS) and Multi-Ethnic Study of Atherosclerosis (MESA). We correctly linked 90–95% of proteomes to their correct genome and for 95–99% we identify the 1% most likely links. The linking accuracy in subjects with African ancestry was lower (~ 60%) unless training included diverse subjects. With larger profiling (SomaScan 5K) in the Atherosclerosis Risk Communities (ARIC) correct identification was > 99% even in mixed ancestry populations. We also linked proteomes-to-proteomes and used the proteome only to determine features such as sex, ancestry, and first-degree relatives. When serial proteomes are available, the linking algorithm can be used to identify and correct mislabeled samples. This work also demonstrates the importance of including diverse populations in omics research and that large proteomic datasets (> 1000 proteins) can be accurately linked to a specific genome through pQTL knowledge and should not be considered unidentifiable. Although initially controversial, the DNA-fingerprinting technique was rapidly and widely adapted by forensic scientists and within a decade was in the public’s vernacular. Soon thereafter the results of the Human Genome Project were published3 and it is now recognized that there are millions of single nucleotide polymorphisms (SNP) which can distinguish individuals within large populations. Identifying individuals by genomics is a rising concern in research because advances in genotyping and sequencing have resulted in large genetic databases  for both research and commercial use. The existence of newer genotyping technologies and large genomic databases has created concerns among policy makers regarding discrimination in health insurance and employment and resulted in new laws that address genetic information  as well as privacy protection efforts such as the Global Alliance for Genomics and Health, which has created frameworks to ensure responsible and secure sharing of genomic and health-related data. A key feature of these policies in the United States is that they explicitly addressed genomic  data only. Despite these policies, there have been multiple instances of “deidentified” personal information linked back to individual genetic profiles4, including well publicized individuals such as Henrietta Lacks5. There have also been methods proposed which can link expression data to genotype through eQTLs6.Nearly four decades ago Jeffreys et al.8 have genetic quantitative trait loci (QTLs), but current practice is to consider these datasets as deidentified data. In this manuscript we show that even limited proteome profiles without peptide sequencing can be linked to specific individuals by using prior independent knowledge of these QTLs and we provide a bioinformatic solution which obfuscates reidentification, yet still preserves at least some biomarker-phenotype relationships. These findings suggest an immediate need to change policy regarding non-genomic data used for research or commercial use.Although lagging behind genotype and sequencing advances by 5–10 years, exponential technological advances in high throughput proteomics are leading to the creation of similar large databases with sensitive personal information. Concurrently there are studies which demonstrate that many proteinsAll study participants provided written informed consent approved by institutional review boards (IRBs). COPDGene and Jackson Heart Study (JHS) cohorts were randomly split into training and testing datasets and training subjects were not included in the testing cohort. Other independent cohorts used for testing included Subpopulations and Intermediate Outcome Measures in COPD Study (SPIROMICS) and Multi-Ethnic Study of Atherosclerosis (MESA). Race was self-reported. Characteristics of subjects used for training and test are shown below with summary demographics in Table 9. Subjects were genotyped using an Illumina HumanOmni Express10. 1184 subjects from the enrollment visit (P1) participated in an ancillary study in which they provided p100 (BD) fresh frozen plasma used for SomaScan 1.3K proteomic profiling which measured 1305 proteins. An additional 547 independent subjects, who only had SomaScan profiling at a 5-year follow up visit (P2) and not used in the training dataset, were used as an independent testing cohort. 5292 also had SomaScan 5K (v4.0) proteomes using plasma from a P2 visit and were randomly split into training and testing to assess whether scaling improved identification accuracy. COPDGene has been approved by the BRANY IRB.The NIH-sponsored multicenter Genetic Epidemiology of COPD ) enrolled 10,263 non-Hispanic white (NHW) and Black (AA) individuals from January 2008 until April 2011 (Phase 1) who were aged 45–80 with ≥ 10 pack-year smoking history and no exacerbations for > 30 days and 457 age and gender matched healthy individuals with no history of smoking were enrolled as controlsThe NIH-sponsored  enrolled 5306 African American residents living in the Jackson, MS, metropolitan statistical area (MSA) of Hinds, Madison, and Rankin Counties. 2055 gave consent for genetic research and also had SomaScan 1.3K proteomic profiling. Genotypes were extracted using TOPMed whole genome sequencing Freeze 8 to create a synthetic Illumina HumanOmniExpress genotype panel. The Jackson Heart Study (JHS) Institutional Review Board (IRB) Working Group (WG) is responsible for Overseeing and monitoring all JHS Institutional Review Board (IRB) activities Facilitating collaborative communications and transfer of information among the IRBs regulating the JHS: Jackson State University, University of Mississippi Medical Center, and Tougaloo College.11 enrolled 2984 subjects who were genotyped using the Illumina HumanOmniExpress genotyping platform12 of which 258 subjects underwent SomaScan 1.3K proteomic profiling using Visit 1 plasma. Additional SomaScan 7K data (version 4.1) were available for 2401 subjects from visit 1, 2, 4, and 5 . SPIROMICS has been approved by by the IRB at the University of North Carolina at Chapel Hill.The NIH-sponsored Subpopulations and Intermediate Outcome Measures in COPD study (SPIROMICS) study 13) which include University of Washington, University of Vermont (biospecimen repository), Columbia University, Johns Hopkins University, Northwestern University, University of California, Los Angeles, University of Minnesota, and Wake Forest University.The NIH-sponsored Multi-Ethnic Study of Atherosclerosis (MESA) study  recruited 6418 participants from four race/ethnic groups: Caucasian, African American, Hispanic, and Chinese. Whole genome sequencing (WGS) was performed at the Broad Institute of MIT and Harvard. SomaScan proteomics 1.3K profiling was performed at the Broad Institute and Beth Israel Proteomics Platform (HHSN268201600034I). The MESA study was approved by its six participating IRBs  at all participating institutions: University of North Carolina at Chapel Hill IRB, Johns Hopkins University IRB, University of Minnesota IRB, and University of Mississippi Medical Center IRB.The Atherosclerosis Risk in Communities (ARIC) study initially enrolled 15,792 participants aged 45–64 years at four study centers in the United States: Washington County, MD; Forsyth County, NC; northwestern suburbs of Minneapolis, MN; and Jackson, MS between 1987 and 1989, aiming to investigate cardiovascular disease and its risk factors. Participants have undergone nine clinical visits. For current analysis, proteomic profiles were obtained from SomaLogic, via SomaScan 5K (v4.0) assay using freshly frozen blood plasma collected at ARIC visit 2 (1990–1992). Genotyping was performed using Affymetrix 6.0 array and imputed using TOPMed Freeze 5b datasets Details of genotyping and imputation quality control methods were previously described15. Normalization follow SomaLogic’s guidelines for data processing encompass three sequential levels of normalization, namely Hybridization Control Normalization (Hyb) followed by Median Signal Normalization (Hyb.MedNorm) and Interplate Calibration . There are no missing data on the platform. SomaScan 5K v4.0 (4776 proteins) was performed by SomaLogic and we used Adaptive Normalization by Maximum Likelihood (anmlSMP). For pQTL discovery, we used a rank-based inverse normal transformation to align protein levels to a normal distribution; however, for estimating genotype probabilities and associations with smoking, we used log transformed protein values.Proteomic profiles for 1305 proteins were generated using SomaScan v 1.3K . Description of the SomaScan 1.3K assay is further described in16. Each model assessed direct association between protein level and genotype, with no adjustment for covariates. Protein quantitative trait loci (pQTLs) were considered significant at FDR corrected p-value < 0.05. The pQTL assessments in JHS and COPDGene were performed independently. After merging the two sets of pQTLs from the two training cohorts, we reduced the set to obtain a list of uniquely associated protein and SNP combinations. For each unique protein in the pQTL set, we kept only the highest significance SNP pQTL as determined by the p-value for the training cohorts  greater than 5% in the sample population were included for analysis. Both datasets were aligned to GRCh38. SNP-by-proteins associations were assessed in separately in both the COPDGene and JHS discovery cohorts using linear regression assuming an additive model by genotype. Analysis was performed using the R package ‘MatrixEQTL’ (version 2.2)g using the genotype specific mean (µ) and standard deviation (σ) estimated from training data. This is similar to an approach used in genotype estimation from eQTLs6. To combine the training estimates from COPDGene and JHS we used the GaussianNB model from scikit-learn (version 0.23.2) for this estimation6. During training, we use the partial_fit method to calculate µ and σ parameters on a single dataset. The same method can be used to update parameters µ and σ, allowing us to train a model on multiple datasets by sharing the trained model. Since each SNP is biallelic, we calculate three probabilities corresponding to the three possible genotypes.g and σg are the estimated mean and variance respectively of the protein levels x for subjects with genotype g. Under the naïve Bayes framework, we estimate the probability of the subject possessing each of the three genotype classes, given an observed protein level  belongs to the subject whose protein levels were observed, we consider this a match. We also tested whether the true match was among the three highest odds (top 3) and 1% highest odds (in top 1%).For predicting the probability of genome matching we use a Naïve Bayesian method Fig.  which esA T-test was used to assess whether proteins (log transformed) were associated with current smoking (smoking cigarettes in the past 30 days).https://github.com/BowlerLab/reidentify_code).All analyses were run in R (version 3.6.11) and Python (version 3.7). The code used in this manuscript is available on GitHub , we then tested prediction accuracies in 4 cohorts  using independent subjects that had not been used for training, including accuracies based on race and ethnicity Table . The truTo determine whether newer and larger proteome assays were more or less accurate at identifying genetic profiles, we randomly split 5292 COPDGene subjects (71% NHW and 29% AA) who had SomaScan v4.0 5K data (4776 proteins) into training and testing groups using a 50/50 train-test split  or 7K (SPIROMICS) data. We show this by calculating Euclidean distances in N-dimensional space and show that this distance is the shortest for the same subjects over years compared to unrelated individuals  of proteomes did not exactly match their genome. In 8 of 18 proteomes the subject had multiple visits which generated proteomes, many of which matched to the same genome of a different person’s DNA, suggesting that the DNA was mislabeled and came from a different person. In 4 of 18 proteomes, all but one of the proteomes matched correctly to the genome and the mismatched proteome had a corresponding mismatched sample from the same visit. This suggests that a plasma sample was swapped between two subjects at a single visit  to link an individual protein profile to a single genetic profile among thousands of subjects and that it is accurate even with imputed genotypes. Additionally, our results suggest that using diverse subjects for selecting the most influential proteins improves overall accuracy, particularly among those with African ancestry and underscores the importance of including diverse subjects in Omics research. We show that proteomic data can identify behavioral features  even after removing the features that allow matching to genomes. The ability to accurately identify someone by linking their proteome to a genome, identify risk for protein related disease such as alpha-1 antitrypsin deficiency18, infer sex, genetic ancestry, or relatedness and also characterize other characteristics such as body fat, renal function, fitness, smoking, alcohol consumption, diabetes, cardiovascular risk19, and age20 implies that proteomic data should have at least the same (if not more rigorous) privacy protections as genetic and genomic datasets.De-identification of data is a key concept for shared research and privacy protection but is not yet used in large scale proteomic studies. While small proof of concept studies have suggested that mass spectrometry can identify missense variants  which can suggest specific SNPsThe two main technological breakthroughs that have facilitated accurately matching an individual proteome to a specific genome are improvement in high throughput proteomic technologies and large scale pQTL studies. Until the last few years, there were no proteomic platforms that could simultaneously and accurately measure more than 100 proteins and there was little known about which of those proteins had strong pQTLs. While our study used three different SomaScan platforms, lack of privacy (de-identification) should be implied for any platform that can simultaneously measure thousands of proteins even when mass spectrometry is not used. The logical continuation of this principle is that proteomic data could be used to discriminate based on identifying the sex of a subject, ancestry, or paternity. A protein profile could even be used to identify close relatives for forensic purposes.The ability to link proteomes to genomes is not always a bad thing, particularly when cleaning data. For instance, we used matching to identify when genomes or proteomes are likely to have been mislabeled in large cohort databases. When more than 2 omics data sets are available from subjects, use of multiple pairwise matching can even pin-point which data entry is mislabeled. In our work we demonstrate examples of both plasma and DNA samples that are likely to have been swapped and have proposed corrections to the labeling of data. When used in a judicious manner, this matching technique can give confidence and improve the quality of multi-omic databases.21. These methods range from industry level data obfuscation/masking and secure data outsourcing techniques such as substitution, shuffling, numeric variance and null-out/mask-out, to more rigorous statistical data obfuscation methodologies used in Hippocratic Databases22, and privacy-preserving data mining23 such t-Closeness24, differential-privacy25 based methods. Machine learning26 and deep learning27 are also being used in proteomic feature identification and we may be able to leverage these same methods to isolate and "cloak" identifiable omics features while maintaining desirable statistical properties of the data for downstream application. We also believe new omics-specific privacy-preserving methods must be introduced to preserve privacy with omics data against model evasion attack methods that can target both traditional profiling models (such as NB) and modern deep learning-based profiling models.De-identification and privacy protection by informatics is a growing field. We acknowledge that our proposed privacy-preserving measures are only applicable when Naïve Bayes (NB) is used for profiling and we recognize the large body of emerging literature on alternative data obfuscation methods to protect privacy of many types of data28 and our work demonstrates that this day has come even for proteomic technologies that do not rely on peptide sequencing. Unfortunately, most governmental policies do not yet apply to newer omics data such as proteomics . We suggest biomedical research policies be clarified or amended to include any omics data  in which genotype can be ascertained29, but also that there be consideration beyond genotype equivalents to include all features of omics . Because data protection is imperfect and frequently breached, a complementary solution to maintaining privacy might include bioinformatic and identity preserving adjustments to proteomic data. We demonstrated that adjusting out the genetic effects on protein measurements protects privacy by obfuscating the genetic effects, but it still does not change non-genetic associations (such as smoking). This strategy is simple and can be reversed, if necessary, when a researcher has the accompanying genetic information. A disadvantage to removing genetic coding of the proteome is that it could remove associations in which genotype mediates protein affect. Another caveat from our work is that if training the method does not include diverse populations, the identification methods may not be generalizable outside European ancestry. While lower identifiability may be beneficial, future privacy protection algorithms may suffer if identifying features in underserved population are not fully known.Bioethicists had anticipated that other omics data such as proteomic data might one day be identifiable and create privacy concernsSupplementary Tables.Supplementary Figures.Supplementary Information 1.Supplementary Information 2.


Pannexin1 (Panx1) channels release cytosolic ATP in response to signaling pathways. Panx1 is highly expressed in the central nervous system. We used four antibodies with different Panx1 anti-peptide epitopes to analyze four regions of rat brain. These antibodies labeled the same bands in Western blots and had highly similar patterns of immunofluorescence in tissue culture cells expressing Panx1, but Western blots of brain lysates from Panx1 knockout and control mice showed different banding patterns. Localizations of Panx1 in brain slices were generated using automated wide field mosaic confocal microscopy for imaging large regions of interest while retaining maximum resolution for examining cell populations and compartments. We compared Panx1 expression over the cerebellum, hippocampus with adjacent cortex, thalamus, and olfactory bulb. While Panx1 localizes to the same neuronal cell types, subcellular localizations differ. Two antibodies with epitopes against the intracellular loop and one against the carboxy terminus preferentially labeled cell bodies, while an antibody raised against an N-terminal peptide highlighted neuronal processes more than cell bodies. These labeling patterns may be a reflection of different cellular and subcellular localizations of full-length and/or modified Panx1 channels where each antibody is highlighting unique or differentially accessible Panx1 populations. However, we cannot rule out that one or more of these antibodies have specificity issues. All data associated with experiments from these four antibodies are presented in a manner that allows them to be compared and our claims thoroughly evaluated, rather than eliminating results that were questionable. Each antibody is given a unique identifier through the NIF Antibody Registry that can be used to track usage of individual antibodies across papers and all image and metadata are made available in the public repository, the Cell Centered Database, for on-line viewing, and download. In situ hybridization imaging demonstrated high expression levels of Panx1 mRNA in the central nervous system  numerous antibodies pass all tests of specificity except the final test in KO mouse tissue, (2) some antibodies pass all tests of specificity by Western blot, but not when using brain tissue, and (3) numerous antibodies pass all rigorous tests of specificity by Western blot, but fail when used for immunohistochemistry  to each other to look for conserved and variable features. We believe that consistency among imaging and cell culture controls with recombinant proteins are also important for antibody validation. Tests of these antibodies on tissue lysates from two different Panx1 KO mice demonstrate that they recognize Panx1 bands, however additional bands are labeled in the KO for some of the antibodies and these vary between KO animals. Thus, it is still unclear whether differences seen between the four antibodies are due to different epitopes being recognized, non-specificity, or the presence of some residual Panx1 protein in KO tissues. Nevertheless, scientific papers continue to be published with these antibodies and/or these KO mouse models, so we discuss strategies for publishing this kind of data by recommending several standards.For immunofluorescence experiments, we used either goat anti-Myc  or mouse anti-Myc  for co-labeling with either mouse monoclonal anti-Panx1 or rabbit polyclonal anti-Panx1 antibodies, respectively. Both antibodies were used at a 1:250 dilution. GFAP, an astrocytic marker, was labeled using a Guinea pig anti-GFAP  at a 1:800 dilution.CkDia, Diatheva, Fano, Italy; NIF antibody registry number: AB_10013320), (2) chicken anti-Panx1 , (3) rabbit anti-Panx1 , and (4) mouse anti-Panx1 . All antibodies were affinity purified using standard procedures.The four Panx1 antibodies used in this study were (1) a commercial chicken anti-Panx1 CkDia: chicken polyclonal antibody CkDia generated against a peptide containing amino acids corresponding to 135–165 of the cytoplasmic loop of the mouse Pannexin1. This antibody was previously characterized in mouse brain by Zappala et al. Ck4515: chicken polyclonal anti-human Panx1 antibody  raised against a peptide containing the most C-terminal amino acid residues (EKNSRQRLLNPS) in the human Panx1 sequence Rb57: rabbit polyclonal antibody Rb57 generated for our laboratory by Abgent, Inc.  against a peptide containing amino acids 148–164 (KVYNRAIKAAKSARDLD) of the first intracellular loop. This sequence is invariant among mouse, rat, and human sequences.(4)Mo503: monoclonal antibody Mo503 generated by Abgent, Inc.  for our laboratory against a peptide containing amino acids 17–31 (LKEPTEPKFKGLRLE) of the N-terminus of Panx1 and denoted as Mo503. This sequence is invariant among mouse, rat, and human sequences.The characterization of the Laird laboratory antibody, denoted RbCT-395, is described in Penuela et al. . Its epi2. Transient transfections were carried out using 2 mL of Lipofectamine reagent  and 0.5 mg of DNA encoding Panx1-myc fusion protein in a pRK5 plasmid  or fixed with ice cold 100% methanol for 10 min then rinsed into PBS to rehydrate (Rb57 and Mo503). The different fixation methods were used because while the CkDia, Ck4515, and Mo503 nicely label Panx1 in tissue culture cells and brain tissue using 2–4% paraformaldehyde, in these dual labeling experiments, the goat anti-Myc antibodies gave significant background with paraformaldehyde fixation. For these experiments, anti-Panx1 antibodies were used at 1:250 dilutions with an overnight incubation at 4°C, and all secondary antibodies were used at 1:100 for 1–2 h at room temperature. The cells were briefly permeabilized with 0.1% Triton X100, then blocked with 1% BSA, 3% normal donkey serum, 50 mM glycine, and cold water fish gelatin in PBS. A summary of conditions for each of these antibodies is shown in Table HeLa cells  were cultured in Cellgro DMEM  medium supplemented with 10% FBS in a 37°C incubator with 10% COGuidefor the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the University of California San Diego. The animal welfare assurance number is A3033-01. Rats were fully anesthetized and Ringer’s solution containing xylocaine and heparin was perfused transcardially for 3 min followed by 4% paraformaldehyde for 10 min. Here, we show images obtained from an 8-week-old female Sprague-Dawley, however, mosaic images from other rat brains we examined had the same Panx1 patterns. The brain was removed and post-fixed in 4% paraformaldehyde overnight at 4°C. Sagittal sections were cut on a Leica vibratome at a thickness of 75 microns and stored at −20°C in cryoprotectant solution  until processed.All experiments involving vertebrate animals conform to the National Institutes of Health Before immunolabeling, each sagittal section was severed into the four regions to be imaged  to facilitate optimal flatness when mounting on slides for subsequent wide scale imaging. Tissue sections to be labeled with the Rb57 anti-Panx1 antibody underwent antigen retrieval using heat and 10 mM citric acid pH 6.5 for 10 min  using sequential line scanning and then a maximum intensity projection of each stack is computed to make the individual tiles. The tiles were stitched together post-data acquisition using National Center for Microscopy and Imaging Research (NCMIR) developed ImageJ plugins to project, flat field, normalize, align, and combine into a mosaic  is viewable at full resolution and annotatable without downloading using the Web Image Browser , and showed almost complete overlap with an antibody for the epitope tag. For immunofluorescence experiments, HeLa cells transiently transfected with Panx1-myc demonstrated the specificity of each of the four anti-Panx1 antibodies based on the overlap of Panx1 with commercial and well-characterized myc antibodies Figure . A negatright hand column). These cells provided a very strong signal for detecting Panx1 in Western blots and have two strong bands ∼48 and ∼52 kDa in size that are labeled by all four antibodies. It has been previously established that Panx1 in SDS-PAGE/Western Blots runs as three bands that are correlated with their glycosylation status . We analyzed the rodent brain lysates after SDS-PAGE and Western blotting and the Panx1 banding patterns comparing rat to mouse brain were similar. Although each of the four antibodies gave highly similar banding patterns in tissue culture cells, there were variations in the banding patterns in Westerns of the same rat and mouse brain lysates using these four Panx1 antibodies. We also performed Western blots of rat cerebellum lysates, however the banding pattern for each antibody looked the same as the total rat brain lysates.As additional controls for validating our antibodies using Western blots, we analyzed banding patterns between wild type and KO mice generated independently by two different labs  or downloaded onto a reader’s computer via the CCDB portalExamples of the cerebellar cell types labeled by the anti-Panx1 antibodies are shown in Figures Examples of hippocampal cell types  labeled by the anti-Panx1 antibodies are shown in Figures The anti-Panx1 antibodies shown in Figure in situ hybridization studies showed staining with Panx1 mRNA probes in the mouse thalamus and in particular, reticular thalamic neurons  is due to various reasons. It is hard to find a single antibody that can perform well by recognizing the Panx1 protein under all Western blot and immuno-microscopy conditions, which has been shown for other protein targets as well especially in brain tissue  of them overlapping. As such, these two antibodies had similar domain and subcellular labeling patterns, in that they tended to highlight the neuronal cell bodies as well as astrocytes, and endothelial cells in most of the brain regions we imaged. Some images are suggestive of plasma membrane staining, particularly with the Mo503 antibody. However, electron microscopy of these slices will be necessary to unequivocally determine this, since it is sometimes difficult to determine proteins or protein complexes that lie just underneath or within the plasma membrane. The Ck4515 antibody, a very commonly used antibody within the pannexin field, has an expression pattern that is closer to the CkDia and Rb57 labeling patterns than the Mo503 labeling. Our results in hippocampus with the Ck4515 antibody were similar to those of Santiago et al.  who usedOther considerations in understanding, Panx1 labeling include post-translational modifications or differentially translated Panx1 that could complicate various banding patterns that were unanticipated from tissue culture control experiments. Using the same tissue lysate, the Western blot protein patterns varied depending on the antibody and presented a more complex situation than tissue culture cells. Our Western blots of the same rat and mouse brain lysates varied from two to eight major bands depending on the probing antibody. In other published studies, bands were shown at ∼43, ∼52, and ∼96 kDa in Western blots of mouse and rat brain lysates  in the CCDB in their full size, resolution and complexity rather than presenting only down-sampled and/or redacted images that one typically finds in publications. By doing so, this unrestricted available data rich resource promotes further data mining and explorations by the public. Other resources like the Allen Brain Atlas and GENSAT may be useful for interpreting our datasets. The Allen Brain Atlas, a publicly available image database for In summary, because this situation occurs more frequently than is documented, we recommend the following protocol: (1) Entire data sets should be made available on-line, (2) Reagents need to be specified, (3) In order to clarify who else uses these reagents, each antibody should have a unique URI, like those utilized by NIF. Reproducibility of reagents and protocols are critical. A recent editorial in Nature highlighted significant reproducibility issues in clinical drug trials because of insufficient or selective reporting (Begley and Ellis, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The genetic component was suggested to contribute to the development of chronic obstructive pulmonary disease (COPD), a major and growing public health burden. The present review aims to characterize the evidence that gene polymorphisms contribute to the aetiology of COPD and related traits, and explore the potential relationship between certain gene polymorphisms and COPD susceptibility, severity, lung function, phenotypes, or drug effects, even though limited results from related studies lacked consistency. Most of these studies were association studies, rather than confirmatory studies. More large-sized and strictly controlled studies are needed to prove the relationship between gene polymorphisms and the reviewed traits. More importantly, prospective confirmatory studies beyond initial association studies will be necessary to evaluate true relationships between gene polymorphisms and COPD and help individualized treatment for patients with COPD. IntroductionGene polymorphisms and COPD susceptibilityAlpha 1-antitrypsin geneTumour necrosis factor genesMicrosomal epoxide hydrolase geneGlutathione S-transferases genesTransforming growth factor-beta(1) geneOther genesGene polymorphisms and disease severityGene polymorphisms and extent of emphysemaGene polymorphisms and exacerbations of COPDGene polymorphisms and lung function declineGene polymorphisms and COPD-related phenotypesGene polymorphisms and COPD-related cardiovascular changesGene polymorphisms and drug effectsPerspectivesChronic obstructive pulmonary disease is characterized by the development of airflow limitation that is progressive and not fully reversible The relationship between gene polymorphisms and COPD susceptibility has been paid special attention and was explored in a large number of studies on hundreds of genes, although the results varied between studies and populations of people, as listed in Table e.g. neutrophil release and activation versus G/G; OR = 1.330, for allele A versus allele G) Tumour necrosis factor α (TNFα) was proposed to play a critical role in COPD pathogenesis, Interestingly, the association of TNF-308 polymorphism with asthma has only been reported in the U.K./Irish population Microsomal epoxide hydrolase (EPHX1) plays an important role in the metabolism of highly reactive epoxide intermediates formed in cigarette smoke. The population was classified into fast, normal, slow and very slow groups of EPHX1 phenotypes, based on reported polymorphisms within the coding region of EPHX1 genes tyrosine/histidine 113 or histidine/arginine 139 e.g. one mutant mEPHX exon-3 allele, GSTM1-null and homozygous isoleucine 105 GSTP1 genotypes, could be valuable indicators of susceptibility to COPD Glutathione S-transferases (GSTs) are key players to detoxify various aromatic hydrocarbons found in cigarette smoke. The proportion of GST mu 1 (GSTM1)-null genotypes was significantly higher in patients with COPD than in controls in a Taiwanese population Abnormalities in the TGF-β1 gene were found to be associated with COPD susceptibility. Two SNPs (rs2241712 and rs1800469) in the promoter region of TGF-β1 and one SNP (rs1982073) in exon 1 of TGF-β1 were discovered to be significantly associated with COPD Numerous other gene polymorphisms are also studied with relation to COPD susceptibility. The Gly16 polymorphism of the beta(2)-adrenoceptor (ADRB2) gene might be associated with the susceptibility to the development of COPD in a Chinese population Some studies failed to find an association between certain polymorphisms and COPD risk. Neither IL-1beta polymorphisms at position −511 base and at the amino acid residue 105 nor IL-1 receptor antagonist polymorphisms in intron 2 were associated with susceptibility to Japanese with COPD Genes might be related to each other functionally or connected in certain pathways or networks; however; little was known about the combined effects of two or more gene polymorphisms on COPD susceptibility, which may be a developing direction for future study.Although several studies including some large-population and well-performed studies have demonstrated the relationship between certain gene polymorphisms and COPD susceptibility, there have been little prospective confirmatory studies to further confirm whether certain polymorphisms could truly increase or decrease COPD risk. Although it will take many years for patients to develop COPD and make follow-up work much tougher, prospective studies are more meaningful and might point to a new direction for future study.e.g. patients with mild COPD have the forced expiratory volume in one-second (FEV1) of ≥80% of the predicted value, moderate with 50%≤FEV1<80% predicted, severe with 30%≤FEV1<50% predicted, or very severe patients have FEV1<30% predicted. Chronic obstructive pulmonary disease patients with AA homozygous at position −308 on the TNFα gene had less reversible airflow obstruction and a significantly higher mortality on a 2-year follow-up Spirometric assessment based on airflow limitation is one of the most important components to evaluate the severity of COPD, On the other hand, some studies failed to find an association between polymorphisms and COPD severity. Some meta-analysis-based studies demonstrated that there was no difference of mean FEV1 between PI MM and PI MZ individuals n = 84) n = 267) and the −28G allele was inversely associated with the CT score n = 389) and smoking controls from the Normative Aging Study  A visual scoring system was used to evaluate the ratio of the low attenuation area to the corresponding lung area in patients with COPD (Mannose-binding lectin (MBL) is a pattern-recognition receptor in serum that assists innate immunity by binding to exposed sugars on the surface of invading bacteria Clinical studies demonstrated that the distribution of the IL-1beta and IL-1 receptor antagonist gene haplotypes was different between Canadian smokers with a rapid decline in lung function and smokers with no decline in lung function, although the genotypes were not associated with the rate of decline of lung function Some gene polymorphisms were indicated not to be related to lung function decline. For example, Decorin (an extracellular matrix proteoglycan) SNPs, TGF-β1 SNPs and their haplotypes were not associated with accelerated FEV1 decline in 1390 cases in the Netherlands Gene polymorphisms of hematopoietic cell kinase (Hck) were found to be associated with COPD-related phenotypes in a Canadian population There are also negative results concerning the relationship between gene polymorphisms and COPD-related phenotypes. For example, a cross-sectional study revealed no significant relationship between common SERPINA1 polymorphisms  and the emphysematous type of COPD e.g. angiotensinogen (M235T), angiotensin-converting enzyme (I/D), and angiotensin II type 1 receptor (A1166C) were associated with right ventricular hypertrophy diagnosed by electrocardiography was investigated in 87 patients with severe COPD e.g. IL-6, monocyte chemoattractant protein-1, and IL-1β, were hypothesized to be associated with the risk for pulmonary hypertension in COPD Whether polymorphisms of the renin angiotensin system, A randomized, double-blind and crossover study enrolling 36 COPD patients found that the mean pulmonary arterial pressure, pulmonary vascular resistance (PVR), and lactate concentration after exercise were lower in patients with II or ID genotypes of angiotensin-converting enzyme after the treatment with captopril, rather than those with the DD genotype Certain gene polymorphisms relating to COPD susceptibility will lead to the development of individualized medicine. The AAT coding gene polymorphisms were used to diagnose patients with severe AAT deficiency, leading to the therapy different from other COPD patients. Gene polymorphisms have also been found to contribute to inter-individual differences in the response to cigarette smoke, which could lead to more targeted anti-smoking interventions. Gene polymorphisms might be used to predict disease severity, complications, prognosis and drug effects for COPD patients individually. Furthermore, it is also important to identify and validate gene polymorphisms-specific biomarkers to trace and define the biological effects of genetic factors. Clinical bioinformatics, as an emerging discipline Numerous studies have shown that gene polymorphisms may contribute to COPD susceptibility, severity, extent of emphysema, acute exacerbations, lung function, phenotypes, cardiovascular changes and drug effects, even though results are still conflicting. Most of these studies were association studies, rather than confirmatory studies. More large-sized and strictly controlled studies are needed to prove the relationship between gene polymorphisms and the reviewed traits. More importantly, prospective confirmatory studies beyond initial association studies will be necessary to evaluate true relationships between gene polymorphisms and COPD. Those studies will help individualized treatment for COPD patients.


Sapovirus is a genus of caliciviruses that are known to cause enteric disease in humans and animals. There is considerable genetic diversity among the sapoviruses, which are classified into different genogroups based on phylogenetic analysis of the full-length capsid protein sequence. While several mammalian species, including humans, pigs, minks, and dogs, have been identified as animal hosts for sapoviruses, there were no reports of sapoviruses in bats in spite of their biological diversity. In this report, we present the results of a targeted surveillance study in different bat species in Hong Kong. Five of the 321 specimens from the bat species, Hipposideros pomona, were found to be positive for sapoviruses by RT-PCR. Complete or nearly full-length genome sequences of approximately 7.7 kb in length were obtained for three strains, which showed similar organization of the genome compared to other sapoviruses. Interestingly, they possess many genomic features atypical of most sapoviruses, like high G+C content and minimal CpG suppression. Phylogenetic analysis of the viral proteins suggested that the bat sapovirus descended from an ancestral sapovirus lineage and is most closely related to the porcine sapoviruses. Codon usage analysis showed that the bat sapovirus genome has greater codon usage bias relative to other sapovirus genomes. In summary, we report the discovery and genomic characterization of the first bat calicivirus, which appears to have evolved under different conditions after early divergence from other sapovirus lineages. Vesivirus, Lagovirus, Norovirus, Sapovirus and Nebovirus. They possess a non-segmented, polyadenylated, positive-sense ssRNA genome of about 7.5 to 8.5 kb in length, enclosed in an icosahedral capsid of 27 to 40 nm in diameter. Among them, noroviruses and sapoviruses (SaVs) are well known to cause enteric disease in a range of mammals, including humans, while vesiviruses and lagoviruses cause systemic diseases in specific animal hosts. Nebovirus is the most recently established genus in the family CaliciviridaeRecovirus, has been proposed for a novel calicivirus detected in stool specimens from rhesus monkeys Valovirus, has been proposed for a novel group of swine caliciviruses known as the St-Valérien-like viruses The caliciviruses are a family of small non-enveloped viruses, and can be classified into five genera: Sapovirus currently contains only one recognized species, the Sapporo virus, which was discovered in 1977 in Sapporo, Japan The genus As mentioned above, both noroviruses and SaVs generally cause mild to asymptomatic enteric infections in human and animal hosts Bats  constitute a significant portion of biological diversity in many ecosystems and have a wide geographical distribution Hipposideros pomona, and genome sequences were obtained for three strains of the bat SaV. Sequence analysis indicated that the novel virus possesses several genomic features atypical of SaVs, and phylogenetic analysis revealed that it descended from a lineage that had diverged early from other SaV.In the present study, we investigated the presence of unknown calicivirus diversity in bats by targeted RT-PCR screening. Novel SaV sequences were amplified from several faecal samples of the bat species A total of 728 anal swabs from different bat species in Hong Kong were obtained. No obvious signs of enteric disease, like anorexia and diarrhoea, were observed in the bats during the brief period of captivity needed for sampling.RT-PCR using broadly reactive degenerate primers for a 185 nt fragment in the 3D-like RNA-dependent RNA polymerase (RdRp) region of the calicivirus ORF1 gene was positive in two specimens. Repeated screening using more sensitive specific primers revealed three additional positive specimens. Further information on the species and RT-PCR screening results are presented in Complete or nearly full-length genome sequences (with incomplete 5′ ends) were obtained for three positive samples using the sequencing strategy as described in the GPPGIGKT), VPg at residues 958 (KGKTK) and 972 (SEYEE), 3C-like protease at residue 1183 (GDCG), 3D-like RNA-dependent RNA polymerase at residues 1520 (GLPSG) and 1568 (YGDD), and VP1 at residue 1867 (PPG). It undergoes proteolytic processing to produce the nonstructural viral proteins and the major capsid protein VP1. Based on comparison with the ORF1 cleavage map of SaV/Mc10 AUGGA) just upstream to the cleavage site, which has also been reported in other caliciviruses The complete ORF1 is 6855 nt long, and encodes a large precursor polyprotein with an estimated molecular mass of 246.8 kDa. The polyprotein contains characteristic amino acid motifs conserved in caliciviruses: 2C-like NTPase at residue 482  could be identified at 24 nt upstream of the ORF2 start codon. Sequence identities for VP2 with other SaVs vary from 15.5% to 19.9%  branch support values as calculated by PhyML.Sapovirus in the family Caliciviridae.Although the phylogenetic positions of the novel bat virus are slightly divergent in the three trees, they generally show the bat SaV as being most closely related to the SaVs. In our opinion, there is insufficient ground for proposing a new genus for the novel virus under the current framework of taxonomic classification. The ORF1 polyprotein and VP1 capsid protein sequences of the novel bat virus showed obvious phylogenetic clustering with other SaV sequences. It should also be noted that the VP2 protein sequences are shorter and more divergent, and therefore are considered to be less useful in the phylogenetic classification of caliciviruses c) (c for background nucleotide composition (Nc′) did not significantly affect the observed difference in codon usage bias.As genomic nucleotide composition is strongly associated with codon usage bias in viruses, we examined the codon usage in the genomes of the novel bat SaV and other SaVs given their different nucleotide composition. The bat SaV genome was found to have significantly greater codon usage bias than the other SaV genomes, as measured by their effective number of codons (Nc) . AdjustiCG and ρGC) and the CpG/GpC ratio were calculated to assess the degree of CpG suppression. Results confirm the presence of significant CpG suppression (ρCG≤0.78) in examined SaV genomes, with the only exception being the bat SaV genome . The RNA was eluted into 50 µl RNase-free water and was used as the template for RT-PCR.5′-GAYTAYTCNMRRTGGGAYTC-3′ and 5′- GGCATNCCNGAKGGNAYNCC -3′) were designed from the multiple sequence alignment of the available calicivirus gene sequences in NCBI GenBank. First-strand cDNA synthesis was performed using SuperScript III kit (Invitrogen) according to manufacturer's instructions. The PCR mixture (25 µl) contained cDNA, PCR buffer , 200 µM of each dNTP and 1.0 U AmpliTaq Gold polymerase (Applied Biosystems). PCR cycling conditions were as follows: hot start at 94°C for 7 min, followed by 50 cycles of 94°C for 1 min, 50°C for 1 min and 72°C for 1 min with a final extension at 72°C for 10 min in an automated thermal cycler (Applied Biosystems). Standard precautions were taken to avoid PCR contamination and no false-positive signal was observed in the negative controls. The PCR products were gel-purified using a QIAquick gel extraction kit (Qiagen). Both strands of the PCR products were sequenced twice with an ABI Prism 3730xl DNA Analyser (Applied Biosystems), using the two PCR primers.Screening was performed by amplifying a 185 nt fragment in the RdRp region of the ORF1 gene of caliciviruses. Conserved degenerate primers . Standard precautions were taken to avoid PCR contamination and no false-positive signal was observed in the negative controls. PCR product purification and sequencing were performed as above.Additional RT-PCR screening was performed on the same samples using specific primers designed from the RdRp nucleotide sequences of bat SaVs obtained from previous rounds of RT-PCR and sequencing, as RT-PCR screening with specific primers usually offers higher sensitivity than a comparable screening with consensus degenerate primers. Sequences of the specific primers are as follows: forward primer Purified PCR products were cloned into a pCR2.1-TOPO vector (Invitrogen) according to manufacturer's instructions. The vector was then used to transform the competent Escherichia coli strain DH5α by electroporation. Positive transformants were identified by blue–white screening, and eight colonies were selected for DNA sequencing of the construct using the M13 forward and reverse primers according to the manufacturer's instructions. Sequencing reactions were performed as described above.Viral genome sequences were obtained using strategies we had previously used for other RNA viruses http://www.ncbi.nlm.nih.gov/projects/gorf/). Annotation of the predicted proteins was performed by BLAST sequence similarity search against annotations in the NCBI RefSeq database. Multiple sequence alignments were constructed using MUSCLE version 3.8.31 ORFs were located using the ORF Finder tool at NCBI (c and Nc′) were calculated using the INCA package version 2.1 c is the effective number of codons in the coding regions of the genome c′ is the effective number of codons adjusted for background nucleotide composition The full-length ORF1 and ORF2 coding sequences were extracted from selected SaV genomes and concatenated for codon usage analysis Click here for additional data file.Figure S2Neighbor-joining tree of partial RdRp nucleotide sequences. The tree was constructed based on the length of the nucleotide sequence in the RdRp region obtained from bat SaV/TLC72.(TIF)Click here for additional data file.Figure S3Hipposideros pomona is in the drainage at Tai Lam – Shek Kong.Photo showing (TIF)Click here for additional data file.Figure S4Hipposideros pomona possesses a small nose leaf.Photo showing (TIF)Click here for additional data file.Table S1Epidemiology of the tested bat specimens.(DOC)Click here for additional data file.Table S2Amino acid identity of the Bat SaV/TLC58/HK with representative caliciviruses of other genera.(DOC)Click here for additional data file.


Principal component analysis revealed that whereas the gene expression patterns in the embryonic tissues varied according to the donor cell type, those in extraembryonic tissues were relatively consistent across all groups. Within each group, the embryonic tissues had more differentially expressed genes (DEGs) (>2-fold vs. controls) than did the extraembryonic tissues (P<1.0×10–26). In the embryonic tissues, one of the common abnormalities was upregulation of Dlk1, a paternally imprinted gene. This might be a potential cause of the occasional placenta-only conceptuses seen in SCNT-generated mouse embryos (1–5% per embryos transferred in our laboratory), because dysregulation of the same gene is known to cause developmental failure of embryos derived from induced pluripotent stem cells. There were also some DEGs in the extraembryonic tissues, which might explain the poor development of SCNT-derived placentas at early stages. These findings suggest that SCNT affects the embryonic and extraembryonic development differentially and might cause further deterioration in the embryonic lineage in a donor cell-specific manner. This could explain donor cell-dependent variations in cloning efficiency using SCNT.The great majority of embryos generated by somatic cell nuclear transfer (SCNT) display defined abnormal phenotypes after implantation, such as an increased likelihood of death and abnormal placentation. To gain better insight into the underlying mechanisms, we analyzed genome-wide gene expression profiles of day 6.5 postimplantation mouse embryos cloned from three different cell types . The embryos retrieved from the uteri were separated into embryonic (epiblast) and extraembryonic  tissues and were subjected to gene microarray analysis. Genotype- and sex-matched embryos produced by  Somatic cell nuclear transfer (SCNT) is a unique technology that produces a cloned animal from a single donor somatic cell nucleus. This technology is potentially promising for many practical applications in the fields of medicine and drug manufacturing, the animal industries and in conservation of genetic resources Xist, a gene responsible for X chromosome inactivation Magea and Xlr gene clusters, localized in chromosomal regions XqF3 and XqA7.2–7.3, respectively The presence of such epigenetic abnormalities related to insufficient or erroneous reprogramming in SCNT-derived embryos has prompted researchers to investigate their gene expression profiles in depth. For bovine and mouse embryos, microarray technology in conjunction with linear amplification of mRNA from individual embryos has been employed extensively to clarify the gene expression patterns specific to SCNT Xist-specific short interfering (si) RNA indicated that many of the developmental failures during the early postimplantation period could be rescued by correction of aberrant Xist expression before implantation Xist knockout embryos still show higher incidences of developmental failure and of SCNT-specific abnormal phenotypes such as placental development lacking a fetus and placental hyperplasia (unpublished data). Therefore, there must be some Xist-independent mechanisms that affect the postimplantation development of SCNT-derived embryos. To gain better insight into this etiology, here we analyzed the genome-wide gene expression profiles of embryonic day (E)6.5 mouse postimplantation embryos cloned from three different cell types . As we inferred in our previous papers, ectopic Xist expression in SCNT-derived embryos is a consequence of the discrepancy of Xist regulation between somatic cells and early embryos, but is not a result of abnormal genomic reprogramming. Therefore, we expected that by using Xist gene knockout we could narrow the gene expression profiles that might more exactly reflect the cell type-specific reprogramming pattern at the time of SCNT. Therefore, in this study we generated cloned embryos of both sexes from Xist knockout donor cells. Embryos retrieved from the uteri of recipient foster mothers were separated into the embryonic (epiblast) and extraembryonic  tissues and the data were compared with those from genotype- and sex-matched control embryos produced by in vitro fertilization (IVF).Despite the abnormal epigenetic and gene expression profiles described above, many mouse SCNT-derived embryos reach the blastocyst stage and subsequently undergo implantation Xist knockout mice Xist knockout B6 female mice with DBA/2 male mice C57BL/6N (B6), DBA/2 and (C57BL/6N × DBA/2)F1 (BDF1) strains of mice were purchased from Japan SLC, Inc.  and ICR mice were purchased from CLEA Japan, Inc. . For somatic cell nuclear donors, heterozygous Eight- to 10-week-old BDF1 females were induced to superovulate by injections of 7.5 IU of pregnant mare serum gonadotropin  and 7.5 IU of human chorionic gonadotropin  at an interval of 48–50 h. Mature MII oocytes were collected from the oviducts 15 h after hCG injection and released from cumulus cells by treatment with 0.1% bovine testicular hyaluronidase  in potassium simplex optimized medium (KSOM).Xist knockout mice with a BDF1 genetic background (see above). Cumulus cells were collected from cumulus–oocyte complexes of superovulated adult female mice after treatment with KSOM containing 0.1% hyaluronidase. Neonatal Sertoli cells were prepared as a testicular suspension as described 2 in air for 1–2 weeks until the fibroblasts expanded and became confluent.All three types of nuclear donor cells  were prepared from heterozygous 4 and 0.1 mg/ml polyvinyl alcohol. After 1 h culture in KSOM, the reconstructed oocytes were activated with 3 mM SrCl2 for 1 h and cultured in KSOM containing 5 µg/ml cytochalasin B for 5 h. They were further cultured in KSOM for 12–18 h until they reached the 2-cell stage. BDF1 embryos generated by conventional IVF were used as controls.Nuclear transfer from cumulus or neonatal Sertoli cells was carried out using a Piezo-driven micromanipulator  as described To determine whether the method of nuclear transfer (microinjection or membrane fusion) could cause differences in the transcriptomes of SCNT embryos, in a preliminary study we reconstructed embryos with cumulus cell nuclei either by injection or fusion and compared their gene expression profiles by microarray. As shown in Figure S1 in Embryos that reached the 2-cell stage were transferred into the oviducts of ICR recipient mice at day 1 of pseudopregnancy . On day 7 of pregnancy, corresponding to E6.5, the implanted embryos were recovered carefully from the uteri and isolated from decidua using fine forceps and a needle under a dissecting microscope. After incubation in PBS with 0.25% pancreatin and 0.05% trypsin for 10 minutes at 4°C, embryos were transferred to DMEM containing 10% FBS and then the visceral endoderm layer was detached from epiblast or extraembryonic ectoderm by being gently aspirated through a mouth pipette several times. Thereafter, the epiblast was separated from the extraembryonic ectoderm using a fine tungsten needle Microarray analyses for gene expression were performed as described Total RNA was isolated using ISOGEN  from embryonic and extraembryonic tissues of the E6.5 embryos. First-strand cDNA was synthesized using a SuperScript III First-Strand Synthesis System  with random hexamer primers. All qPCR runs were performed using QuantiTect SYBR Green PCR kits  and ABI Prism 7900HT Sequence Detection System  with a condition of 40 cycles of 95°C for 20 s, 60°C for 20 s and 72°C for 20 s, following a denaturation step at 95°C for 10 min. Primers used are listed in t tests with a 5% significance level and a 2.0-fold filter was applied to identify differentially expressed genes (DEGs) between SCNT-derived embryo samples and sex- and tissue-matched IVF-derived control samples. Microarray data were MIAME (Minimum Information About a Microarray Experiment) compliant and have been deposited in the GEO public database (GSE49173) (http://www.ncbi.nlm.nih.gov/geo/). Other statistical analyses are indicated in the text as appropriate.Statistical analyses of microarray data were performed using Gene Spring GX 12.5 software (Agilent Technologies). All raw data were subjected to quintile normalization. To reduce background noise, the genes of a microarray flagged with “not detected,” both in the SCNT and control samples, were removed from the data set. Consequently, about 30000 genes (probes) (27662 to 32001 out of 41267) were used for the statistical analysis (see Results). Normalized values were analyzed by moderated Pou5f1 , Nanog, Fgf5, Nodal and Cripto genes, which are expressed dominantly in the epiblast Cdx2, Elf5, Fgfr2, Plet1 and Gjb5, which are predominantly expressed in extraembryonic tissues Pgk1 gene was stably detected in both tissues  to determine how the gene expression patterns were affected by the donor cell genome. For the embryonic tissues, the expression profiles could be clearly separated into three donor cell groups . The extraembryonic samples contained fewer DEGs  compared with embryonic samples (901–4642 genes) in all of the donor cell types  cells Magea and Xlr genes were strongly repressed to the basal level  was also repressed in the extraembryonic tissues, so there was no difference between the control and SCNT-derived embryos  were expressed in both IVF- and SCNT-derived embryos, indicating that the expression of Xlr had recovered in SCNT-derived embryos after implantation  and Ascl1  Among the five common DEGs in the extraembryonic tissue, there were no candidate genes that could account for the abnormal placental development in SCNT-derived embryos. We assumed that this might have arisen, at least in part, from unexpectedly high variations in the expression profiles in the control IVF-derived extraembryonic tissues. It is known that SCNT-derived placentas at early to midgestation stages are characterized by a decrease in undifferentiated diploid cells and an increase in trophoblastic giant cells Xist knockout mice as nuclear donors. As we have shown previously, the Xist gene when expressed ectopically in mouse SCNT-derived embryos causes a global dysregulation of many development-related genes Xist knockout mice as donors, the birth rates increased to about 10%, reflecting extensive correction of the global gene expression patterns Xist was probably caused by the lack of a Xist-repressing epigenetic imprint in the donor genome, but not by reprogramming errors within the recipient ooplasm Xist from the microarray analysis would help towards better understanding of SCNT-associated abnormalities from the viewpoint of genomic reprogramming errors.It is very common that abnormal phenotypes appear predominantly after implantation following SCNT cloning in mammals. The present study was undertaken to determine whether there were any SCNT-specific aberrations in gene expression shortly after implantation that might explain such abnormal phenotypes. There have been many studies analyzing gene expression profiles in SCNT-derived embryos before implantation in different species, including mice, cattle and pigs Xlr family genes, which are strongly repressed at the blastocyst stage in a Xist-independent manner One of the interesting findings in this study was the differential responsiveness of embryonic and extraembryonic tissues to SCNT. As clearly demonstrated by PCA , genes iCdx2, Esrrb and Eomes) Tpbpa) were downregulated, while those essential for differentiation into giant cells (Hand1) Here we sought to identify the genes responsible for placental abnormalities associated with mouse SCNT. It is known that cloned mouse placentas are characterized by hypoplasia of diploid trophoblastic cell layers in early stages and hyperplastic trophoblast cell proliferation in later stages Esx1 mutants In contrast to the poor placentation at earlier stages, cloned placentas at term are typically two or three times larger than normal, primarily because of extraordinarily enlarged spongiotrophoblast and glycogen cell layers Dlk1 as one of the upregulated genes in the embryonic tissue of the SCNT-derived conceptuses. This finding was confirmed by our qPCR analysis  and four maternally expressed noncoding RNAs  Dlk1 in this cluster . This might have been because of their relatively lower expression levels at early postimplantation stages Dlk1–Dio3 cluster and aberrant expression of imprinted genes in this cluster including Dlk1 and Gtl2 (unpublished data). Indeed, overexpression of Dlk1 is known to have deleterious effects on embryonic development Dlk1Our statistical analysis for DEGs common to three SCNT groups identified The present study was undertaken under defined genetic conditions in terms of the genotypes of the donor cells (B6D2F1), the recipient oocytes for nuclear transfer (B6D2F1) and the recipient females for embryo transfer (ICR). Such availability of strains with defined genetic backgrounds is one of the biggest advantages of the use of laboratory mice as models, although it does not always ensure that results can be extrapolated to mammals in general. The set of strains we used has been recognized to be standard in mouse SCNT experiments since the first birth of cloned mice in 1998 File S1File containing supporting figures S1–S4. Figure S1. Comparative global gene expression analysis between the two SCNT methods. Principle component analysis (PCA) clearly shows that global gene expression profiles of cumulus cell-derived clones generated by cell fusion (purple) or nuclear injection (red) were not distinguishable. Dark- and light-gray circles represent male and female IVF-derived control embryos, respectively. Figure S2: Expression of epiblast and extraembryonic markers  and extraembryonic marker genes  extracted from the microarray data. Nanog, Fgf5 and Nodal were detected significantly in embryonic tissues, but were scant in extraembryonic samples: Krt7, Phlda2 and Dnmt3L showed the opposite pattern. Em, embryonic samples; Ex, extraembryonic samples; IVF, control samples from in vitro fertilized embryos; CC, cumulus cell-derived clone; FC, fibroblast-derived clone, SC: Sertoli cell-derived clone. Figure S3: PCA of global gene expression profiles with an equivalent analysis of IVF-derived embryos (continued from Dlk1–Dio3 imprint region. Gene expression levels between cumulus cell-derived clones (CC) and IVF-derived control samples were compared by qPCR. In a similar fashion to Dlk1 gene expression was upregulated in embryonic samples of CC (P = 0.018 by one-sided Student's t test), though not in extraembryonic samples. There were no significant differences between the CC and IVF-derived control samples in the Gtl2, Dio3 and Rian genes. Each dot represents the expression level relative to the Gapdh gene. Bars represent the means of samples in the same group.(PDF)Click here for additional data file.Table S1Primer list for qPCR.(XLSX)Click here for additional data file.Table S2Overlapping DEGs among the three SCNT groups.(XLS)Click here for additional data file.


The objective of the current study was to determine the effects of in utero versus neonatal exposure to a low dose  of second-hand smoke on adult atherosclerotic lesion development using the apolipoprotein E null mouse model. Consequently, apolipoprotein E null mice were exposed to either filtered air or second-hand smoke: (i) in utero from gestation days 1–19, or (ii) from birth until 3 weeks of age . Subsequently, all animals were exposed to filtered air and sacrificed at 12–14 weeks of age. Oil red-O staining of whole aortas, measures of mitochondrial damage, and oxidative stress were performed. Results show that both in utero and neonatal second-hand smoke exposure significantly increased adult atherogenesis in mice compared to filtered air controls. These changes were associated with changes in aconitase and mitochondrial superoxide dismutase activities consistent with increased oxidative stress in the aorta, changes in mitochondrial DNA copy number and deletion levels. These studies show that in utero or neonatal exposure to second-hand smoke significantly influences adult atherosclerotic lesion development and results in significant alterations to the mitochondrion and its genome that may contribute to atherogenesis.Cardiovascular disease is a major cause of morbidity and mortality in the United States. While many studies have focused upon the effects of adult second-hand smoke exposure on cardiovascular disease development, disease development occurs over decades and is likely influenced by childhood exposure. The impacts of  For these studies, it was found that both in utero and neonatal SHS exposure resulted in significantly increased adult atherosclerotic lesion development and changes within the mitochondrion that heighten CVD susceptibility.Hence, to determine whether prenatal versus neonatal SHS exposure had distinct effects upon adult CVD development, hypercholesterolemic apolipoprotein E null (apoE−/−) mice were exposed to SHS All studies were approved by the University of Alabama at Birmingham Animal Resource Program IACUC .ad libitum before and subsequent to, but not during, SHS exposure. Male offspring from pregnant mice were fed chow (PicoLab Rodent Chow 20) diets until 12–14 weeks of age.Apolipoprotein E null (apoE−/−) mice were purchased from Jackson Laboratories  and shipped directly to the University of California-Davis (UC Davis) for use as breeders for the SHS exposures. Apolipoprotein E is an important ligand for lipoprotein receptors in the liver, and thus apoE−/− mice have high plasma levels of cholesterol  and triglycerides compared to wild-type mice in utero or neonatal SHS at a concentration of 1 mg/m3, 4–5 ppm carbon monoxide, and 200–300 µg/m3 nicotine. This dose of SHS falls well within the range for humans observed indoors 3 SHS (6 hrs/day) from gestation days 1–19 (in utero exposure), followed by filtered air exposure until sacrifice at 12–14 weeks of age, or (ii) filtered air or 1 mg/m3 SHS (6 hrs/day) from birth to 21 days of age , followed by filtered air exposure until sacrifice at 12–14 weeks of age .All SHS exposures were performed at the Center for Health and the Environment, University of California-Davis in accordance with institutional guidelines. Mice were exposed to s of age Figure 1Mice were anesthetized by interperitoneal injection of Ketastat/Xylazine (60 mg/kg Ketastat/10 mg/kg Xylazine), and exsanguinated. The aortas  were perfused with cold PBS immediately following removal and then flash-frozen  in liquid nitrogen unless used for oil red-O staining or isolation of mitochondria. Tissues were stored at −80°C until use. Aortas prepared for oil red-o had adventitial tissues removed and were washed with PBS and fixed in formalin.Whole aortas were stained with oil red-O to assess atherosclerotic plaques as previously described Mitochondrial DNA copy number was determined by QPCR of a 163 bp product as previously described 2 and 20 mM isocitrate at 25°C. Aconitase is specifically inactivated by superoxide (O2.−) and peroxynitrite (ONOO−) and hence, decreased activity correlates with increased oxidative stress associated with superoxide (O2. −) and peroxynitrate (ONOO−) − formation is related to O2.− production, decreased aconitase activity can be associated indirectly with increased O2.− generation.Aconitase activity was determined by measuring the transformation of isocitrate to cis-aconitate at 240 nm in 50 mM TrisHCl (pH 7.4) containing MnClTo evaluate the specific activity of aconitase, western blots were performed using the same homogenates used for the enzyme activity. Decreases in enzymatic activity without significant changes in aconitase protein level were interpreted as consistent with decreased enzymatic specific activity. Antibody specific to aconitase was generated based upon the NCBI reported murine mitochondrial aconitase (Acon2) sequences. After analysis of the protein sequence, three peptides were chosen for conjugation and antigen preparation. These sequences are specific to mitochondrial aconitase and are not reported to be present in cytosolic aconitase:N′-HLDDPANQEIERGKTYLRLRPDR-C′N′-DHLIEAQVGGEKDLRRAKD-C′N′-HRMKKYLSKTGRTDIANLAEEFKDH-C′Upon synthesis, peptides were each conjugated to KLH via their N′ and their C′ termini, dialyzed, lyophilized and made ready to immunize two rabbits per peptide. Serum was collected at pre-immune, 6 weeks, 8 weeks and 9 weeks. The sera were pooled, screened by ELISA and Western blot, and later affinity purified against the peptides.To perform immunoblots on homogenized tissue, 50 µg of protein was loaded onto 4–12% Bis Tris PAGE gels , subjected to electrophoresis, transferred to PVDF membrane, blocked in milk, and immunobloted with the anti-aconitase antibody for two hours. Following incubation with the primary antibody, blots were immunoblotted with secondary HRP-goat anit-rabbit IgG antibody. Blots are visualized using ECL Plus Western Blotting Detection Reagent (Amersham).Mitochondrial SOD (SOD2) activity and protein levels were determined as previously described P<0.05) existed, then a Student-Newman Keuls test was used for group comparisons. All statistical analyses were carried out using SigmaStat statistical software.For all endpoints, a minimum of five animals per group were utilized. Results are expressed as mean ± SEM. A Shapiro-Wilk normality test was performed prior to analysis of variance (ANOVA) which tested the null hypothesis that all samples were drawn from a single population. All data within this study passed the normality test. If significant differences . By contrast, in utero SHS exposure was associated with decreased average body weight in adult (12–14 weeks old) mice compared to both control and neonatal SHS exposed mice  diet Figure 32.−) and peroxynitrite (ONOO−) in utero or neonatal SHS compared to filtered air controls; furthermore, aconitase protein levels were not significantly different between exposure groups (2.− and ONOO−).Because oxidative stress is known to play an important role in atherosclerosis initiation and progression 2.− related oxidative stress in the vasculature. Quantification of mitochondrial superoxide dismutase activity  from aortic homogenates showed that while no differences existed in total SOD2 activity, differences were observed in SOD2 protein levels in the adult mice exposed to developmental SHS, also resulting in decreased SOD2 specific activity in adult animals exposed to SHS in utero or as neonates  exposed to 25 mg/m3 TSP 3 TSP Atherosclerosis is a chronic, pro-inflammatory disease resulting from the accumulation of lipids, macrophages, T-cells, and vascular smooth muscle cells in the sub-intimal space. Cigarette smoke exposure is one of the strongest predictors of CVD development and it has been estimated that exposure to second-hand smoke (SHS) increases the risk of CVD by 30% −/− mice as both in utero and neonatal exposure showed comparative increases in oil red-O staining. Similar effects were observed in the vasculature regarding measures of oxidative stress such as aconitase activity, SOD2 specific activity, mtDNA copy number, and the number of distinct mtDNA deletions. Interestingly, the accumulation of mtDNA deletions was different depending on the timing of the SHS exposure; while in utero SHS exposure increased the overall amount of mtDNA deletions, neonatal exposure to SHS was not associated with such an increase in the adult. This difference may be temporal, in that in utero SHS exposure contributed to deletion formation at an earlier developmental period compared to neonatal exposure. During embryogenesis rapid growth and cell division occur, and early introduction of a mtDNA deletion will result in a greater accumulation of these deletions over time in the adult tissues. The observation that the number (frequency) of mtDNA deletions is similar between in utero and neonatal exposures, yet the levels of mtDNA deletions are higher in the in utero animals, is consistent with the increased accumulation of deletions over time −/− animals compared to unexposed controls and eventually become distinct. This may also suggest that it is the number of different types of mtDNA deletions that influence atherogenesis. In addition, empirically it appears that both the in utero and neonatal exposure tissues appear to have a higher number of large mtDNA deletions  has been shown to increase atherogenesis and mtDNA damage Macaca mulatta have been shown to increase aortic mtDNA damage In aortic homogenates, aconitase, a redox sensitive enzyme, was shown to have decreased activity following an et al. have shown impairment of oxidative phosphorylation in SHS in exposed rabbit myocardial mitochondria During development, mitochondria serve a major role in providing the energy required for the rapid growth associated with this phase in life and may play key roles in signaling in utero and neonatal exposure to a low dose of SHS increases adult atherosclerosis, and that certain molecular similarities exist between these two forms of developmental exposure. By contrast, differences in the levels of mtDNA deletions exist between in utero and neonatal exposure, suggesting that developmental stage of exposure can influence levels of mtDNA deletions. However, in absence of a longitudinal study to investigate appearance and progression of atherosclerotic lesion development, these current results also suggest that exposure at different developmental stages can yield similar pathologies in terms of atherosclerosis, and overall suggests that a potentially large window exists during childhood in which environmental exposures can significantly influence adult CVD development and susceptibility. Further studies directed at determining when lesions appear and how they progress will shed light on whether differences exist in these parameters between in utero and neonatal SHS exposure. In addition, whether an interaction exists between developmental exposure to SHS and diet (e.g. high fat western diet) remains to be addressed. However, the current study does clearly suggest that both in utero or neonatal exposure to a low dose of SHS increases adult atherosclerosis, oxidative stress, and mitochondrial damage.These studies show that both


The long bones of -/-Arf mice had increased expression of OB genes while -/-Arf OB showed enhanced differentiation in vitro. Mice transgenic for the Tax oncogene develop lymphocytic tumors with associated osteolytic lesions, while Tax+-/-Arf mice uniformly developed spontaneous OS by 7 months of age. Tax+-/-Arf tumors were well differentiated OS characterized by an abundance of new bone with OC recruitment, expressed OB markers and displayed intact levels of p53 mRNA and reduced Rb transcript levels. Cell lines established from OS recapitulated characteristics of the primary tumor, including the expression of mature OB markers and ability to form mineralized tumors when transplanted. Loss of heterozygosity in OS tumors arising in Tax++/-Arf mice emphasized the necessity of ARF-loss in OS development. Hypothesizing that inhibition of ARF-regulated bone remodeling would repress development of OS, we demonstrated that treatment of Tax+-/-Arf mice with zoledronic acid, a bisphosphonate inhibitor of OC activity and repressor of bone turnover, prevented or delayed the onset of OS. These data describe a novel role for ARF as a regulator of bone remodeling through effects on both OB and OC. Finally, these data underscore the potential of targeting bone remodeling as adjuvant therapy or in patients with genetic predispositions to prevent the development of OS.The ARF tumor suppressor regulates p53 as well as basic developmental processes independent of p53, including osteoclast activation, by controlling ribosomal biogenesis. Here we provide evidence that ARF is a master regulator of bone remodeling and osteosarcoma (OS) development in mice.  ARF in humans, p19ARF in mice) is a tumor suppressor and a key sensor of hyperproliferative signals such as those from the Ras and Myc oncoproteins ARF (p14Arf-deficient mice develop undifferentiated sarcomas (rarely osteosarcomas), lymphomas, and carcinomas with a mean life expectancy of ∼9 months Arf and p53 exhibit a tumor spectrum distinct from that observed in single knockout animals, implying that ARF and p53 can act independently to suppress tumor formation Arf-/- transgenic mice affects the timing and characteristics of the tumors that arise. For example, -/-Arf mice transgenic for Eµ-myc developed aggressive B cell lymphomas with a dramatic acceleration in onset and increased mortality when compared to +/+Arf Eµ-myc mice -/-Arf bone marrow resulted in the rapid development of acute myeloid leukemia Arf-deficient mice succumb to blindness soon after birth. In this case, the lack of programmed cell death within the hyaloid vasculature leads to an accumulation of perivascular cells and destruction of the retina and lens Arf deficiency leads to increased osteoclast (OC) function and accelerated differentiation through nucleophosmin, independent of p53 Arf-deficient mice also develop enhanced osteoblast (OB) function and that the co-regulation of OB and OC in the absence of ARF leads to constitutively increased bone remodeling in vivo.ARF possesses activities that are independent of its role as a tumor suppressor. Arf-/- mice with mice expressing the viral oncogene Tax -/-Arf mice (prone to accelerated bone remodeling) with Tax mice  would exacerbate tumorigenesis and tumor mediated osteolysis. Unexpectedly, the Tax+Arf-/- mice became highly susceptible to development of spontaneous osteosarcoma. Osteosarcoma (OS) is the most common primary bone malignancy, with a peak incidence in adolescence, a time of high bone remodeling, and 5-year survival rates in patients with metastatic or relapsed disease of <20% The role of ARF in bone remodeling carries implications for cancer biology. p53, which is regulated by ARF, has also been demonstrated to regulate OB function -/-Arf mice recapitulates the malignancy described in the p53/Rb OB-specific deletion models.p53 mutations and Rb loss is common in human OS -/-Arf mice contributed to OS development and provide evidence that zoledronic acid, a bisphosphonate inhibitor of bone resorption, prevented or delayed onset of OS in Tax+-/-Arf mice. These data demonstrate that ARF regulates bone remodeling through cell autonomous effects on both OB and OC and introduce Tax+-/-Arf mice as a new model of spontaneous OS. This discovery implicates bisphosphonates as novel therapeutics for targeted prevention of OS after resection of primary OS tumors or in patients with genetic cancer predisposition syndromes.We further hypothesized that the increased bone remodeling in -/-Arf mice on a C57BL/6 background +/+Arf, -/-Arf, Tax+ and Tax+-/-Arf mice on a C57BL/6 x FVB mixed background were used. In some experiments, -/-Arf mice were crossed with TAX-LUC mice, a strain with granzyme B dependent Tax expression driving firefly luciferase via the Human T-cell lymphotropic virus type I long terminal repeat promoter, thereby providing a bioluminescent readout of Tax-dependent early tumor development -/-  mice were used for experiments involving tumor allografts. The use of murine models and tissues in this study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Mice were housed under pathogen-free conditions according to the guidelines of the Division of Comparative Medicine and all experiments were approved by the Animal Studies Committee, Washington University School of Medicine under protocols 20100151 (K.N.W.) and 20100026 (L.R.)Femurs were suspended in agarose and femoral metaphyses were scanned by micro–computed tomography  as described previously Early tumor development and myloperoxidase (MPO) activity were monitored by non-invasive bioluminescence imaging of anesthetized mice  following the administration of D-luciferin or luminol as described  in a 2% sodium bicarbonate solution. Mice were labeled 7 days and 2 days prior to sacrifice. Calveria were fixed in 70% ethanol and embedded in methylmethacrylate and sectioned. This labeling allowed for evaluation of bone surface (BS), single labeled surface (sLS) and double labeled surface (dLS). These measurements were then used to calculate bone formation rate (BFR/BS) per year as previously described Serum was isolated from animals and a sandwich ELISA based assay for murine osteocalcin was performed according to manufacturer's instructions .RNA was isolated from in vitro differentiated OB or TAN OS tumor cells using the Qiagen RNeasy mini kit . To isolate RNA from whole bones or whole OS, bone marrow was removed and bones were mechanically crushed with a mortar and pestle and RNA extracted using TRIzol reagent  according to standard protocol. 2 µg of RNA were subjected to DNase I digestion (Invitrogen) and RT-PCR was carried out using SuperScript III First-Strand Synthesis system (Invitrogen) according to manufactures instructions. 100 ng of cDNA template were used for each reaction. Qualitative RT-PCR was performed using RedTaq  and quantitative RT-PCR was performed using iQ SYBR Green Supermix  on a CFX96 Real-Time PCR detection system (Bio-Rad). A list of primers utilized can be found in 5 cells/well were plated into a 24 well plate in plating media. For RNA isolation, 4×105 cells/well were plated into a 6 well plate. The following day or when cells reached confluence, the plating media was replaced with osteogenic media . Cells were supplied fresh media every 7 days for the duration of the assay. At indicated timepoints, cell monolayers were washed with PBS and fixed in 10% neutral buffered formalin. Alkaline Phosphatase staining was carried out with Fast Blue RR salt (Sigma-Aldrich). 0.4% Alizarin Red solution or Von Kossa staining (0.25 g AgNO3 in 5 ml water) was used to detect mineralized matrix.Whole bone marrow was flushed from the femurs and tibias. The resulting BM of one mouse was plated onto one 150 mm tissue-culture coated dish in αMEM media (GIBCO) containing 20% FBS, L-Glutamine, and 1% Pen-Strep (plating media). Non-adherent cells were removed after 2 days. Adherent bone marrow stromal cells were harvested with trypsin/EDTA at 70% confluence. For differentiation assays, 1×10Development of OS was monitored by serial x-ray imaging . Prior to imaging, mice were anesthetized with a cocktail of Ketamine/Xylazine.Bones or OS were fixed in 4% paraformaldehyde and decalcified for 14–21 days in 14% EDTA. Paraffin-embedded sections were stained with H&E, TRAP, or immunohistochemical stains for Tax Tax+ARF null) tumor cell line was isolated from a spontaneous mandibular OS that arose in a Tax+-/-Arf mouse. All soft tissue was removed and the calcified tumor was mechanically digested into small pieces using bone rongeurs and a razor blade under sterile conditions. The small bone fragments were plated onto tissue-culture coated dishes in 10% RPMI-1640 (Sigma) media supplemented with 10% fetal calf serum and 1% Pen-Strep. After 1 week, non-adherent cells and bone fragments were removed. The adherent tumor cells were propagated.The TAN . Alternatively, 1×106 TAN cells in 100 µL PBS were injected in the tail vein and monitored by radiography.Mice were anesthetized and 1×10-/-Arf mice. ZA dosing was continued on a weekly basis until 9 months of age or until the animal was moribund and sacrificed. This dosing schedule of ZA was designed to produce drug levels similar to those achieved with the clinical dosing regimen of 4 mg Zometa® for the treatment of bone metastases. OS development was monitored by manual palpation and confirmed by radiography.ZA  was administered subcutaneously at a dose of 0.75 µg/mouse/week (approx. 30 µg/kg) t-test or one-way ANOVA in the case of experiments with greater than three experimental groups. In calculating two-tailed significance levels for equality of means, equal variances were assumed for the two populations. Kaplan-Meier plots were analyzed by the Log-rank (Mantel-Cox) Test. Results were considered to reach significance at p<0.05 and are indicated with an asterisk (*).All experiments were analyzed using Student's -/-Arf mice have enhanced OC activity through ARF effects on nucleophosmin and ribosome biogenesis p53-/- mice have also been shown to have enhanced OC activity; however, this is attributed to an indirect (non-cell autonomous) increase in OB function resulting from p53 loss -/-Arf and Arf+/+ littermates demonstrated significant increases in bone mineral density, trabecular bone volume and trabecular number and thickness in -/-Arf mice  . Overall, the bone tumors consisted of well-demarcated, nodular, moderately cellular, unencapsulated masses that originated in bone and expanded into and replaced existing bone. The cells at the outer perimeter of the mass that were responsible for the expansile growth of the tumors were OB-like with a polygonal morphology and amphiphilic cytoplasm. The tumors were composed of trabecular bone interspersed with dense spindle cells that had eosinophilic and lightly vacuolated cytoplasm, ovoid nuclei, moderate anisocytosis/anisokaryosis and a low mitotic index  of the remaining Arf allele in all OS tumors tested from Tax+Arf+/- mice (n = 5), two by promoter methylation and three by genetic deletion . The presence of Tax slightly enhanced OB differentiation when compared to wild-type cells and caused a modest increase in Tax+-/-Arf OB differentiation relative to Arf deficiency alone. To identify and characterize the malignant cell population within the OS we established a cell line, herein referred to as TAN (Tax Arf Null), from a primary mandibular OS of Tax-/-+Arf mice. TAN cells were alkaline phosphatase positive , a drug commonly used for the treatment of osteoporosis and to prevent skeletal complications of bone metastasis, is a amino-bisphosphonate that blocks the activity of farnesyl pyrophosphate synthase (FPPS), a key component in the HMG-CoA reductase pathway, which is necessary for prenylation and lipid modification of a variety of proteins including Ras, Rho and Rac -/-+Arf mice with a weekly injection of ZA from one month of age to 9 months of age. This dosing regimen of ZA effectively reduced OC function and resulted in the development of high bone mass osteosclerosis . In Tax mice, early and late stages of lymphoma can be distinguished by the complete blood counts and differential counts with elevated WBC and immature neutrophils present in the blood in late stage disease -/-Arf mice.OS most commonly occurs in adolescent children during times of bone growth and high bone remodeling. We hypothesized that the increased bone remodeling in the Tax+clerosis . In Tax clerosis . In Tax -/-Arf mice. These findings support both p53-dependent and p53-independent actions of ARF on bone remodeling. OC activity is increased in -/-Arf mice in part through effects on NPM mediated ribosomal biogenesis -/-Arf mice in a p53-dependent manner similar to that observed in the -/-p53 mice Bone remodeling is the continuous process of resorption and new bone formation that occurs in a healthy adult at the rate of about 10% per year -/-Arf mice developed OS compared to 0% of animals with either modification alone. Arf-deficient mice rarely develop spontaneous OS Arf was sufficient to promote increased OB activation, proliferation and differentiation, other genetic hits are likely required to promote OB transformation. p14Arf is repressed by promoter methylation in 47% of human OS Arf mutations and modifications are associated with decreased survival in patients with OS The loss of ARF selectively predisposed Tax transgenic mice to development of OS. In our study, 100% of Tax+p53 is the rate-limiting step in initiation of OS, but that Rb loss cooperates in the steps leading to overt malignancy -/-Arf model is comparable to that reported in these mouse models of OS. The loss of ARF compromises p53 function with OB no longer able to activate p53 in response to oncogenic stimuli -/-p53 or Tax++/-p53 mice ARF is a well-established regulator of p53. Mice with OB specific loss of both p53 and Rb also develop spontaneous OS. Osterix-cre transgenic mice with p53 and Rb deletions in OB lineage cells have revealed that loss of -/-Arf mice (approximately 2%) -/-Arf mice on a Tax-null background Click here for additional data file.Figure S1Tax+-/-Arf osteosarcoma tumors arise primarily in jaw and skull bones. The locations of n = 50 OS tumors arising in Tax+-/-Arf mice. The majority of tumors arose in the mandible, often presenting with malocclusion prior to palpable tumor. Other bones of the skull include the frontal, parietal and premaxillary lobes. Maxillary bones contain the upper incisors and upper molars. Tumors occasionally arise in the long bones of the legs, however, these arise much later than the tumors of the skull and jaw.(TIF)Click here for additional data file.Figure S2The expression of Tax did not cause a further significant increase in Tax+-/-Arf osteoblast differentiation relative to Arf deficiency alone. In vitro differentiation of +/+Arf, Tax+, -/-Arf and Tax+-/-Arf OB from primary bone marrow stromal cells under osteogenic conditions (β-glycerophosphate and ascorbic acid). Cells were co-stained for alkaline phosphatase expression (purple) and mineralization (Alizarin Red) at indicated days. Representative of >3 independent experiments. Note that the experiment presented here is the same presented in (TIF)Click here for additional data file.Figure S3Tax+-/-Arf primary osteosarcoma have reduced levels of Rb transcript but intact levels of p53 transcript. Quantitative RT-PCR for Rb (A) and p53 (B) expression in Arf+/+ tumor-free bone (open bars), Tax+-/-Arf tumor-free bone (closed bars) or Tax+-/-Arf primary mandibular OS (hatched bars). N = 3/group. Data is represented as fold change following normalization to cyclophilin levels. Significance values of Rb: Arf+/+ normal bone vs Tax+-/-Arf OS p = 0.0027; Tax+-/-Arf normal bone vs Tax+-/-Arf OS p = 0.0004.(TIF)Click here for additional data file.