6. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. virus-like particles in an RNA-dependent process requiring the NC basic linker. J Virol 81: 50005013. Wang T, Tian C, Zhang W, Luo K, Sarkis PT, et al. 7SL RNA mediates virion packaging of the antiviral cytidine deaminase APOBEC3G. J Virol 81: 1311213124. Svarovskaia ES, Xu H, Mbisa JL, Barr R, Gorelick RJ, et al. Human apolipoprotein B 19464323” mRNA-editing enzyme-catalytic polypeptide-like 3G is incorporated into HIV-1 virions through interactions with viral and nonviral RNAs. J Biol Chem 279: 3582235828. Khan MA, Kao S, Miyagi E, Takeuchi H, Goila-Gaur R, et al. Viral RNA is required for the association of APOBEC3G with human immunodeficiency virus type 1 nucleoprotein complexes. J Virol 79: 58705874. Lecossier D, Bouchonnet F, Clavel F, Hance AJ Hypermutation of HIV-1 DNA in the absence of the Vif protein. Butein price Science 300: 1112. Mangeat B, Turelli P, Caron G, Friedli M, Perrin L, et al. Broad antiretroviral defence by human APOBEC3G through lethal editing of nascent reverse transcripts. Nature 424: 99103. Zhang H, Yang B, Pomerantz RJ, Zhang C, Arunachalam SC, et al. The cytidine deaminase CEM15 induces hypermutation in newly synthesized HIV-1 DNA. Nature 424: 9498. Harris RS, Bishop KN, Sheehy AM, Craig HM, Petersen-Mahrt SK, et al. DNA deamination mediates innate immunity to retroviral infection. Cell 113: 803809. Mariani R, Chen D, Schrofelbauer B, Navarro F, Konig R, et al. SpeciesSpecific Exclusion of APOBEC3G from HIV-1 Virions by Vif. Cell 114: 2131. Yu Q, Konig R, Pillai S, Chiles K, Kearney M, et al. Single-strand specificity of APOBEC3G accounts for minus-strand deamination of the HIV genome. Nat Struct Mol Biol 11: 435442. Suspene R, Sommer P, Henry M, Ferris S, Guetard D, et al. APOBEC3G is a single-stranded DNA cytidine deaminase and functions independently of HIV reverse transcriptase. Nucleic Acids Res 32: 24212429. Guo F, Cen S, Niu M, Saadatmand J, Kleiman L Inhibition of FormulaPrimed Reverse Transcription by Human APOBEC3G during Human Immunodeficiency Virus Type 1 Replication. J Virol 80: 1171011722. Bishop KN, Holmes RK, Malim MH Antiviral potency of APOBEC proteins does not correlate with cytidine deamination. J Virol 80: 84508458. Mbisa JL, Barr R, Thomas 6178174 JA, Vandegraaff N, Dorweiler IJ, et al. Human immunodeficiency virus type 1 cDNAs produced in the presence of APOBEC3G exhibit defects in plus-strand DNA transfer and integration. J Virol 81: 70997110. Luo K, Wang T, Liu B, Tian C, Xiao Z, et al. Cytidine deaminases APOBEC3G and APOBEC3F interact with human immunodeficiency virus type 1 integrase and inhibit proviral DNA formation. J Virol 81: 72387248. Kaiser SM, Emerman M Uracil DNA glycosylase is dispensable for human immunodeficiency virus type 1 replication and does not contribute to the antiviral effects of the cytidine deaminase Apobec3G. J Virol 80: 875882. Schrofelbauer B, Yu Q, Zeitlin SG, Landau NR Human immunodeficiency virus type 1 Vpr induces the degradation of the UNG and SMUG uracilDNA glycosylases. J Virol 79: 1097810987. Yang B, Chen K, Zhang C, Huang S, Zhang H Virion-associated Uracil DNA Glycosylase-2 and Apurinic/Apyrimidinic Endonuclease Are Involved in the Degradation of APOBEC3G-edited Nascent HIV-1 DNA. J Biol Chem 282: 1166711675. Yang Y, Guo F, Cen S, Kleiman L Inhibition of initiation of reverse transcription in HIV-1 by human APOBEC3F. Virology 365: 92100. Luo K, Ehrlich E, Xiao Z, Zhang W, Ketner G, et al. Adenovirus E4orf6 a

cKO mice die within 6 days of birth. Although the cause of death in MFN2 deficient animals is uncertain, they are underweight, lack milk spots and exhibit an unsteady gait. A similar phenotype has been described in mice lacking MFN2 in the cerebellum and, as the Pax2 promoter selected in the present study is also expressed in the mid- and hind-brain, extra-renal loop-out of the MFN2f allele in the central nervous system is get PTK/ZK likely the cause of the early death in our mice. Although Pax2-Cre+/ MFN2f/+ mice survived and exhibited intermediate body weight, highly variable mitochondrial morphology was observed, precluding further studies in Pax2-Cre+/MFN2f/+ mice or cells harvested from their kidneys. As a result, our in vivo model of MFN2deficiency is limited as we are unable to analyze the role of MFN2 in mature collecting duct cells that is only seen in three to four week old mice. We are therefore unable to study the susceptibility of MFN2 cKO to acute or chronic kidney disease. The small blood volume collected from four-day old pups also precludes measurement of creatinine, a second estimate of GFR that could confirm our BUN data. We recognize that MFN1 and MFN2 may have partially redundant functions and while the marked fragmentation 5 January 2012 | Volume 7 | Issue 1 | e31074 MFN2 in Renal Stress observed in MFN2-deficient cells both in vivo and in vitro may suggest that MFN2 is the major mediator of mitochondrial fusion in renal epithelial cells, we have not addressed the role of MFN1 in our studies. The kidney is somewhat unique in that it operates in a relatively hypoxic environment and is therefore remarkably susceptible to ischemic injury. Mitochondrial fragmentation and fusion are recently reported to be involved in both acute and chronic cellular stress responses in the kidney. Renal ischemia-reperfusion injury in vivo causes a marked shift from filamentous to fragmented mitochondria and mice treated with a Drp1 inhibitor, which prevents fragmentation, are protected from ischemia- injury. Similarly, MFN2 over-expression that promoted “2987739 mitochondrial fusion has been suggested to delay the onset of chronic diabetic nephropathy in mice. In keeping with these in vivo data, Hela cells over-expressing MFN1 or MFN2, as well as rat proximal tubule cells expressing dominant negative Drp1, have filamentous mitochondria and are protected from azide or cisplatin-induced apoptosis. In contrast, Mouse embryonic fibroblasts from MFN1 or 2 knockout mice have increased mitochondrial fragmentation and are more prone to injury-induced cell death. In the kidney, we show that mouse proximal tubule cells, a primary target of acute and chronic renal injury, are more susceptible to metabolic stress when MFN2 expression is reduced and mitochondria fragmented. MFN2-deficiency did not affect cell survival at baseline or mitochondrial energetics but markedly increases the leakage of both cytochrome c and AIF from the outer mitochondrial membrane following ATP depletion, demonstrating that MFN2 plays an important role in protecting renal tubule cells from stress-induced apoptosis. Our data suggest that targeting mitochondrial dynamics may be an important therapeutic option to ameliorate tubular cell death following acute or chronic renal insults. How fission and fusion mediate susceptibility to renal cell death is presently “2987731 unclear. Given that BCL proteins regulate mitochondrial injury during in vitro metabolic stress and following ischemia-reperfusion inju

tate, “1446712 major end-products of the human probiotic propionibacteria. Apoptosis 12: 573591. 21. Herve C, Fondrevez M, Cheron A, Barloy-Hubler F, Jan G Transcarboxylase mRNA: a marker which evidences P. freudenreichii survival and metabolic activity during its transit in the human gut. Int J Food Microbiol 113: “2674416 303314. 22. Lan A, Bruneau A, Philippe C, Rochet V, Rouault A, et al. Survival and metabolic activity of selected strains of Propionibacterium freudenreichii in the gastrointestinal tract of human microbiota-associated rats. Br J Nutr 97: 714724. 23. Lan A, Bruneau A, Bensaada M, Philippe C, Bellaud P, et al. Increased induction of apoptosis by Propionibacterium freudenreichii TL133 in colonic mucosal crypts of human microbiota-associated rats treated with 1,2-dimethylhydrazine. Br J Nutr 100: 12511259. 24. Leverrier P, Fremont Y, Rouault A, Boyaval P, Jan G In vitro tolerance to digestive stresses of propionibacteria: influence of food matrices. Food Microbiol 22: 1118. 25. Saxelin M, Lassig A, Karjalainen H, Tynkkynen S, Surakka A, et al. Persistence of probiotic strains in the gastrointestinal tract when administered as capsules, yoghurt, or cheese. Int J Food Microbiol 144: 293300. 26. Thierry A, Deutsch SM, Falentin H, Dalmasso M, Cousin FJ, et al. New insights into physiology and metabolism of Propionibacterium freudenreichii. Int J Food Microbiol 149: 1927. 27. Bortner CD, Oldenburg NBE, Cidlowski JA The role of DNA fragmentation in apoptosis. Trends Cell Biol 5: 2126. 28. Chou TC, Talalay P Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 22: 2755. 29. Cousin FJ, Mater DDG, Foligne B, Jan G Dairy propionibacteria as human probiotics: A review of recent evidence. Dairy Sci Technol 91: 126. 30. Sohn D, EW-7197 Schulze-Osthoff K, Janicke RU Caspase-8 can be activated by interchain proteolysis without receptor-triggered dimerization during druginduced apoptosis. J Biol Chem 280: 52675273. 31. Fischer U, Stroh C, Schulze-Osthoff K Unique and overlapping substrate specificities of caspase-8 and caspase-10. Oncogene 25: 152159. 32. Fulda S Caspase-8 in cancer biology and therapy. Cancer Lett 281: 128133. 33. Hopkins-Donaldson S, Bodmer JL, Bourloud KB, Brognara CB, Tschopp J, et al. Loss of caspase-8 expression in highly malignant human neuroblastoma cells correlates with resistance to tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis. Cancer Res 60: 43154319. 34. Eggert A, Grotzer MA, Zuzak TJ, Wiewrodt BR, Ho R, et al. Resistance to tumor necrosis factor-related apoptosis-inducing ligand -induced apoptosis in neuroblastoma cells correlates with a loss of caspase-8 expression. Cancer Res 61: 13141319. 35. Kaminskyy V, Surova O, Vaculova A, Zhivotovsky B Combined inhibition of DNA methyltransferase and histone deacetylase restores caspase-8 expression and sensitizes SCLC cells to TRAIL. Carcinogenesis. 36. Hinnebusch BF, Meng SF, Wu JT, Archer SY, Hodin RA The effects of short-chain fatty acids on human colon cancer cell phenotype are associated with histone hyperacetylation. J Nutr 132: 10121017. 37. Grunstein M Histone acetylation in chromatin structure and transcription. Nature 389: 349352. 38. Lu Y, Zhang BY, Jia ZX, Wu WJ, Lu ZQ Hepatocellular carcinoma HepG2 cell apoptosis and caspase-8 and Bcl-2 expression induced by injectable seed extract of Coix lacryma-jobi. Hepatobiliary Pancreat Dis Int 10: 303307. 39. Slee EA, Harte MT, Kluc

calves became partially immune after 4 drug-attenuated infections based on pre-defined parasitological and immunological parameters, including a significant reduction in worm burden, an increase in the percentage of larvae and a change in cytokine expression profile. During the final drugattenuated infection, three calves were drug treated and allowed to rest for 34 weeks and then orally dosed with a tap water placebo. These calves were used as controls. The remaining three calves, which also underwent 4 rounds of infection-treatment-resting procedures and allowed to rest 34 weeks between the treatments, were orally infected with a single-dose of 105 L3 infective larvae for 14 days. At the end of the experiment, calves were sacrificed, and the abomasal contents were collected. The abomasal luminal pH was measured using a standard pH meter. The sample was snap frozen in liquid nitrogen prior to storage at 280uC until DNA was extracted. Fecal egg count was monitored during the repeat infection experiment using zinc sulfate double centrifugation, and parasite burdens were determined as previously described. Roche/454 Pyrosequencing The abomasal microbiota was characterized by two sequencing approaches using the Roche/454 GS FLX Titanium chemistry, the 16S rRNA gene and the whole genome shotgun. For the first approach, unidirectional sequencing of amplicon libraries was performed according to the manufacturer’s instructions with a modification. This modification, using a specific fusion primer design, accommodates amplification using the GS FLX Titanium emPCR Kits. Five hundred ng of DNA were used to generate libraries using the GS FLX Titanium Rapid Library Preparation method ” for WGS sequencing. Therefore, emulsion PCR was carried out using a Lib-L kit for both approaches. Pyrosequencing was conducted using a GS FLX Titanium System following the manufacturer’s protocol. Sequence analysis, protein prediction and annotation 16S rDNA raw sequence reads were first decoded based on sample-specific 8-bp bar codes; their quality was checked, and artifacts “2987739 were removed. Sequence reads shorter than 200 bp were excluded. The sequence read that passed the quality filters were analyzed using the RDP classifier at both 80% and 95% confidence threshold levels for Salianic acid A chemical information taxonomic classification and phylogenetic inference. The 16S rDNA sequences were then analyzed using CD-HITOTU for the abomasal microbial composition at the species level. This algorithm uses a greedy incremental clustering process to identify OTU from 16S rDNA tags, which involves 3 major steps: raw read filtering and trimming, selection of error-free reads, and clustering selected representative reads into individual OTU at a user-specific cutoff. The program avoids over estimation of OTU, a common problem for many existing programs, and results in a rapid and more accurate estimation of microbial diversity in complex microbial ecosystems. OTU identified were then annotated using FR-HIT against the GreenGene database. The 16S rDNA sequences were further analyzed using Fast UniFrac. Briefly, the core set of the 16S GreenGene database was downloaded, and the input 16S sequences were analyzed using MegaBLAST. The resultant hit table was input into the Fast UniFrac server for Principal Coordinates Analysis. Several quality control filters were applied to WGS raw reads before analysis. First, host contaminants were removed using FR-HIT against the bovine genome Btau 4.0. The possible artifacts wer

mbo J “1446712 15: 15961606. Schanda P, ” Kupce E, Brutscher B SOFAST-HMQC experiments for recording two-dimensional heteronuclear correlation spectra of proteins within a few seconds. J Biomol NMR 33: 199211. 10 October 2011 | Volume 6 | Issue 10 | e25981 Chromatin Immunoprecipitation: Revisiting the Efficacy of Sample Preparation, Sonication, Quantification of Relebactam Sheared DNA, and Analysis via PCR Pamela D. Schoppee Bortz1, Brian R. Wamhoff1,2 1 Cardiovascular Division, Department of Medicine, University of Virginia Health System, Charlottesville, Virginia, United States of America, 2 Robert M. Berne Cardiovascular Research Center, University of Virginia Health System, Charlottesville, Virginia, United States of America Abstract The “quantitative”ChIP, a tool commonly used to study protein-DNA interactions in cells and tissue, is a difficult assay often plagued with technical error. We present, herein, the process required to merge multiple protocols into a quick, reliable and easy method and an approach to accurately quantify ChIP DNA prior to performing PCR. We demonstrate that high intensity sonication for at least 30 min is required for full cellular disruption and maximum DNA recovery because ChIP lysis buffers fail to lyse formaldehyde-fixed cells. In addition, extracting ChIP DNA with chelex-100 yields samples that are too dilute for evaluation of shearing efficiency or quantification via nanospectrophotometry. However, DNA extracted from the MockChIP supernatant via the phenol-chloroform-isoamyl alcohol method can be used to evaluate DNA shearing efficiency and used as the standard in a fluorescence-based microplate assay. This enabled accurate quantification of DNA in chelex-extracted ChIP samples and normalization to total DNA concentration prior to performing real-time PCR. Thus, a quick ChIP assay that can be completed in nine bench hours over two days has been validated along with a rapid, accurate and repeatable way to quantify ChIP DNA. The resulting rtPCR data more accurately depicts treatment effects on protein-DNA interactions of interest. Citation: Schoppee Bortz PD, Wamhoff BR Chromatin Immunoprecipitation: Revisiting the Efficacy of Sample Preparation, Sonication, Quantification of Sheared DNA, and Analysis via PCR. PLoS ONE 6: e26015. doi:10.1371/journal.pone.0026015 Editor: Yamini Dalal, National Cancer Institute, United States of America Received July 25, 2011; Accepted September 15, 2011; Published October 25, 2011 Copyright: 2011 Schoppee Bortz, Wamhoff. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding for this research was provided by the National Institutes of Health and by an American Heart Association Scientist Development Grant to BRW. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. E-mail: [email protected]; [email protected] Introduction Molecular biologists commonly use the chromatin immunoprecipitation assay is a tool to study protein-DNA interactions in healthy and diseased biological systems. As a result, numerous variations of the original approach to ChIP are present within the peer-reviewed literature and on molecular biology protoco

naive, chronically HIV-infected individuals. The high rate of X4 and D/M tropism seen in this study may be explained at least partly by the fact that most of the samples in this study were derived from MSM individuals. It is noteworthy that the prevalence of X4 and D/M tropism among 7 October 2011 | Volume 6 | Issue 10 | e25869 ~ Analysis of HIV-1 Strains in Sao Paulo, Brazil our MSM group is high compared to the 3.2% reported in the study of 126 recently infected MSM from six major cities in the USA. This high prevalence should seriously be considered when decisions are made about initial regimens for therapy-naive individuals, and HIV-1 coreceptor usage should be screened before initiation of any chemokine receptor CCR5 antagonists in clinical settings. These suggestions are in agreement with the conclusions of Frange et al. that noted that X4/DM strains can heavily fuel the cellular HIV-1 reservoir leading to viral persistence over a long period complicating future therapeutic options, including CCR5 antagonists. The assessment of HIV tropism in our study was limited to sequence- based algorithms rather than using phenotypic methods. Although phenotypic assays still have an edge over genotypic methods, genotypic predictors prove to be highly concordant with phenotype data and can reliably be used to determine viral tropism with better results in PBMC than in plasma samples. In this study, we used geno2pheno because it allows for an 8 October 2011 | Volume 6 | Issue 10 | e25869 ~ Analysis of HIV-1 Strains in Sao Paulo, Brazil Sample ID Resistance mutations “23303071 PI NRTI M41L, D67N, T215Y NNRTI HIV-1 subtype Tropism Risk 04BR 1050 06BR 1123 05BR 1105 04BR 1055 03BR 1020 05BR 1077 05BR 1088 05BR 1075 05BR 1107 03BR 1042 03BR 2018 04BR 1053 04BR 1067 05BR 1111 L24I,M46I,F53L, I54V, V82S D30N, M46I D30N, M46I D30N G73S V82A B B B B B B X4 R5 ND R5 R5 R5 X4 R5 R5 ND R5 X4 R5 X4 MSM MSM MSM MSM MSM MSM HETM MSM MSM MSM HETM MSM HETM MSM V75A M184V M184I M184I M184V K103N K103N K103N K103N K103N B “ 21526763 B B B BF1 B BF1 B HET; heterosexual man, MSM; men who have sex with men. doi:10.1371/journal.pone.0025869.t003 M 9 October 2011 | Volume 6 | Issue 10 | e25869 ~ Analysis of HIV-1 Strains in Sao Paulo, Brazil adjustable cutoff, and it can determine HIV-1 co-receptor usage in all viral genotypes. This method has shown a similar performance to the Trofile phenotypic assay, the most often used tropism method. Moreover, the method has been shown to achieve higher sensitivity while retaining high level of specificity when compared with the performance of different algorithms. Our study represents the largest analysis of the NFLG of the HIV-1 genomes undertaken to date from well-characterized recently infected patients and provides recent data on the molecular characterization of HIV-1 in treatment-naive patients residing in Sao Paulo, Brazil. Overall, our results demonstrate a ~ cocirculation of 3 group M viral subtypes, 3 URFs, and 2 CRFs. However, these data need to be interpreted with some caution, as the samples recruited may not fully represent the general population of Sao Paulo because they disproportionately represent men who ~ reported having sex with men. The existence of various HIV-1 subtypes in Brazil will invariably challenge existing diagnostic tests and/or interpretation algorithms. Depending on future findings related to the transmis- CF-101 sibility, pathogenicity, and treatment implications of various subtypes, these variant subtypes may also play

NA confirms the presence of EGFRvIII. C. Real-time amplification plot showing EGFRvIII-positive OSCC patient and the positive control. doi:10.1371/journal.pone.0031723.g003 Four samples were excluded from the analysis either due to failed amplification of the reference genes or having Ct values greater than 38 for one or both of these reference genes. All other samples successfully amplified both reference genes. Our real time PCR assay revealed that only one patient was positive for EGFRvIII mRNA expression. Retesting of this sample confirmed EGFRvIII positivity. In addition, both direct cDNA sequencing and conventional RTPCR were performed on this sample, however both methods failed outright. This failure may be attributed to formalin fixationinduced degradation and modification of DNA/RNA and further highlights the limitations of conventional methods when using FFPE tissue. We also measured total EGFR protein levels for all samples by quantitative fluorescent immunohistochemistry using the HistoRx AQUAH 9004-82-4 platform . EGFR AQUA scores, representing the concentration of EGFR protein, showed a range of expression from 426 to 1696 in normal oral cavity squamous epithelium with a median score of 1151 and a standard deviation of 6406. We used the median EGFR AQUA score plus one standard deviation as our definition for EGFR over-expression. Using this definition, we found that tumors from 22 of 50 patients over-expressed EGFR. Comparison of EGFR AQUA scores with EGFRvIII expression showed that the tumor sample with the highest EGFR AQUA score was the EGFRvIII-positive case identified by our real-time RT-PCR assay. In order to address tumor heterogeneity, an additional FFPE tumour ” block was randomly selected from 22 patients in the cohort and was tested for EGFRvIII expression. The additional samples included a tumor block from the patient that had tested positive for EGFRvIII. EGFRvIII transcript was not present in any of these additional tumor samples. Furthermore, AQUAnalysisH of the EGFRvIII-negative sample obtained from the single EGFRvIIIpositive patient showed this sample to have significantly lower wild-type EGFR protein expression. Taken together, these results suggest that EGFRvIII expression in OSCC is a rare event and most likely to be present in tumors which express very high levels of EGFR protein. Discussion We have developed a highly sensitive and specific real-time RTPCR assay for EGFRvIII detection. We validated our assay in a cohort of glioblastoma patients and compared its efficiency to conventional PCR and direct sequencing. Furthermore, in light of the conflicting reports regarding the ” frequency of EGFRvIII in HNSCC, we investigated the frequency of EGFRvIII in OSCC, the most prevalent form of HNSCC, using our novel technique. Despite the highly sensitive nature of our assay, we only detected a single EGFRvIII-positive patient in our OSCC cohort of 50 patients. This discrepancy between our novel real-time RTPCR assay results and the reported frequency of EGFRvIII in HNSCC may be due to differing EGFRvIII mutation frequencies in specific HNSCC subsites or selection bias inherent in early phase clinical trials. Patient eligibility for such trials was based on recurrent or metastatic disease status or failure of first-line therapy. Since EGFRvIII-positive tumors are expected to be less responsive to conventional therapies than EGFRvIII-negative tumors, it would be expected that EGFRvIII-positive tumors would be over-represent