GATEWAY-compatible lentiviral tetracycline-regulated RNAi (GLTR) systems. The combination of a modified RNA-polymerase-III-dependent H1 RNA promoter (designated ‘THT’) for conditional shRNA expression with different lentiviral delivery vectors allows (1) the use of fluorescent proteins for colour-coded combinatorial RNAi or for monitoring RNAi induction (pGLTR-FP), (2) selection of transduced cells (pGLTR-S), and (3) the generation of conditional cell lines using a one vector system (pGLTR-X). All three systems were found to be suitable for the analysis of essential genes, such as CDC27, a component of the mitotic ubiquitin ligase APC/C, in cell lines and primary human cells.RNA interference (RNAi) has become an essential technology for functional gene analysis. Its success, however, depends on the effective expression of RNAi-inducing small double-stranded interfering RNA molecules (siRNAs) in target cells. In many cell types, RNAi can be achieved by transfection of chemically synthesised siRNAs, which results in transient knockdown of protein expression. Expression of double-stranded short hairpin RNA (shRNA) provides another means to induce RNAi in cells that are hard to transfect. To facilitate the generation of stable, conditional RNAi cell lines, we have developed novel one- and two-component vector  RNA interference (RNAi) has advanced into an essential tool for functional gene analysis The RNAi pathway can be activated by two means; delivery of synthetic siRNAs, which induces a transient knockdown of protein expression, or by expression of dsRNA precursor molecules that are processed by the cellular RNAi machinery into siRNAs, which results in longer lasting gene knockdown All three systems are widely used for RNAi experiments that include genome-wide loss-of-function screens in selected human cell lines and the establishment of transgenic model organisms for functional gene analysis. The success of an RNAi experiment crucially depends on the choice of the target sequence as well as the efficacy of siRNA expression, which has to be optimised for each cell line and adapted for experimental requirements. Thus, while for certain experiments in some cell lines transient transfection of synthetic siRNAs is the optimal strategy, expression of shRNAs might be more suitable in other circumstances and the best RNAi strategy has often to be determined experimentally. To overcome the limitations of transfection technologies, shRNAs are frequently expressed from viral vectors, including adeno-, retro- and lentiviral vectors, which also allow the generation of stable RNAi cell lines Several different conditional RNAi systems have been developed over the past decade All chemicals were obtained from Sigma , enzymes from Promega  and oligonucleotides from MWG Biotech  or Microsynth AG , unless stated otherwise.5′-CTGCAGGAATTCGAACGCTGACG-3′ and TCTCTATCACTGATAGGGACTTATAAGATTCCCAAATCCAAAG-3′5′-TATAGA to introduce a TetR binding site (underlined) downstream of the TATA box, and subcloning into the episomal expression vector pEPU, a derivative of pCEP-Pu The THT promoter was constructed by first subcloning the H1-RNA gene promoter as a SmaI-HinDIII fragment of pSUPER  into the respective sites of pUHD10-3 , followed by PCR amplification using primers 5′-TATAGGATCCGCCACCATGGCTAGATTAGATAAAAGTAAAGTGATTAACA-3′ and 5′-CCACATCGCCGCAGGTCAGCAGGCTGCCGCGGCCTTCACCACCGCCGTCG-3′ in the first and 5′-TATAGGATCCCCCGGGCCCGGGTTTTCTTCCACATCGCCGCAG-3′ in the second reaction (which introduced a T2A sequence between TetR and eGFP). The PCR product was digested with BamHI and sublconed into pHR-SIN-CSGW upstream of eGFP. After sequencing, a DEST cassette was inserted as described above. For further modifications of pGLTR-X-FP, a KpnI-NdeI fragment containing the ‘NLS-T2A-eGFP’ region was subcloned into KpnI-NdeI digested pUC19, generating pUC19-NLS-T2A-eGFP. The pac gene fragment was PCR amplified from pGLTR-S-PURO with primers 5′ATATACCGGTCGCCACCATGGCCATGACCGAGTACAAG-3′ and 5′- ATATGCGGCCGCTTCAGGCACCGGGCTTGCGGG-3′ and digested with AgeI and NotI to replace the eGFP fragment. In a second step, the NLS-T2A-Puro containing KpnI-NdeI fragment from pUC19-NLS-T2A-Puro was subcloned back into pGLTR-X-FP, resulting in pGLTR-X-PURO. pENTR-THT-CDC27 was created by cloning 5′-end phosphorylated and annealed oligos GCCAGATCCTGACCAAACATTCAAGAGATGTTTGGTCAGGATCTGGCTTTTTGGAAA-3′5′- GATCCCC and 5′- AGCTTTTCCAAAAAgccagatcctgaccaaacatctcttgaatgtttggtcaggatctggcGGG-3′ into BglII-HinDIII digested, dephosphorylated pENTR-THT-I (the sense target sequence is underlined). After isolation of recombinant plasmids, the insert was amplified using primers 5′-CTGGAGGAATTCGAACGCTGACG-3′ and 5′-TGTAAAACGACGGCCAGT-3′, and DNA sequenced on an AB 5500 XL Solid Sequencer . The THT-shRNA expression cassette was subsequently transferred into GLTR vectors using standard LR recombinase reactions (Invitrogen). The retroviral expression vector pLIB-TetR-KRAB-IRES-BLAS was constructed by subcloning TetR-KRAB from pEF-TetR-KRAB 5′-TATAAGATCTGGATCCACCATGGCTAGATTAGATAAAAGTAAAGTG-3′ and 5′-TATAGATATCTCAGGCACCGGGCTTG-3′. All described plasmids are deposited at the plasmid distribution platform Addgene (www. addgene.com).The lentiviral GATEWAY destination vector pHR-DEST-GFP ( = pGLTR-FP-GFP) was generated by inserting a DEST cassette into the blunt-ended EcoRI site of pHR-SIN-CSGW . Plasmid pHR-DEST-dtTOMATO ( = pGLTR-FP-RFP) was made by exchanging the GFP cassette in pHR-DEST-GFP with that for dtTOMATO . The selectable lentiviral construct pHR-DEST-PURO ( = pGLTR-S-PURO) was constructed by exchanging GFP with the puromycin N-acetyl transferase (pac) gene U2OS (ATTC:HTB-96), HEK293T, HEK293A  and the PHOENIX amphotropic retroviral packaging cell line Retro/lentiviral infection of target cells was performed as described previously Total cell lysates were prepared by lysing 10∧6 cells in 100 µl SDS-sample buffer containing 5% 2β-mercaptoethanol. After boiling, 10 µl were separated by denaturing gel electrophoresis transferred to nitrocellulose membrane using semi-dry electroblotting and analysed by using antibodies against CDC27 , α-tubulin , BIM , or GAPDH  followed by enhanced chemiluminescence using HRP-conjugated secondary antibodies.Life cell images were taken on inverse Zeiss Axiovert 200M  or Olympus IX70  microscopes using 20x and 40x objectives. For sorting, 10∧6 PREB697/EU3 cells were infected simultaneously with lentiviral vectors expressing shRNA targeting Bim To develop a conditional RNAi system, we constructed the THT-promoter by flanking the human H1-RNA gene promoter with Tet operator sequences, i.e., binding sites for bacterial TetR. First, a heptamerised Tet-operator was introduced upstream of the H1-promoter, which was then further modified by substituting the spacer between the TATA box and the transcription start site with a single TetR binding site . These mThe unmodified H1- as well as the THT-promoters were then subcloned into GATEWAY ENTR vectors to obtain pENTR-H1 (kanamycin resistance), pENTR-THT (kanamycin) as well as pENTR-THT-III (gentamycin) as shown in To evaluate the conditional RNAi system, we chose to target CDC27 (APC3), a subunit of the essential mitotic ubiquitin ligase anaphase promoting complex/cyclosome (APC/C) GATEWAY-compatible lentiviral tetracycline-regulated RNAi  (pGLTR-FP) for tracking transduced cells, for colour coded combinatorial RNAi and to have a proxy for RNAi induction in cells expressing TetR-KRAB. The second type of vectors was created to allow selection of transduced cells (pGLTR-S), while the third system was designed as a one vector approach for GFP labelling or puromycin resistance and conditional RNAi .Because RNAi experiments can require different gene delivery or expression strategies, we constructed three different types of vectors for Ai GLTR, . The firTo test the functionality of our two-vector system (pGLTR-FP and pHR-SFFV-TetR-KRAB-IRES-PURO), we used GATEWAY-recombination to generate pGLTR-FP-GFP-CDC27, in which GFP expression is controlled by the constitutive SFFV-promoter and CDC27 shRNA by the conditional THT promoter. Since TetR-KRAB is known to silence genes within up to 10 kb of its binding site To test this prediction, we first generated U2OS cells constitutively expressing TetR-KRAB and then transduced these cells with lentiviral pGLTR-FP-GFP-CDC27 particles. As can be seen in The efficacy of RNAi is dose-dependent and efficient lentiviral expression of shRNAs may, therefore, require multiple viral integrations, which can be achieved by high lentiviral titres or multiple rounds of infection. In both cases, selection of transduced cells might be required to establish efficient RNAi clones. To establish conditional and selectable RNAi, we investigated the use of TetR as a specific regulator of THT-promoter dependent shRNA gene expression that would not affect the expression of adjacent genes.First, we generated U2OS cells constitutively expressing TetR by lentiviral infection and selection for Blasticidin S resistance. Resistant U2OS-TetR cells were then superinfected with pGLTR-FP-GFP-CDC27 and cultured in the presence of increasing amounts of doxycycline. In these cells, GFP expression was constitutive , while CTo demonstrate that the above described pGLTR-FP vectors can be used for flow cytometry-based enrichment, we chose the hard to transfect preB leukemic cell line PREB697/EU3. Since one round of infection is often insufficient for efficient gene knockdown in this cell line (unpublished observations), we generated the lentiviral vector pGLTR-FP-RFP by exchanging the GFP marker gene of pGLTR-GFP with the gene for red fluorescent dtTOMATO To evaluate dual colour-coded RNAi, we infected PREB697/EU3 cells with lentiviral RNAi vectors that target the pro-apoptotic BH3-only protein BIM The above described conditional RNAi systems are based on two components, the THT-shRNA expression cassette and a tetracycline-dependent repressor, each encoded by separate viral vectors (two-vector system). To overcome the need for sequential or co-infection of target cells, we designed pGLTR-X, which contains a GATEWAY-DEST cassette for uptake of the THT-shRNA gene and an expression cassette for a TetR variant with a C-terminal nuclear localisation signal (NLS) followed by a T2A sequence fused to eGFP driven by the constitutively active SFFV promoter. During translation, the T2A sequence induces ribosomal ‘skipping’ that causes stop codon independent peptide release and re-initiation of translation at the T2A site To examine whether our single vector system was sufficient for the generation of conditional RNAi cell lines, we transduced U2OS cells with pGLTR-X-GFP-CDC27 and analysed CDC27 levels upon doxycycline treatment. Similar to the two vector system, CDC27 levels were efficiently reduced in a time- and dose-dependent manner . As expeNext, we exchanged the GFP cassette with a puromycin resistance sequence to enable selection and enrichment of infected cells. Infection of U2OS cells with pGLTR-X-PURO-CDC27 and induction with doxycycline resulted in mitotic arrest  and CDC2The success of RNAi experiments critically depends on the expression levels of shRNAs or siRNAs, respectively. Too low expression levels may result in difficult-to-interpret hypomorphic phenotypes, while too high levels can interfere with the processing of endogenous small non coding RNAs, such as miRNAs To satisfy these requirements, we generated a novel modular RNAi system for stable and conditional RNAi. This system uses GATEWAY recombination-mediated transfer of a conditional promoter for shRNA expression into a set of novel lentiviral delivery vectors that can be used for establishing stable RNAi cell lines, combinatorial RNAi as well as conditional RNAi. To achieve conditional RNAi, we generated an H1-RNA gene derived promoter, THT, which enables conditional expression of transduced shRNAs in target cells expressing tetracycline-sensitive repressors, such as TetR-KRAB or TetR. The comparison of TetR-KRAB and TetR regulated shRNA expression suggested that both molecules are equally efficient to tightly control the induction of RNAi by repressing the activity of the THT promoter. Because TetR-KRAB induces silencing by recruiting HDACs to the THT promoter it can be used to monitor the induction of RNAi in cells transduced with lentiviral pGLTR-FP vectors. However, the use of TetR-KRAB has to be considered carefully because lentiviral integration is random and TetR-KRAB might also silence genes near the viral integration site Due to the spreading silencing effect of the KRAB domain, a selection gene to enrich for transduced cells can also not be used together with TetR-KRAB. In contrast, TetR, which acts by steric hindrance of RNA-polymerase-III-dependent shRNA transcription, effectively represses the THT promoter and allows the selection or enrichment of transduced cells if used in combination with pGLTR-S or pGLTR-FP vectors, respectively. Of course, all pGLTR-FP and pGLTR-S vectors can be used for constitutive, long-term RNAi experiments. The generation of pGLTR-FP vectors encoding different FACS-compatible fluorescence marker  further increases their applicability in hard to subclone or transfect cells or to enable combinatorial RNAi.To facilitate the establishment of conditional RNAi cell lines in primary cells, which have a limited capacity to proliferate in vitro, we established a one vector system, which combines GFP labelling or puromycin resistance with TetR-based conditional RNAi. This approach enabled the generation of conditional RNAi in primary HUVEC cells after a single round of infection using pGLTR-X encoded viral particles. Since lentiviral particles can transduce non-proliferating cells, this system might be useful for any kind of conditional RNAi experiments in primary cells.


Homologous recombination (HR)-mediated repair of DNA double-strand break (DSB)s is restricted to the post-replicative phases of the cell cycle. Initiation of HR in the G1 phase blocks non-homologous end joining (NHEJ) impairing DSB repair. Completion of HR in G1 cells can lead to the loss-of-heterozygosity (LOH), which is potentially carcinogenic. We conducted a gain-of-function screen to identify miRNAs that regulate HR-mediated DSB repair, and of these miRNAs, miR-1255b, miR-148b*, and miR-193b* specifically suppress the HR-pathway in the G1 phase. These miRNAs target the transcripts of HR factors, BRCA1, BRCA2, and RAD51, and inhibiting miR-1255b, miR-148b*, and miR-193b* increases expression of BRCA1/BRCA2/RAD51 specifically in the G1-phase leading to impaired DSB repair. Depletion of CtIP, a BRCA1-associated DNA end resection protein, rescues this phenotype. Furthermore, deletion of miR-1255b, miR-148b*, and miR-193b* in independent cohorts of ovarian tumors correlates with significant increase in LOH events/chromosomal aberrations and BRCA1 expression.DOI:http://dx.doi.org/10.7554/eLife.02445.001 The DNA in a cell is damaged thousands of times every day. One of the most serious types of damage involves something breaking both of the strands in the double helix. Such a double-strand break can delete genes or even kill the cell. In fact, conventional cancer therapy kills cancer cells by causing irreparable double-strand breaks. Conversely, a normal cell that is constantly exposed to DNA damaging agents can become a tumor if double-strand breaks are incorrectly repaired. An efficient and accurate double-strand break repair system needs to be in place to prevent this transformation. Therefore, an in-depth understanding of double-strand break repair and the factors involved are important for both gaining insight into the cause of cancer and to improve cancer therapy.Cells have evolved several different ways to detect and repair double-strand breaks. A method called homologous recombination, for example, uses an undamaged DNA molecule as a template that can be copied to make new DNA. Since it needs a readily available DNA template, this method only works in phases of the cell growth cycle where there are many copies of DNA—that is, in the post-DNA replication phases. In particular, homologous recombination does not work during the pre-replication, G1 phase. If homologous recombination is attempted during G1, it will block the other methods employed by cells to repair broken strands of DNA.An important challenge is to understand how homologous recombination is restricted to particular parts of the cell cycle. Although certain proteins associated with the early stages of double-strand repair are thought to determine the type of DNA repair that occurs, the details of this process are not fully understood.One group of molecules that are thought to be involved are microRNAs, which normally limit the number of proteins produced from certain genes. However, since a single microRNA molecule can be associated with several proteins, and since a single protein can be associated with several microRNA molecules, it has proved difficult to establish the exact effects of a specific microRNA molecule.Choi et al. now show that seven microRNA molecules can control homologous recombination, and three microRNAs in particular restrict homologous recombination during the G1 phase of the cell cycle. If these microRNAs are inhibited during the G1 phase, which allows homologous recombination to start, and counter-intuitively more double-stranded breaks are seen. However, if a gene involved in starting homologous repair–called CtIP—is silenced while the microRNAs are inhibited, then the DNA breaks are repaired. Exactly, how the microRNA molecules produce different effects during different phases of the cell cycle will be need to be investigated by future studies.DOI:http://dx.doi.org/10.7554/eLife.02445.002 Double-stranded DNA break (DSB)s are deleterious for cell health and accurate repair of DSBs is critical for maintaining genome stability. Two major mechanistically distinct pathways, homologous recombination (HR) and non-homologous end joining (NHEJ) have evolved to repair DSBs . HR requThe molecular mechanism of restricting HR to S/G2 phases of the cell cycle is of paramount importance in cancer biology. Ectopic HR leading to LOH may play a critical role in carcinogenesis as recessive oncogenic mutations are revealed and/or tumor suppressor function is lost . The regMicroRNAs (miRNAs) are abundant small (∼20–22 nts) non-coding RNAs that typically dampen gene expression at the post-transcriptional level  and are In this study, we used PARP inhibitor sensitivity as a marker for HR deficiency to conduct a functional screen to identify miRNAs that down-regulate HR. Over-expression of seven miRNAs significantly reduced HR-mediated DSB repair. Based on their expression in a panel of breast and ovarian lines, we focused on characterizing the mechanism and physiological relevance of miR-1255b, miR-193b*, and miR-148b*. Despite lacking canonical binding sites, miR-1255b, miR-193b*, and miR-148b* associate with the BRCA1, BRCA2, and RAD51 transcripts and regulate their expression. Remarkably, the miRNA-mediated regulation of these genes is cell cycle dependent and inhibition of miR-1255b, miR-193b*, and miR-148b* leads to enhanced expression of BRCA1, BRCA2, and RAD51 specifically in the G1 phase. A functional consequence of inhibiting these miRNAs is a basal increase in DSBs in G1 cells. This phenotype can be reversed by silencing CtIP (the BRCA1-associated DNA end resection factor) which initiates HR-mediated DSB repair. Furthermore, deletion of these miRNAs in two independent cohorts of high-grade serous ovarian tumors correlates with increase in LOH.Z-score thresholds [Z < −2]). Prior to individual validation experiments, we utilized the results from the screen performed with the Qiagen library to confirm the impact of these miRNAs on ABT888 sensitivity. Our results show that 12 of the 13 miRNAs had at least a 25% impact on ABT888 sensitivity. Next, we confirmed the impact of these miRNAs on PARP inhibitor sensitivity using two clinical PARP inhibitors, ABT888 and olaparib . miRNA mimics are 20–22 nt, chemically modified double-stranded RNA molecules designed to mimic endogenous mature miRNAs. The miRNA mimics from Qiagen and Applied Biosystems have proprietary chemical modifications that cause inactivation of the ‘passenger’ strand, allowing the ‘active’ strand to associate with target transcripts and regulate gene expression. The goal was to identify miRNA mimics that sensitize breast tumor cells, MDA-MB231 to the clinical PARP inhibitor, ABT888 . The miRolaparib  in two bolaparib . TogetheSensitivity to PARP inhibitors directly correlates with HR deficiency, but it is plausible that other pathways may also contribute to this phenotype. To test whether expression of the 12 selected miRNAs impacts HR, we used a functional assay that assesses HR-mediated repair of an I-SceI-induced DSB . U2OS ceγ-H2AX formation is a marker for DSBs, and PARP inhibitor treatment of BRCA-deficient cells cause accumulation of γ-H2AX . To asseAll our results so far have been derived by artificially introducing miRNAs in cells. To explore the physiological relevance of miR-1231, miR-1255b, miR-148b*, miR-876-3p, miR-221*, miR-193b*, and miR-185*, we first identified cells where these miRNAs are endogenously expressed. From the perspective of cancer biology, the HR pathway is extremely relevant for breast tumors, specifically triple negative breast cancer (TNBC) , and cliWe rationalized that the 3 miRNAs identified in the TNBC lines above  are likely to influence HR by regulating the expression of genes involved in the DNA damage response (DDR). We used a two-pronged approach to identify these genes. First, we used a collection of target prediction algorithms to identify genes targeted by these 3 miRNAs and compared it to the list of DDR proteins . Second, we used a candidate-based approach using the prediction algorithm RNA22 to screen all the genes implicated in DDR for miRNA recognition element of miR-1255b, miR-148b*, and miR-193b*. This gave us a list of 18 proteins which have been previously implicated in HR-mediated repair of DSBs. We then assessed the impact of over-expressing miR-1255b, miR-148b*, and miR-193b* on the mRNA level of these 18 genes . There wmiRNAs typically suppress gene expression by direct association with target transcripts. To test for association of miR-1255b, miR-148b*, and miR-193b* with their respective target transcripts, we adopted a recently described method for capturing miRNA–mRNA complexes using streptavidin-coated beads from cells transfected with biotinylated forms of the miRNA mimics . The amoWe next scanned the BRCA1, BRCA2, and RAD51 transcripts for miR-1255b, miR-148b*or miR-193b* binding sites or miRNA recognition element (MRE)s. Canonical MREs are predicted based on the seed rule, that is, the target site within 3′ UTR forms Watson–Crick pairs with bases at positions 2 through 7 or 8 of the 5′ end of the miRNA . HoweverTo verify further that BRCA1, BRCA2, and RAD51 are targets of miR-1255b, miR-148b*, and miR-193b* and to confirm that the interaction is mediated by the predicted MREs, we used the luciferase reporter assay which is a surrogate for target protein. The MREs were cloned in the 3′UTR of the luciferase gene, and expression monitored in cells transfected with mimics for miR-1255b, miR-193b*, and miR-148b*. As antiThe luciferase reporter assays with MREs provide important information regarding the miRNA/mRNA association but have limited physiological relevance. To determine the functional significance of non-canonical MREs in the BRCA1, BRCA2, and RAD51 transcripts we generated expression constructs without the MREs by either deletion (MREs in 3′UTR) or mutation (MREs in CDS) of them. Next, MDA-MB231 cells were co-transfected with (i) miR-1255b and BRCA1 or BRCA2 expression plasmid lacking miR-1255b binding sites; (ii) miR-193b* and BRCA1 or BRCA2 or RAD51 expression plasmid lacking miR-193b* binding sites; (iii) miR-148b* and a RAD51 expression plasmid lacking miR-148b* binding sites. First, the BRCA1, BRCA2, and RAD51 expression constructs lacking the specific MREs completely restored the expression of these genes in the presence of the corresponding miRNA mimic further validating the predicted MREs  utilizes cell cycle-dependent degradation of Cdt1 and Geminin to mark G1 and S/G2/M cells by fusing the red (mKO2) and green (mAG) fluorescent proteins to Cdt1 and Geminin, respectively (illustrated in RPA2 foci mark the presence of single-strand DNA (ssDNA), a consequence of end resection. Consistent with resection of DNA ends, and production of ssDNA, we observe a significant increase in RPA2 foci in the G1 phase of cells transfected with antagomirs for miR-1255b, miR-193b* and miR-148b* and again the depletion of CtIP negates the formation of RPA2 foci in these cells . To furtThe two major mechanisms proposed to cause LOH are deletion of chromosomal fragments/chromosome loss and mitotic recombination between homologous alleles . MitoticWe compared the number of LOH events for a cohort (417 tumors) of high-grade serous ovarian tumors that have deletions of miR-1255b, miR-148b*, and miR-193b* and those that are copy neutral at those loci. We observed that the number of LOH events is significantly higher in tumors with deletions of miR-148b* and miR-193b* relative to the tumors that are diploid copy neutral at these loci. The number of LOH events was also moderately higher  in tumors with deletion of miR-1225b compared to the copy neutral ones . It is nTo validate our analysis in a second cohort, we obtained processed LOH and somatic copy number alteration data for 47 high-grade serous ovarian tumors, which had been processed at Dana-Farber/Harvard Cancer Center (DF/HCC). The details of these samples have been previously described . We compContrary to other cellular processes and signaling pathways, there has been limited investigation of miRNAs in DSB repair . In the The choice of DSB repair pathway is critical for maintaining genomic stability . SpecifiBRCA1 MRE1 and BRCA2 MRE2 located in the gene coding sequence were mutated by site-directed mutagenesis. The primers used are as follows:BRCA1 MRE1-1-US, AAGTTTCTATCAGGCAAAGTATGGGCT, BR1_Res1-1-DS, AGCCCATACTTTGCCTGATAGAAACTT; BR1_Res1-2-US, CTAAAAGATGAAGAAACTATCAGGCAAA, BR1_Res1-2-DS, TTTGCCTGATAGTTTCTTCATCTTTTAG; BR2_Res2-US, AAATAGTCATATAAGGGCTCAGATGTTATTT, BR2_Res2-DS, AAATAACATCTGAGCCCTTATATGACTATTT.miRNA screen was carried out in triplicates using miRNA mimic libraries from Applied Biosystems  and Qiagen , both the libraries were based on miRbase 14. MDA-MB231 cells were plated on 384-well plate at 1000 cells/well density and reverse transfected with 50 nM control miRNA, miRNAs, or BRCA2 siRNA using Lipofectamine RNAiMax transfection reagent before being treated with 20 µM ABT888 next day. In 3 days, cells were subjected to viability test using Celltiter Glo . The average luminescence and Z-score was calculated. % viability control was calculated for each miRNA and the cut off was set at 75% control viability and Z-score of positive control, BRCA2 siRNA. Z-score was calculated by the following equation where x is luminescence value for each miRNA, μ is negative control mean, and σ is negative control standard deviation: z = (x−μ)/σ.Clonogenic assays and colorimetric assays with MDAMB231 or 21NT cells treated with PARP inhibitors were done as previously described . We modiHR assay was performed as described in Immunofluorescence in MDAMB231 cells were done as previously described Total RNA was prepared and expression was analyzed by qRT-PCR as described in Mre11A-F, TCTGCCTTTAGTGCTGAT GAC, R, GCTCTTCCTCTTTGAGACCC;RAD50-F, GTGCGGAGTTTTGGAATAGAG, R, GAGCAACCTTGGGATCGTGT;NBS1-F, CACTCACCTTGTCATGGTATCA, R, CCGTCCTGACAGATCAACATT;ATM-F, AAGGCTATTCAGTGT GCGAG, R, GGCTCCTTTCGGATGATGGA;ATR-F, CCAGCATTCTCCAGGTGACA, R, AGCCAGCATTCTCCAACCA T;ATRX-F, CAAGGTCTGCAAAGAAAGCAG, R, TGGAATCATCATTTTCATCTTCC;CtIP-F, GGCTTATGTGATCG CTGTGC, R, ACAGCATCAAGCAGCTGAGC;RPA2-F, TTCACAGGTCACTAT TGTGGG, R, GAACAAAAAGA GCCTGGTAGC;TOPBP1-F, TTTCCGTGCTGTGG TCTCAC, R, GAAACTCCAGGAC GTCCCAG;Abraxas-F, TGCA GGAGCATTTTTCAAACC, R, GTATGTCCACTGGTTTTAGCC;RAD52-F, TA CAATGGCTGGGCACACT C, R, TCCTTCCTTGCCTTCTCCAA;MDC1-F, GAGACATCTGAGGAAATACAAG, R, TCTTTCTGGTAGCAGTTTCTCA;BRCA1-F, AGGCAACTTATTGCAGTGTGG, R, ACTTTTCTGGATGCCTCTCAG;EXO1-F, AGCCAAAGGTGAACCTACTGA, R, AGCTTCCGAGACTTTCCCCT;RAD51-F, GGGTCGAGGTGAGCTTTCAG, R, GG GCGATGATATTTCCTCCA;BRCA2-F, GCCAAGTCATGCCACACATT, R, TGTGC CATCTGGAGTGCTT T;PALB2-F, TTGTTTGTCTCAGCAGGATCTC, R, CTTGGGTGTCATCTGTTCTTT G;BRIP1-F, ATGAACC AAGGAACTTCACGTC, R, CTGCTGTGTAGTTTCTAAGGGTC.Gene-specific primers used for qRT-PCR are as follows:We adopted unbiased target prediction algorithms for miRNA-1255b, miRNA-193b*, and miRNA-148b* and each algorithm predicted a list of targets as follows:Comparative target prediction analysis for miRNA-1255b, miRNA-193b*, and miRNA-148b*:http://sciencepark.mdanderson.org/labs/wood/dna_repair_genes.html#Human%20DNA%20Repair%20Genes; http://cm.jefferson.edu/rna22v2/) (http://cbcsrv.watson.ibm.com/rna22.html) and PITA (http://genie.weizmann.ac.il/pubs/mir07/mir07_data.html), to analyze the Human DNA Repair Gene list that resulted in a list of DDR genes predicted as targets of miRNAs of our interest. 18 of them are implicated in HR-mediated repair and they were examined in The prediction list was compared with an updated list of Human DNA Repair Genes (The immunoblots were done as described previously  with BRC6 cells/well on 6 well-plate overnight and transfected with biotinylated control miRNA (C. elegans miRNA [Bi-cel-miR-67]) or biotinylated miR-1255b, miR-193b*, or miR-148b* (Sigma). The cells were harvested 6 hr after transfection in 700 µl lysis buffer (20 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 0.3% IGEPAL CA-630) containing freshly added 300U RNaseOUT  and Protease Inhibitor Cocktail  and incubated on ice for 20 min. After centrifugation for 15 min at 10000×g, 4°C, a 50-ml aliquot of supernatant was taken as input for subsequent RNA extraction. The remaining supernatant was incubated with activated Streptavidin-Dyna beads  for overnight at 4°C. Reaction mixture was centrifugated to remove unbound material. The beads were washed five times with lysis buffer and treated with 10U DNase I in lysis buffer for 10 min at 37°C. The beads were washed and treated with 50 µg/100 µl Protease K in 10% SDS containing lysis buffer for 20 min at 55°C. After centrifugation, the supernatant was taken for RNA extraction using acid phenol-choloroform (Applied Biosytems). RNA was subjected to qRT-PCR using gene specific primers. The analysis was done as follows: miRNA pull-down/control pull-down (‘A’), miRNA input/control input (‘B’); fold enrichment = A/B.MDAMB231 cells were plated at 0.3 × 10Renilla luciferase. Luciferase assay in MDAMB231 cells using WT and Mt MRE constructs was done as described previously  or mutant (Mt) miRNA recognition elements (MREs) of target genes were synthesized as oligo sequences, annealed and cloned in psiCHECK2 (Promega) downstream to eviously . The oliMt BRCA1 MRE1+2+3-F, TCGATTGATGAAGAA ACGAGCAGGCAGAAGTGGTA CAAGAGGCGAGCAGGCTGAAGCCTGGTGAGGAAACGAGTAGGC, R,GGCCG CCTACTCGTTTCCTCACCAGGCTTCAGCCTGCTCGCCTCTTGTACCACTTCTGCCTGCTCGTTTCTTCATCAA;Mt BRCA2 MRE1-F, TCGACAATGAACAAAATGGGCAGGCA,R, GGCCTGCCTGCCCATTTTGTTCATTG;Mt BRCA1 MRE4+5+6-F, TCGAAGTGAGCAGGAGTTTTAAAGCCGAAGGGTAG AGCGAGAGGGAACCCCTGAAGCAGTCGA ACAGGGTTTTAGGGC, R,GGCCGC;CCTAAAACCCTGTTCGACTGCTTCAGGGGTTCCCTCTCGCTCTACCCTTCGGC TTTAAAACTCCTGCTCACT;Mt BRCA2 MRE2-F, TCGAGTAAAATAGGCATTTTAGGGCT, R, GGCCAGCCCT AAA ATGCCTATTTTAC;Mt RAD51 MRE1-F, TCGATAGGGCAAGGAGTTTTAAGGCC, R, GGCCGGCCTT AAAACTCCT TGCCCTA;Mt RAD51 MRE2+3-F, TCGATCATATGTTGCCTTGAGAACTAGAAGACTCTACA TATTATCCTGAACT, R, GGCCAGTT CAGGATAATATGTAGAGTCTTCTAGTTC TCAAGGCAACATATGA.The oligonucleotides for mutant MREs are as follows:6 cells/well and treated with 500 µM mimosine for 24 hr. The cells were washed and released into growth media and collected for FACS analysis and RNA extraction after indicated time intervals. For FACS, the cell were fixed in 70% ethanol, washed with PBS buffer, and analyzed in PI/RNase staining buffer . MDAMB231 cells transfected with miRNA antagomirs were similarly synchronized 48 hr after transfection. The siRNA for CtIP was obtained from Dharmacon, Pittsburgh, PA (#J-011376-06).MDAMB231 or MCF10A cells were seeded at 0.2 × 10We obtained processed LOH and copy number calls for serous ovarian cancer samples from TCGA (2011) . In brieThe processed LOH calls and per SNP copy number calls were previously described (GSE39130) . We focu review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  Criteria for selection of positives in the screenIn the first paragraph of the Results section, the authors claim “we screened two independently synthesized commercially available miRNA mimic libraries and focused on overlapping 'hits'.” Having said that, it is not entirely clear what criteria was used to select those miRNAs that scored positively in this screen.not selected?The authors selected miR1255b, miR-148b* and miR-193b* for further studies. Maybe, I am missing something here, but why were miR-185*, miR-367*, and miR-1204 Also, why were miRNAs that caused a loss of viability equal to BRCA2 siRNA were not considered?2) In 3) In 4) The problem of HR silencing during G1 is not solved here but pushed back one step: i.e., there is no information as to how the levels of miRNAs are regulated in a cell cycle dependent manner.5) The manuscript itself meanders around such that the reader can lose interest.6) Much of the data is borderline in quality: i.e., affinity pull downs, luciferase assays, etc.7) In mRNAs apparently regulated by more than one miRNA, why wasn't the combined effect measured?8) Many of the experiments would have been much more convincing if done in synchronized or FACS-sorted cells. We thank the reviewers for their constructive and insightful suggestions and comments. Addressing these comments has significantly improved the manuscript.Specific comments:1) Criteria for selection of positivesin the screenIn the,first paragraph of the Results section, the authors claim “we screened two independently synthesized commercially available miRNA mimic libraries and focused on overlapping 'hits'.” Having said that, it is not entirely clear what criteria was used to select those miRNAs that scored positive in this screen.First of all, I would like to apologize for the confusion regarding the screen and our criterion for selecting specific miRNAs. We have completely re-written this section to clarify these issues. To systematically identify miRNAs that impact PARP inhibitor sensitivity we screened two commercially available miRNA mimic libraries (Qiagen and Applied Biosytems). MiRNA mimics are 20-22 nts, chemically modified double-stranded RNA molecules designed to mimic endogenous mature miRNAs. The miRNA mimics from Qiagen and Applied Biosystems have proprietary chemical modifications that cause inactivation of the ‘passenger’ strand, allowing the ‘active’ strand to associate with target transcripts and regulate gene expression. We used the Applied Biosytems library as our primary screen, and this yielded a rank ordered list of the top 13 miRNAs based on viability percentage that sensitized cells to PARP inhibitor. Prior to individual validation experiments we utilized the results from the screen performed with the Qiagen library to confirm the impact of these miRNAs on ABT888 sensitivity. Basically the screen with the Qiagen library was used to validate the ‘hits’ from the Applied Biosystems library, before we did any focused experiments.Also,why were miRNAs that caused a loss of viability equal to BRCA2 siRNA were not considered?BRCA2 siRNA served as a positive control for each plate as the original discovery of PARP inhibitors was made using BRCA2 mutant cells. Depletion of BRCA2 dramatically sensitizes cells to PARP inhibitors. However, BRCA2 is an important gene for repair of replication-induced DNA damage and depletion of BRCA2 has an impact on viability even in the absence of PARP inhibitors . Our goaThe authors selected miR1255b, miR-148b* and miR-193b* for further studies. Maybe, I am missing something here, but why were miR-185*, miR-367* and miR-1204 not selected?The results in 2) In, it would be important to include a few miRNAs that scored negatively in their screen.The rationale for Figure3A was not clearly explained in the original version of the manuscript, and we suspect that this comment may be due to this lack of clarity on our part. ∼880 miRNAs scored ‘negatively’ in our screen. The expression level of a large majority of these miRNAs is not known in TNBC lines. I’m not sure what criterion we would utilize to select the ‘negative’ miRNAs and look at their expression in TNBC lines, and more importantly how that would add to our story. However, we did take the reviewers’ comment in mind to further substantiate this figure. Of the seven miRNAs we initially selected based on impact on HR-mediated DSB repair, miR-1231, miR-876-3p, miR-221*, and miR-185* were not expressed in the TNBC lines. However, it is important to show that our detection method is valid for these miRNAs, and their lack of expression in TNBC lines is not an artifact. Therefore we have included data showing that these miRNAs are expressed in other cell lines.3) In, it would be important to show the luciferase assays with the wild-type 3'UTR in the presence of an inhibitor of the miRNA under study (e.g. miR-1255b on the upper panel).We thank the reviewers for this suggestion, and have conducted the luciferase assays in the presence of the inhibitors . Consist4) The problem of HR silencing during G1 is not solved here but pushed back one step: i.e., there is no information as to how the levels of miRNAs are regulated in a cell cycle dependent manner.We completely agree with the reviewers and we have mentioned this point in the Discussion. Looking at how the miRNAs are regulated in a cell cycle dependent is beyond the scope of this study, and would definitely be an important follow-up of this work.5) The manuscript itself meanders around such that the reader can lose interest.One issue here is that we have covered significant ground in this one manuscript, and gone from an unbiased screen in cell lines, to molecular mechanism of multiple ‘hits’ and then to clinical analysis. That being said, we have revised the text to improve how the manuscript reads.6) Much of the data is borderline in quality: i.e., affinity pull downs, luciferase assays, etc.For the affinity pull down assays we have added additional data using combination of biotinylated miRNA mimics with overlapping targets to confirm and improve the results presented. For the luciferase assays based on the reviewers comments we have used the loss-of-function approach to further substantiate our previous results.7) In mRNAs apparently regulated by more than one miRNA,why wasn't the combined effect measured?We thank the reviewers for this suggestion. We have now done a combination of these miRNAs with overlapping targets and the results are very consistent. First for the affinity pulldown with biotinylated miRNA mimics we used a combination of miR-1255b and miR-193b*, and a combination of miR-148b* and miR-193b* in 8) Many of the experiments would have been much more convincing if done in synchronized or facs sorted cells.In


Rag1 or Rag2 genes, the protein products of which are critical members of the cellular apparatus for V(D)J recombination. In this report, we describe an infant with Omenn syndrome with a previously unreported termination mutation (p.R142*) in Rag1 on one allele and a partially characterized substitution mutation (p.V779M) in a “core” region of the other Rag1 allele. Using a cellular recombination assay, we found that while the p.R142* mutation completely abolished V(D)J recombination activity, the p.V779M mutation conferred a severe, but not total, loss of V(D)J recombination activity. The recombination defect of the V779 mutant was not due to overall misfolding of Rag1, however, as this mutant supported wild-type levels of V(D)J cleavage. These findings provide insight into the role of this poorly understood region of Rag1 and support the role of Rag1 in a post-cleavage stage of recombination.Omenn syndrome is a primary immunodeficiency disorder, featuring susceptibility to infections and autoreactive T cells and resulting from defective genomic rearrangement of genes for the T cell and B cell receptors. The most frequent etiologies are hypomorphic mutations in “non-core” regions of the  During the development of B cells and T cells, a diverse repertoire of antigen receptor genes are assembled from multiple component gene segments in a process known as V(D)J recombination . Each ofSince antigen receptor gene assembly is required for the proper development of B cells and T cells, mutations that disrupt V(D)J recombination can lead to impaired immune function. Omenn Syndrome OMIM 603554) is an autosomal recessive variant of severe combined immunodeficiency (SCID) with distinctive clinical features of generalized erythodermia, hepatosplenomegaly, and lymphadenopathy -thymidine incorporation during the last 24 hours of a 5-day culture. The control numbers for proliferations refer to an internal laboratory control; shipping controls were comparable. This study was approved by the Institutional Review Board at Boston Children’s Hospital. Both parents gave written informed consent.Immunoglobulin levels and PRP and tetanus titers were determined by nephelometry at the Boston Children’s Hospital Chemistry Lab. Pneumococcal titers were determined by the Luminex method at ARUP Reference Laboratory. T cell subsets were determined in the Boston Children’s Hospital Immunology Lab by flow cytometry. T cell proliferations were determined at the University of Iowa using [TCR spectratype  was performed with 24 TCR Vβ subfamily specific primers as well as CB primers as described previously. ScoringRag1 are from NIH RefSeq NM_000448.2 and NP_000439.1, respectively, and Rag2 from NM_000536.3 and NP_000527.2, respectively. Nucleotide numbering starts with 1 at the A of the ATG translation initiation codon.Gene sequencing was performed using standard techniques . Sequences were analyzed using Sequencher software (Gene Codes Corporation). The reference DNA and protein sequences for Rag1, c.2459A>G which codes for p.Lys820Arg (NIH RefSNP accession rs2227973). Two homozygous, non-synonymous, coding-region SNPs were found on Rag2, p.Val154Ala (RefSNP accession rs17852002) and p.Met322Thr (RefSNP accession rs17856658).Other sequencing results were identified in this patient: A homozygous single nucleotide polymorphism (SNP) was identified in Antibodies used in this study were: anti-HA ; anti-FLAG ; anti-alpha Tubulin ; and sheep anti-mouse IgG HRP-linked whole antibody . pGG49, pGG51, p3xFLAG-Rag2, and pcDNA6-myc-hisA-Rag1 were described previously . Point mR/CamR colonies to the number of AmpR colonies [V779M, and Rag1R142* were transiently expressed using the pcDNA6-myc-hisA vector (Invitrogen). Expression of all proteins was confirmed by Western analysis.Extrachromosomal V(D)J recombination assays were performed in Br3neo human fibroblastoid cells as described previously , using ecolonies . Full-leV779M were purified from Escherichia coli as described previously [Recombinant FLAG-tagged full-length Rag2 was expressed by transient transfection of 293T cells and purified as described previously . Recombieviously .V779M were used for in vitro V(D)J cleavage assays as described previously [2, and 0.25 pmol of 32P-labeled DNA substrate. Reaction mixtures were incubated at 30°C for 2 hours, stopped with 95% formamide loading dye, separated by super-denaturing gel electrophoresis, and visualized by autoradiography.Rag1 and Rag1eviously . BrieflyP1 was born at term to non-consanguineous parents. Starting at one month of age, he developed recurrent, waxing and waning, scaling erythroderma with recurrent skin infections requiring oral antibiotics. At approximately 4 months, due to progression of his rash, he was referred to a dermatologist and underwent a skin biopsy, which showed acute micro-vesiculating, spongiotic dermatitis, dermal inflammation, and eosinophils. At 4.5 months of age, physical examination revealed global erythroderma, sparse, friable hair, generalized lymphadenopathy, and hepatosplenomegaly. Comèl-Netherton syndrome was ruled out by microscopic examination of scalp and eyelash hairs. Laboratory evaluation revealed hypoalbuminemia, peripheral eosinophilia, profound hypogammaglobulinemia, elevated IgE, absent isohemaglutinnins, and lack of specific antibody production . Lymphoc2/dose) prior to transplant for prophylaxis against graft rejection. Stem cells were depleted of T cells by CD34-positive selection followed by rosetting with sheep red blood cells. He received a total of 30.4 x 106 CD34 cells/kg and 3.2 x 103 CD3 cells/kg. Early post-transplant complications consisted of mucositis and persistent skin rash, which resolved by one month following HSCT. At approximately one-year after HSCT, he developed isolated severe myositis, presumed to reflect chronic GVHD, which required prolonged mechanical ventilation and treatment with steroids and cyclosporine A. He ultimately recovered, was able to breathe on his own, and was weaned off mechanical ventilation and immunosuppressive therapy. He has had no recurrence of his myositis. He was clinically well at age 5 years, when he was lost to follow-up. He has normal in vitro T cell function and specific antibody production following vaccination for Tetanus, Haemophilus influenzae, Pneumococcus, and polio virus.At age 6.3 months, this patient underwent a T cell-depleted, HLA single-antigen mismatched, paternal peripheral blood transplant at Memorial Sloan-Kettering Cancer Center. Chemotherapeutic cytoreduction was achieved with intravenous busulfan (16 mg/kg) and cyclophosphamide (50 mg/kg). He received two doses of alemtuzumab (10 mg/mRag1 and Rag2 genes. In addition to previously characterized polymorphisms (see Rag1 (c. 424C>T)   resultin 424C>T) . SequencArg 142 is located within basic region I (aa 142–147) in the N-terminal “non-core” portion of Rag1. Assuming canonical start codon usage , truncatIn addition to the maternal nonsense mutation, P1 also harbored a heterozygous missense mutation in Rag1 (c.2335G>A) on the paternal allele , resultiR142* and Rag1V779M to perform V(D)J recombination in cultured human cells using an extrachromosomal V(D)J recombination assay[V779M, or Rag1R142*, plus both full-length Rag2 and an exogenous recombination substrate to detect either signal joints (pGG49) or coding joints (pGG51) [V779M exhibited a dramatic deficiency (>99%) in V(D)J recombination activity J recombination.To test whether the p.R142* and p.V779M mutations impairs the function of Rag1, we examined the ability of Rag1ion assay. Fibrobl (pGG51) . Despiteactivity . Nonetheactivity . Althougy J recombination activity, we wanted to test whether the p.V779M mutation impairs V(D)J cleavage. To generate a DNA double-strand break at a recombination signal sequence, the Rag1/2 complex must bind to the RSS and then catalyze both nicking and hairpinning. To assay for a defect in one of these steps of V(D)J recombination, we tested the ability of recombinant wild-type and mutant core Rag1 proteins (aa 387–1011) to catalyze V(D)J cleavage in vitro. The recombinant proteins, which expressed and purified equally well J cleavage at wild-type levels, generating both nicked and hairpinned products  and a missense mutation on the paternal allele (p.V779M). Functional analysis of these Rag1 alleles revealed that the p.R142* mutation creates a null allele, while the p.V779M substitution is a hypomorphic mutation that severely impairs V(D)J recombination in vivo. However, biochemical analysis demonstrated that the p.V779M substitution does not impair V(D)J cleavage in vitro, indicating that this mutation is not a catalytic mutation.We present here an infant boy with Omenn Syndrome bearing compound heterozygous mutations of in vitro—for example, mutants that are impaired in Rag1-Rag2 complex formation [in vitro but impaired V(D)J recombination in vivo—for example, mutants that are impaired in chromatin-binding [Over the past 15 years, many different Rag mutations have been shown to cause Omenn Syndrome ,32,33,34ormation ,36,37, Dormation , or RSS ormation ; those t-binding ,39 or in-binding ,41,42,43in vitro and V(D)J recombination in vivo.P1’s nonsense mutation on the maternal allele (p.R142*) would, if translated, remove all of the functional regions in Rag1 described above. As such, it either produces a severely truncated, nonfunctional protein or the message is deleted by nonsense-mediated decay. Therefore, the p.R142* mutation clearly falls in the first category of mutations that impair both V(D)J cleavage in vitro V(D)J cleavage assays demonstrate that Rag1V779M is fully functional in V(D)J cleavage, arguing against any substantial defect in protein folding. While a previous study suggested that Rag1V779M might be modestly impaired in 12-RSS nicking in vitro [in vitro, but impaired V(D)J recombination in vivo. It is possible that Rag1V779M is deficient in V(D)J recombination because it fails to interact with some key regulatory factor in vivo, and is therefore deficient in coupled cleavage, analogous to the Rag2 mutations described in other Omenn Syndrome patients [V779M impairs the rejoining of the broken DNA ends generated during V(D)J cleavage. Along these lines, it is interesting to note that Rag1V779M is more severely impaired in coding joint formation than it is in signal joint formation (V779M may be a joining-deficient mutant that affects the post-cleavage stage of V(D)J recombination, analogous to previously identified Rag mutants [P1’s missense mutation on the paternal allele (p.V779M) is more interesting in that it appears to leave the previously described functional regions of Rag1 intact , and yetin vitro , we founpatients ,45. Alteormation , suggest mutants ,41,42,43in vivo, it does not affect any of the known functional elements in this region. Does V779 lie within a new functional region of the Rag1 C-terminal domain? A recent study took a systematic approach to correlate the recombination activity of mutant human Rag1 proteins to the clinical and immunological presentation of the patients harboring these mutations [in vivo: p.R764P; p.Y768*, p.E770K, p.R778W, and p.R786L. In light of these findings, it is tempting to speculate that V779 lies within a functionally significant region of the Rag1 C-terminal domain, possibly facilitating the proper repair of signal ends and coding ends via the classical non-homologous end-joining pathway.Previous studies using limited proteolysis have identified two potential domains within the core portion of human Rag1: the central domain (aa 531–763); and the C-terminal domain (aa 764–983) . There autations . Intrigu


HLA-G allelic variants and serum soluble HLA-G (sHLA-G) levels on risk of non-small-cell lung cancer (NSCLC). We analyzed 191 Caucasian adults with NSCLC and 191 healthy subjects recruited between January 2009 and March 2014 in Ariana (Tunisia). Serum sHLA-G levels were measured by immunoassay and HLA-G alleles were determined using a direct DNA sequencing procedures. The heterozygous genotypes of HLA-G 010101 and -G 010401 were associated with increased risks of both NSCLC and advanced disease stages. In contrast, the heterozygous genotypes of HLA-G 0105N and -G 0106 were associated with decreased risks of NSCC and clinical disease stage IV, respectively. Serum sHLA-G levels were significantly higher in patients with NSCLC and particularly in those with advanced disease stages compared to healthy subjects. The area under the receiver-operating characteristic (ROC) curves was 0.82 for controls vs patients. Given 100% specificity, the highest sensitivity achieved to detect NSCLC was 52.8% at a cutoff value of 24.9 U/ml. Patients with the sHLA-G above median level (≥ 50 U/ml) had a significantly shorter survival time. This study demonstrates that HLA-G allelic variants are independent risk factors for NSCLC. Serum sHLA-G levels in NSCLC patients could be useful biomarkers for the diagnostic and prognosis of NSCLC.Human leukocyte antigen (HLA)-G acts as negative regulator of the immune responses and its expression may enable tumor cells to escape immunosurveillance. The purpose of this study was to investigate the influence of  Lung cancer is the most common malignancy and the leading cause of cancer mortality in the world ,2. The pThe host immune system plays a crucial role in the detection and elimination of cancer cells by a process called the cancer immune surveillance . During HLA-G alleles have been reported to be associated with the risk of various types of tumors, including hepatocellular, esophageal, bladder, uterine cervical, childhood neuroblastoma and breast cancers [HLA-G alleles and lung cancer has not yet been studied. It remains to be elucidated whether the expression of HLA-G in the context of lung cancer depends on the grade/type of tumor or genetic background of patients or both.HLA-G exhibits low polymorphism with only 51 alleles described so far and HLA-G expression can be modulated by allelic variants . HLA-G a cancers –19. HoweA total of 191 Caucasian adults admitted to Abderrahmen Mami Hospital  and diagnosed with non-small cell lung cancer (NSCLC) were collected between January 2009 and March 2014. Histological diagnosis and tumor stage were determined for all patients in accordance with the World Health Organization criteria and the TNM staging system of international union against cancer. The collection of samples was performed before patients begin their treatment. 191 controls subjects were selected from healthy blood volunteers’ donors, recruited from the National blood transfusion center of Tunisia at the same period. These controls were unrelated to patients with malignancies, and none had a personal history of malignancy at time of ascertainment. Written informed consent was obtained from each individual and the study was approved by the Institutional Ethics Committee of Abderrahmen Mami Hospital.HLA-G alleles were determined by direct DNA sequencing analysis of the nucleotide regions encompassing exons 2–4 using purified DNA from blood samples as described previously [sHLA-G serum levels were measured using a sHLA-G Elisa kit (Exbio), which allows simultaneous detection of HLA-G1 and -G5 soluble proteins without discrimination. eviously .HLA-G alleles with NSCLC or disease status, those subjects who were heterozygous and homozygous were compared separately with subjects who tested negatively for that allele. The association between each of the putative HLA-G alleles and risks of NSCLC and disease progression was investigated using adjusted multivariate logistic regression to derive odds ratios (ORs) and respective 95% confidence intervals (CI) as estimates of relative risks. Since control and patient groups were similar for age, gender and ethnicity, the models were adjusted only for smoking status. The analyses were restricted to those HLA-G alleles found at a frequency above 5% in the study population.All analyses were performed using the Graph Pad PRISM 5.0 for Windows and Epi-Info statistical program 7.0. Participants were classified according to the disease status and descriptive statistics were used to describe pertinent variables. In order to assess the association between each of the Overall patient survival was defined as the time from the date of diagnosis to the date of last follow-up (censored) or date of patient death by any cause (event). Survival probabilities were calculated using the Kaplan–Meier method. The comparison of the survival curves was carried out using the Log-rank test. The feasibility of using sHLA-G in serum as a potential biomarker for differentiating NSCLC patients from normal controls was assessed using the receiver-operating characteristic (ROC) curve analysis. P-values less than 0.05 were considered statistically significant in all analyses.The distribution of demographic and smoking status variables by disease status are outlined in HLA-G alleles were found in the study population. The wild-type HLA-G*01:01:01 was the most prevalent allele (33.2%) among the controls, followed by G*01:01:02 (21.2%), G*01:03:01 (11.5%), G*01:04:04 (10.7%), G*01:06 (9.4%), G*01:05N (5.5%), G*01:04:01 (3.9%), G*01:01:03 (1.6%), G*01:01:12 (1.6%), G*01:01:14 (1.1%), and G*01:01:17 (0.3%).Eleven HLA-G genotypes by disease status while HLA-G genotypes and NSCLC and disease stages for those with allele frequency of 5% or more. Some HLA-G genotypes were associated with altered risks of both NSCLC and advanced disease stages. The heterozygous genotypes of HLA-G*01:01:01 and G*01:04:01 were significantly associated with increased risks of both NSCLC  and advanced disease stages . In contrast, the heterozygous genotypes of HLA-G*01:05N and G*01:06 were associated with a decreased risks of NSCC (P = 0.044) and clinical disease stage IV (P = 0.005), respectively. HLA-G genotypes and histological types, respectively. The heterozygous genotypes of HLA-G*01:01:01 and G*01:04:01 were associated with increased risks for all histopathological types reaching significant level for the adenocarcinoma lung cancer type which affected 55% of the patients of the present study. The heterozygous genotypes of HLA-G*01:05N and G*01:06 were associated with decreased risks for all histological types but levels were not significant when analyzed separately. The lack of significant associations found when cases are dichotomized by the different histopathological types or clinical disease stages is presumably due to the relatively low number of patients per subgroups of histological types or disease clinical stages. This lack of sufficient power becomes more important with low prevalent alleles such as HLA-G*01:05N and G*01:06.The mean serum level of sHLA-G in NSCLC patients (53.3 ± 4.6 U/ml) was significantly increased compared to that found in controls (8.36 ± 0.4 U/ml), P< 0.0001 . The meaROC curves were used to evaluate the performance of serum sHLA-G in detecting NSCLC.The area under the curves was 0.82 for controls vs patients . Given 1The most common immune-escape mechanism in experimental or spontaneous tumors is the downregulation or lack of expression of classical HLA class I molecules (HLA-A and -B), which directly affects cytotoxic T cell function against tumor cells . In the HLA-G gene [HLA-G alleles and serum sHLA-G levels are significantly associated with altered risks of NSCLC and clinical disease stages in Tunisians.Previous studies have demonstrated HLA-G expression in lung cancer tissue –11 and rA-G gene ,24. HoweHLA-G*01:01:01 and G*01:04:01 alleles increased risks, whereas the HLA-G*01:05N and G*01:06 alleles conferred protection against both NSCLC and advanced disease stages. Moreover, in agreement with previous studies [HLA-G*01:04:01 allele have increased blood sHLA-G levels, whereas those harboring the HLA-G*0105N allele, which does not encode a functional HLA-G1 protein, have reduced blood sHLA-G levels [HLA-G is under the control of the genetic background of the patients.The  studies –14, seruG levels . This fiet all [The ROC curve analyses were performed to evaluate the feasibility of sHLA-G as a diagnostic marker for NSCLC. Similarly to the study of Cao et all  this anaet all –14.HLA-G allelic variants are an independent risk factor for NSCLC in the Tunisian population. Serum sHLA-G levels in NSCLC patients could be useful biomarkers for the diagnostic and prognosis of NSCLC.In conclusion, this study demonstrates that S1 TableN: number, NSCC: none small cell carcinoma. Selected HLA-G genotypes are those with a frequency above 5% in the study population.(DOCX)Click here for additional data file.S2 TableCI, confidence interval; N.A: none applicable; NSCC: none small cell carcinoma OR, Odds ratio. Selected HLA-G genotypes are those with a frequency above 5% in the study population. Adjusted ORs for smoking status(DOCX)Click here for additional data file.S1 FigsHLA-G in NSCLC patients with all histopathological types were significantly increased when compared to controls as determined by the Mann-Whitney U test (*** P< 0.0001).(DOCX)Click here for additional data file.


T = 3 Grouper nervous necrosis virus-like particle (GNNV-LP) is determined by the ab initio method with non-crystallographic symmetry averaging at 3.6 Å resolution. Each capsid protein (CP) shows three major domains: (i) the N-terminal arm, an inter-subunit extension at the inner surface; (ii) the shell domain (S-domain), a jelly-roll structure; and (iii) the protrusion domain (P-domain) formed by three-fold trimeric protrusions. In addition, we have determined structures of the T = 1 subviral particles (SVPs) of (i) the delta-P-domain mutant (residues 35−217) at 3.1 Å resolution; and (ii) the N-ARM deletion mutant (residues 35−338) at 7 Å resolution; and (iii) the structure of the individual P-domain (residues 214−338) at 1.2 Å resolution. The P-domain reveals a novel DxD motif asymmetrically coordinating two Ca2+ ions, and seems to play a prominent role in the calcium-mediated trimerization of the GNNV CPs during the initial capsid assembly process. The flexible N-ARM  appears to serve as a molecular switch for T = 1 or T = 3 assembly. Finally, we find that polyethylene glycol, which is incorporated into the P-domain during the crystallization process, enhances GNNV infection. The present structural studies together with the biological assays enhance our understanding of the role of the P-domain of GNNV in the capsid assembly and viral infection by this betanodavirus.Betanodaviruses cause massive mortality in marine fish species with viral nervous necrosis. The structure of a  Nodaviridae and cause the mortality of numerous larval-stage fish species. Here we report protein crystal structures of a piscine betanodavirus, the Grouper nervous necrosis virus (GNNV), in four different forms. Highlights are two structural features that contribute to the viral molecular mechanisms of the T = 3 and T = 1 capsid assembly: a calcium-associated protrusion domain and a functional arginine-rich motif. These results also shed insights into the structural basis for evolutionary lineage of the family Nodaviridae.Betanodaviruses belong to the family  Nodaviridae is a family of positive-sense single-stranded RNA viruses with a non-enveloped T = 3 capsid. These viruses are characterized by a viral genome comprising two RNA molecules–RNA1 and RNA2. RNA1 (3.1 kb) encodes protein A, which is a RNA-dependent RNA polymerase (RdRp) responsible for viral RNA replication [lication .Nodaviridae [Macrobrachium rosenbergii Nodavirus (MrNV) and Penaeus vannamei Nodavirus (PvNV), which cause muscle necrosis in prawns [Nodaviridae includes not only the known types but also other members with a wide distribution.Alphanodaviruses and betanodaviruses are the major genera in the family aviridae . Alphanoaviridae . VNN is aviridae . Betanodaviridae . Based oaviridae . One repn prawns . These fNodaviridae, an assemblage of 180 CPs form a T = 3 capsid of diameter ~29−35 nm. CP is typically composed of the core jelly-roll topology, forming a face-to-face β-sandwich with two pairs of anti-parallel β-sheets [In the family β-sheets . During β-sheets . Proteinβ-sheets ,16; it aβ-sheets . The strβ-sheets . HoweverT = 3 GNNV-like particle (GNNV-LP) at 3.6 Å resolution; (ii) T = 1 subviral particles (SVPs) of the delta-P-domain mutant at 3.1 Å; (iii) the N-ARM deletion mutant at 7.0 Å; and (iv) the individual P-domain of GNNV CP at 1.2 Å. The crystal structure of GNNV-LP demonstrates several significant and distinct variations in capsid architecture and molecular mechanisms of capsid assembly compared to the genus alphanodavirus and other RNA viruses. In particular, we have identified the conserved structural characteristics of the shell domain on GNNV. Various forms of the T = 3 and T = 1 GNNV capsids show that the N-terminal arginine-rich motif (N-ARM) acts as a molecular switch. Second, the P-domain, with its DxD motif together with two bound Ca2+ ions, plays a pivotal role in the trimerization of the GNNV CP and the particle assembly. These high-resolution structural details contribute further to our in-depth understanding of the molecular mechanisms of viral assembly and infection, and should provide the structural basis for studying the evolution of the family Nodaviridae.In this report, we describe the crystal structure of the grouper nervous necrosis virus (GNNV) of the genus betanodavirus in various forms: (i) a complete Escherichia coli (E. coli) and the GNNV-LPs are self-assembled in vitro. Based on the EM images, the morphology of GNNV-LP shows a T = 3 capsid with a diameter of 30~35 nm  averaging and refine the structure to 3.6 Å  (residues 34−51), the shell domain (S-domain) (residues 52−213), the linker region (residues 214−220) and the protrusion domain (P-domain) (residues 221−338) . The ordquasi three-fold (Q3) axes to form 60 protrusions on the particle surface  of the family Caliciviridae also contains three ordered N-arms from the C1, C10 and C12 subunits located near the I3 axis similar to GNNV, but oriented toward another direction  axis and the subunit-C/C dimer along the I2 axis during T = 3 particle assembly [T = 3 quaternary organization [6 through hydrogen bonds to stabilize the subunit-C/C dimer.The CP subunits adapt to the assembly . The bennization ,16. The 2+ ions located at interfaces between pairs of subunits within each of the S-domains, which are coordinated with side chains of Asp130 and Asp133 to form the 130DxxDxD135 motif at the E-F loop, Gln100 at the C-D loop, Ser170 at the G-H loop and Glu213 near the linker region from the neighboring subunit (DxDxxD) [DxxD) [DxExxD motif and incorporate one or two Ca2+ along the Q3 axis in its CP [Divalent metal ions, such as calcium, are typically associated with metal-coordinating residues for particle formation, stability and infectivity . The GNN subunit . There a(DxDxxD) –30 and S) [DxxD) . In contn its CP ,32.T = 3 PaV  of alphanodavirus . Our cryTo gain more complete and detailed structural information, we have determined the crystal structure of the truncated P-domain (residues 214−338) at high resolution 1.2 Å; . Since i2+ ions near the non-crystallographic three-fold axis, which are coordinated with the C’-D’ loop to stabilize the trimeric structural fold  to bind to the cell surface and to enhance virus infection [An inspection of the structure of the truncated P-domains reveals additional electron densities in several pockets on the surface. We consistently find three glycerol (GOL) molecules located between the B’-C’ and F’-G’ loops of the three P-domains, and one polyethylene glycol (PEG) molecule at the interface between the two F’-G’ loops from neighboring subunits . The B-fnfection . On the T = 3 GNNV-LP, the N-terminus of the CP contains the disordered N-ARM for putative RNA interactions at the inner cavity of the particles. The next N-arm is ordered along the I2 interface only on subunit C. The P-domains of GNNV-LP show an independent trimeric organization, which is different from that of the S-domains. We therefore speculate that the N-ARM or P-domain of GNNV-LP might act as a major molecular switch in regulating T = 3 or T = 1 assembly. To address this issue, we have constructed two sub-clones, including (i) the delta-P-domain mutant (residues 35−217) and (ii) the N-ARM deletion mutant (residues 35−338), and have determined their structures . Based on the failure of VLP formation in the presence of single mutations of either C115A or C201A, the existence of a disulfide-bond linkage between Cys115 and Cys201 was previously postulated [Despite significant variations in amino-acid sequences, the structure of the GNNV S-domain exhibits a jelly-roll topology, similar to other structural viral CPs . Divalenomavirus , rotaviromavirus , tombusvomavirus –30, sobeomavirus ,31,40 anomavirus ,32. In G viruses –31,40,43 viruses . Investistulated . Howeverstulated  but diffstulated   are negatively charged components of the cell surface and play a role in virion attachment to host cells and binding to secondary host receptors during viral infection. For instance, the surface L-protein of HBV is reported to bind to glycosaminoglycans (GAGs) on the host-cell surface; its GAG-dependent binding is enhanced by PEG to facilitate viral infection . Our stuT = 3 capsid assembly. However, the bent conformation of the subunit dimer leads to the disordered N-arm lying at the inner cavity of the T = 1 delta-P-domain mutant without β-annulus formation. This observation indicates that the N-ARM of GNNV CP makes an essential contribution to the organization of the β-annulus along the I3 axes. We propose that the β-annulus on T = 3 GNNV might be an outcome of T = 3 capsid assembly rather than a profound effect on switching structural symmetries.The organization of the N-terminus and encapsidated RNA have been implicated in providing a dynamic equilibrium of the dimeric subunits between “bent” and “flat” conformations during viral assembly . The N-tT = 1 architecture. Comparatively, in alphanodavirus, a complete N-ARM deletion (delta residues 1−54) leads to the inhibition of particle assembly. Conversely, the partial N-ARM deletion (delta residues 1−31) was shown to cause the formation of highly heterogeneous particles, including small bacilliform-like and irregular structures [T = 3 VLPs and two SVPs of T = 2 and T = 1 architectures in vitro [Nodaviridae shows that the Orsay virus CP contains a basic-charged N-terminus but lacks the N-ARM  of National Cheng Kung University (IACUC #100065).E. coli BL21-CodonPlus(DE3)-RIL (Stratagene), and the cells were cultured in Luria Bertani (LB) broth (Merck) containing chloramphenicol (34 μg/ml) and ampicillin (100 μg/ml) until the OD reached 0.6–0.7 at 600 nm at 37°C. IPTG  (Bioshop) was added to a final concentration of 0.5 mM and cultures were incubated overnight at 18°C. The cells were harvested and disrupted by sonication in lysis buffer (50 mM Tris HCl (pH 8.0), 0.25 M NaCl, 20 mM imidazole, 5 mM β-mercaptoethanol and 1 mM EGTA). CP was purified through a Ni-NTA column . The SUMO-tag was cleaved using SUMO protease that was later removed with a Ni-NTA column.A consensus CP DNA sequence from the orange-spotted grouper nervous necrosis virus (OSGNNV) RNA2 (GenBank accession no KT071606) was amplified by PCR and cloned into a modified pET32-Xa/LIC vector carrying 6×histidine residues and yeast SUMO (SMT3) as the N-terminal fusion tag . This co4)2SO4 (750 mM) was added to the dialysis, and GNNV CP was finally dialyzed against the GNNV-LP formation buffer (20 mM Tris HCl (pH 8.0), 0.2 M NaCl, 1% (v/v) glycerol and 2 mM CaCl2). The size of GNNV-LP was measured by size-exclusion chromatography on a Superose 6 10/300 GL column . The purified GNNV-LP was concentrated to 30 mg/ml and stored at 4°C.The purified GNNV CP was diluted to a concentration of 0.3 mg/ml and dialyzed overnight at 4°C against lysis buffer without EGTA or β-mercaptoethanol at a ratio of 1:150. (NHThe truncated P-domain (residues 214−338), delta-P-domain mutant (residues 35−217) and N-ARM deletion mutant (residues 35−338) proteins were prepared using the same methods described above for GNNV CP. The truncated P-domain (20 mg/ml) and S-domain (30 mg/ml) proteins were stored in a buffer containing 300 mM NaCl and 50 mM Tris HCl (pH 7.5), whereas the N-ARM deletion mutant (30 mg/ml) was stored in the GNNV-LP formation buffer at 4°C.The purified GNNV-LP, the N-ARM deletion mutant and the delta-P-domain mutant were all diluted to a final concentration of 50 μg/ml and blotted on freshly glow-discharged, carbon-coated 200 mesh copper grids . Grids were negatively stained with 5 μl of 2% (w/v) uranyl acetate solution and screened using the H-7650 transmission electron microscope (Hitachi High-Technologies Co.) operated at 80 kV. All images were acquired using a 1024 x 1024 pixels CCD camera  and recorded at a magnification of 100,000 ×.3). All diffraction data were processed with HKL2000 [C2 space group with unit-cell dimensions of a = 477 Å, b = 422 Å, c = 337 Å, and β = 134°. The diffraction data of GNNV-LP crystals contained 499,184 reflections and was 98% complete at a resolution range from 50 to 3.6 Å. To help initial phase determination by ab initio phasing [The initial GNNV-LP crystallization experiment was performed at 18°C with the hanging-drop vapor-diffusion method. A Mosquito liquid-handling robot (TTP Labtech) was used for high-throughput crystallization condition screening. The initial condition of 0.2 M sodium formate (pH 7.2) and 20% (w/v) PEG3350 was obtained from the PEG/Ion Screen I kit (Hampton Research). This condition was further optimized to improve the diffraction quality and resolution of the crystals. Crystals appeared within 1−2 weeks. All crystals were cryoprotected with 25~30% (w/v) PEG3350 and frozen in liquid nitrogen before data collection. X-ray diffraction data were collected on BL44XU at SPring-8  with a CCD detector  using X-ray wavelength of 0.9 Å. All images were collected with an oscillation angle of 0.3° per frame with an exposure time of 3 s and a crystal-to-detector distance of 600 mm. A total of 600 frames were recorded on different positions from one crystal , 30% (w/v) PEG400; and 0.1 M NaCl, 0.1M lithium sulfate, 0.1 M MES (pH 6.5), 30% (w/v) PEG400, respectively. All crystals appeared within one week. X-ray diffraction data of the truncated P-domain and delta-P-domain mutants were collected on BL15A1 with a CCD detector  of NSRRC in Taiwan at a wavelength of 1.0 Å. The N-ARM deletion mutant was collected on BL44XU at SPring-8  with a CCD detector  at a wavelength of 0.9 Å. The diffraction data were processed with HKL2000 [The initial crystallizations of the truncated P-domain, delta-P-domain mutant and N-ARM deletion mutant proteins were performed with similar approaches as for the GNNV-LP. The initial crystallization conditions of the truncated P-domain, the delta-P-domain mutant and the N-ARM deletion mutant were 0.2 M Ca acetate, 0.1 M MES (pH 6.5), 10% (w/v) PEG8000; 0.2 M MgCl HKL2000 . All datT = 3 GNNV-LP were determined by the ab initio method using icosahedral non-crystallographic symmetry (NCS) averaging [κ = 72°, 120° and 180° hemispheres were analyzed with Molrep [T = 3 GNNV-LP particles in the monoclinic unit cell with one two-fold NCS axis of the virus particle coinciding with the crystallographic two-fold axis. The asymmetric unit contained half of the particle or 30 copies of the icosahedrally-related trimeric CPs. The spherical-shell model with uniform density was used as the starting model. The inner and outer radii of 119 and 159 Å, respectively, were chosen as initial parameters of the model [ab initio method, which used NCS-averaging (NCSA) with phase extension, a proper mask was necessary for dividing two regions: the protein region to be NCS-averaged and the solvent region to be flattened. The initial mask for NCSA and solvent flattening was created from the atomic structure of T = 3 FHV (PDB ID: 4FSJ) with a large mask-radius of 11~13 Å around each atoms (RAVE [CCP4 [1/a) (This process is referred as “procedure” hereafter). During cycles of iterations, the R factor and correlation coefficient comparing Fobs and Fcalc were monitored. The interpretable electron density map was successfully obtained . The complete models of the S-domain and the linker region were subsequently built up based on the amino-acid sequence of the GNNV CP manually .For structure determination of the on by DM  was used REFMAC5  and manuC5 [Coot  to fit th MAPROT , the modARP/wARP . Structu [PHENIX . Resolut [PHASER . The cryEpinephelus coioides) collected in Taiwan. The isolated virus was propagated in GF-1 cells and collected when 90% of the cells displayed a cytopathic effect (CPE). GF-1 cells were seeded in 12-well plates at a density of 1 x 105 per well in 2 ml L-15 medium supplemented with 5% (v/v) FBS, and cultured to 80−90% confluence. For infection, GF-1 cells were washed with PBS three times and subsequently infected with GNNV at a titer of 104 TCID50/ml in serum-free medium, 4% (w/v) PEG3000- and 4% (w/v) PEG8000-containing serum-free medium, respectively. After incubation with the virus for 30 min, the cells were washed thrice with PBS and L-15 medium (2 ml) supplemented with 1% (v/v) FBS in the presence or absence of PEG3000 and PEG8000 was added.The GF-1 grouper cell line  was cultAfter infection, GF-1 cells were washed with PBS, and RNA was extracted with the TRIzol reagent (Invitrogen). Reverse transcription and real-time quantitative PCR were performed as previously described . All datT = 3 GNNV-LP, the truncated P-domain (214−338) and the T = 1 delta-P-domain mutant (35−217) have been deposited at the Protein Data Bank (PDB) with accession codes 4WIZ, 4RFU and 4RFT, respectively.Nucleotide sequences of PCR-amplified fragments of OSGNNV RNA2 from have been deposited in the GenBank nucleotide database under the accession code KT071606. Atomic coordinates and diffraction data of the S1 FigT = 3 GNNV-LP  and FHV  and the first  interpretable electron density map at 3.7 Å and the ‘initial’ mask made from the T = 3 FHV (orange). (B) Inner surface view of the density map at 3.7 Å corresponding to the iASU, labeled accordingly with the Cα carbon skeletons of CP of GNNV (blue) and FHV (green) superimposed. RNA-binding α-helices of FHV are removed for clear view. (C) A stereo-view of the α-helix (residues 90–99) in the S-domain directed towards the corresponding ‘initial’ density map at 3.7 Å. (D) Progress of the R factors and correlation coefficients during phase extension. The ‘initial’ means the first interpretable trial by the ab initio method. The ‘final’ means the progress during the phase extension with the revised mask and the refined NCSA matrices.(A) Lateral view of the iASU of (TIFF)Click here for additional data file.S2 FigT = 3 GNNV-LPs; (B) T = 1 SVPs of the N-ARM deletion mutant; (C) the delta-P-domain mutant. Bar: 100 nm.Electron micrographs of negatively stained VLPs used for crystallization. (A) (TIFF)Click here for additional data file.S3 FigT = 1 assembly is well arranged in the corresponding unit cell with dimensions of a = b = 260.8 Å, c = 250.5 Å and γ = 120° in space group P6322. Five neighboring subunits of CP along the I5 axis are labeled as green, wheat, cyan, yellow and magenta colors, respectively. (B) Analyses of self-rotation functions of the T = 1 N-ARM deletion mutant. The NCS relationship was corroborated by the self-rotation functions of κ = 72°, 120° and 180° hemispheres, and calculated with Molrep [(A) Crystal packing of N-ARM deletion mutant in h Molrep .(TIFF)Click here for additional data file.S4 Fig2+ ions (yellow spheres) are incorporated at the interfaces of neighboring subunits A (cyan), B (green) and C (magenta), where Gln100, Asp130, Asp133, Ser170 and Glu213 (orange sticks) participate in Ca2+ coordination. (B) An illustration of the geometric and electrostatic differences on the inner surface between GNNV (left) and PaV (right). The charge distribution around the inner surface of the pentameric capsomers of empty GNNV-LP and PaV has a positive electrostatic potential (blue), and five short ordered encapsidated RNA duplexes are shown along the I2 axes. (C) The distribution of cysteine residues in the S-domains. A superimposition of S-domain structures from GNNV (magenta), SeMV  and Orsay virus  is shown. Cysteine residues are shown in stick, and Cys107−Cys195 of Orsay virus represents the disulfide linkage.(A) S-domains of three subunits per iASU. Three Ca(TIFF)Click here for additional data file.S5 FigThe crystal structures of the P-domain of the GNNV and Orsay viruses (PDB ID: 4NWV), and P1-domain of HEV (PDB ID: 2ZTN) and Calicivirus (PDB ID: 2GH8) are shown as ribbon diagrams. The N’ and C’ terminus of Calicivirus indicate the connecting regions between the P1 and P2 domains.(TIFF)Click here for additional data file.S6 Fig2+ using size-exclusion chromatography (SEC) on a Superdex 75 10/300 GL column . These data were compared to protein standards . (C) SDS-PAGE analysis of oligomerization of three CPs in solution. Full-length GNNV CP (lane 1) and the N-ARM deletion mutant (residues 35−338) (lane 2) exhibit monomeric and trimeric forms concurrently; and monomeric and dimeric forms of the delta-P-domain mutant (residues 35−217) (lane 3) are shown compared to protein standards (lane M).(A) Conservation of water molecules at the interfaces of neighboring subunits in the truncated P-domain. Three sets of two water molecules (blue spheres), at the subunit-interface regions, are coordinated with the conserved residues shown from two neighboring subunits in yellow and green, respectively. (B) The equilibrium properties of the monomeric and trimeric P-domains. The purified GNNV P-domain was analyzed in the absence (solid line) or presence (dashed line) of Ca(TIFF)Click here for additional data file.S7 Fig2+ ions and water molecules bound at the DxD motif. Two residues  coordinating two Ca2+ ions (yellow spheres) and water molecules (blue spheres) are conserved on the P-domain of each neighboring subunit. The |2Fo–Fc| map (orange mesh) around the binding site is shown with a contour level at 3σ. (B) There are three conserved GOL-binding pockets and one PEG-binding site on the electrostatic surface of the trimeric P-domains. Electron density maps of PEG (blue) and GOL (green) are shown with the |2Fo–Fc| coefficient and contour at 1σ around the interaction site. (C) A comparison of the linker regions between the truncated P-domain and the T = 3 GNNV-LP. Each hinge region is identified and color-coded as a rainbow gradient with respect to B-factor values  to depict the relatively rigid and flexible area. The three subunits  are shown with magenta, green and cyan, respectively.(A) A stereo view of the trimeric P-domains with Ca(TIFF)Click here for additional data file.S8 FigNodaviridae. The neighbor-joining phylogenetic unrooted tree was built using Mega5 [Nodaviridae are identified as alphanodavirus, betanodavirus, unassigned nodavirus and Orsay virus, respectively. (B) The sequence alignment of RNA2-encoded CP from different genotypes of betanodavirus. Multiple sequence alignment was performed with sequences of the CPs from OSGNNV, DGNNV, RGNNV, BFNNV, SJNNV and TPNNV using ClustalW. Each domain of GNNV CP is indicated on the top of alignment with colors as in DxxDxD and DxD motifs of GNNV CP are identified in the orange boxes. (C) N-terminal sequence identification of the CPs from different strains of the family Nodaviridae. Basic- and acidic-charged residues are colored in blue and red, respectively.(A) The phylogenetic tree of the family ng Mega5  with a m(TIFF)Click here for additional data file.


To optimize imaging protocols, it's beneficial to understand the factors that influence the measurement. We measured the accuracy and reproducibility of spin echo-spin lock (SESL) T1rho-prepared balanced-steady-state free precession (bSSFP) sequences for myocardial relaxation time mapping. We subsequently performed 2O cylinders with MnCl2 concentrations of 0.01- 0.15 g/mL was used for the comparison of 24 T1rho mapping sequences with various repetition times (TR = 1.8-10 sec), flip angles (0-70 degrees), and numbers of segments (Nseg = 10-70). The scans were performed on a 1.5 T MRI , and reference T1rho measurements were obtained by using gradient echo acquisition with TR = 10 sec, flip angle = 90 degrees, Nseg = 1. The overall scan time for this exam was approximately 1.5 hours. Three fitting methods were used to estimate T1rho recovering times,A phantom containing 8 homogeneous HSL/A + B;linear model: log[S(t)] = -TSL/A); and,2-parameter model: S(t) = B*exp(-TSL/A) + C,3-parameter model: S(t) = B*exp. Two sequences were applied on each subject .For T1rho lower than 50 ms, we found that the 3-parameter model was the most accurate and reproducible. 2-parameter and linear models are preferred for higher T1rho values. As shown in Figure seg increases T1rho underestimation, while higher flip angle and lower Nseg were more accurate and reproducible. SNR is related to the component of amplitude of the transverse magnetization which also varies with flip angle, there's a thus trade-off between T1rho measurement precision and accuracy. Although long TR and small Nseg allows the full relaxation of magnetization producing more accurate results, the resulting long scan time was not very practical in clinical settings.As shown in Figure in vivo T1rho and its error map obtained in a 24-year-old healthy male subjects.Figure We described a number of adjustable factors in T1rho mapping. These factors depend on protocol adjustments, and can potentially influence the accuracy and reproducibility.None.


Two HIV-1 subtype C gp120 protein candidates were the selected antigens for several experimental vaccine regimens now under evaluation in HVTN 100 Phase I/II clinical trial aiming to support the start of the HVTN 702 Phase IIb/III trial in southern Africa, which is designed to confirm and extend the partial protection seen against HIV-1 infection in the RV144 Thai trial. Here, we report the comprehensive physicochemical characterization of the gp120 reference materials that are representative of the clinical trial materials. Gp120 proteins were stably expressed in Chinese Hamster Ovary (CHO) cells and subsequently purified and formulated. A panel of analytical techniques was used to characterize the physicochemical properties of the two protein molecules. When formulated in the AS01 Adjuvant System, the bivalent subtype C gp120 antigens elicited 1086.C- and TV1.C-specific binding antibody and CD4+ T cell responses in mice. All the characteristics were highly representative of the Clinical Trial Materials (CTM). Data from this report demonstrate the immunogenicity of the gp120 antigens, provide comprehensive characterization of the molecules, set the benchmark for assessment of current and future CTM lots, and lay the physicochemical groundwork for interpretation of future clinical trial data. HIV-1 entry is mediated by the envelope glycoprotein (Env), consisting of two non-covalently bound subunits, the receptor binding glycoprotein gp120, and the transmembrane glycoprotein gp41. Env is the only protein on the viral surface exposed to the humoral immune system and is also the target for the binding neutralizing antibodies. Thus, it has been a natural choice for development of antibody-based vaccines against HIV-1. The RV144 clinical trial conducted in Thailand, which showed 31.2% efficacy 3.5 years after vaccination [Over the past decades, a number of highly potent broadly neutralizing antibodies (bNAbs) have been discovered, some of which could suppress HIV-1 replication and entry into CD4+ cells . Viral eDetails about plasmids, stable cell line generation, and clone selection are described in a separate report . Gp120 rN-glycosylated gp120s was determined by LC-MS using a Waters Xevo G2-S QTOF and the MaxEnt1 deconvolution software.Molecular weight (MW) of intact gp120s was measured by MALDI-TOF (Matrix Assisted Laser Desorption Ionization-Time of Flight) using a Bruker UltrafleXtreme MALDI-TOF/TOF instrument. MW of de-B, was a liposome-based formulation containing 50 µg 3-O-desacyl-4′-monophosphoryl lipid A  and 50 µg QS-21  in 500 µL. The animals received 1/10th of the human dose, which means that they received 5 µg MPL and 5 µg QS-21. T cell and antibody responses were characterized at 14 days post-second and third dose. The study was performed at GSK Vaccines laboratory in Rixensart, Belgium and was approved by the Institutional Animal Care and Use Committee conforming to the requirements set forth in the Animal Welfare Act, the ILAR Guide, and all applicable local, state, and federal laws and regulations. Details of the methods are described in the CB6F1 mice  were immunized intra-muscularly at days 0, 14, and 28 with 2 µg each of gp120 proteins without adjuvant or formulated with 50 µg aluminum hydroxide or 50 µL AS01. The AS01 used here, called AS01N-glycosylated by PNGase F (peptide-N-glycosidase F), and then analyzed by RP-HPLC (Reversed Phase-High Pressure Liquid Chromatography) using a C18 column. UV at 215 nm and MS/MS were used for online detection and identification. IEF was performed using the Invitrogen Novex Precast IEF gels (pH 3–7 and pH 3–10) and associated buffers.Gp120 proteins were denatured in Guanidine HCl, reduced by DTT (dithiothreitol), alkylated by Iodoacetamide, de-DSC was performed using a Microcal VP-DSC scanning microcalorimeter. For CD, Gp120 materials were buffer exchanged into 10 mM phosphate at pH 7.0 and then analyzed by a Jasco J-1500 Circular Dichroism Spectrometer . The Contin/LL program in the CDPro Analysis software  was used to deconvolute experimental spectra with reference to the SP43 dataset consisting of soluble proteins.N-glycosylated tryptic peptides of gp120 proteins were analyzed by LC-MS/MS on Xevo G2-S operated under Product Ion Discovery (PID) mode. Briefly, the MS was programmed to fragment and sequence all precursor ions that gave rise to signature sugar peaks  upon collision. The identification of the O-linked glycans was based on the accurate mass of the glycans.Reduced, alkylated, and de-N-linked glycosylation site mapping, reduced and alkylated tryptic peptides were digested by Endo H (endoglycosidase H), Endo F3 (endoglycosidase F3), or PNGase F, and analyzed by LC-MS/MS using a Thermo LTQ Orbitrap MS. Data were analyzed to look for variable modifications of GlcNAc at Asn residues. For N-linked glycoprofiling, gp120 proteins were heated at 90 °C with RapiGest SF (Waters) and de-N-glycosylated by Rapid PNGase F . After reductive amination with 2-AB, labeled glycans were resolved by LC using a Waters Acquity Glycan BEH Amide column with both fluorescent and MS detection. SimGlycan software  was used for glycan identification.For N-glycosylated by PNGase F. Peptides were analyzed by the LC-MS/MS using LTQ Orbitrap with both CID (collision induced dissociation) and ETD (electron transfer dissociation). For detection of non-bonded Cys residues, gp120 was alkylated by iodoacetamide without prior reduction by DTT before trypsin and PNGase F digestion.For disulfide bond analysis, gp 120 proteins were digested by trypsin with or without reduction/alkylation and de-The development reference materials are different from the research materials ; the latN-linked glycans contributing approximately half the molecular mass. A reduced SDS-PAGE gel analysis of both neat and de-N-glycosylated TV1.C and 1086.C gp120 is shown in N-glycosylation by Peptide-N-Glycosidase F (PNGase F). The presence of lower MW bands was due to clipping by proteases (discussed later) during prolonged incubation at 37 °C. Gel mobility can be affected by many factors, such as post-translational modifications and matrix effects. Thus, the apparent MW may not be a true indication of the molecular mass. To better determine MW, mass spectrometric methods were used. Intact neat gp120s were hard to resolve by LC-MS likely due to complexity of glycosylation. Therefore, it was analyzed by MALDI-TOF instead. As shown in N-glycosylated gp120s were analyzed by LC-MS. After deconvolution, the MW of the main species was 57,965 Da for TV1.C and 52,823 Da for 1086.C. In both molecules, glycans accounted for ~45% of the molecular mass. In addition, multiple smaller peaks were also observed with ∆mass of 294 Da and 656 Da, which corresponded to the mass of mono- and oligo-saccharides, and suggested the presence of O-linked glycans on gp120 molecules.The gp120 designation comes from the apparent MW of approximately 120 KDa from band mobility on the SDS-PAGE gels. Gp120s are heavily glycosylated with The calculated isoelectric points (pI) of gp120s were slightly basic, above 8. However, due to extensive glycosylation (many glycans are acidic), the pI was expected to be acidic. This was confirmed by IEF gel analysis C. Also, To gain a low-resolution characterization of gp120 secondary and tertiary structures and set a benchmark for comparison among lots, Circular Dichroism (CD) analysis was performed using near- and far-UV regions  to characterize the thermodynamics of gp120s. The protein melting point (Tm) in a given solvent environment, which is indicative of protein unfolding, is a commonly used measurement of thermal stability of proteins. TV1.C gp120 showed thermal transitions that spanned a wide temperature range with Tm at 61.2 °C. In contrast, 1086.C gp12 showed a sharp and strong main peak transition and a higher Tm at 63.7 °C E. The di+ T cell responses were measured at 14 days post-third immunization with the bivalent Clade C gp120 antigens alone or adjuvanted with Aluminum hydroxide. In contrast, the gp120s/AS01 formulation elicited robust 1086.C- and TV1.C-specific CD4+ T cell responses  14 days post-third dose  of 1973 and 1145, respectively, at 14 days post-third dose. Aluminum hydroxide  adjuvanted gp120 antigens significantly increased binding antibody titers up to GMT of 8807 (anti-1086.C gp120) and 4698 (anti-TV1.C gp120). The AS01-based formulation elicited the highest antibody responses reaching anti-1086.C and anti-TV1.C gp120-specific GMT of 32,936 and 31,860, respectively A,B. Postird dose . TogetheN-glycosylated before peptide mapping experiment was run. Sequence coverage of 92.6% (based on amino acid numbers) was achieved for TV1.C and 96.6% for 1086.C  co-purified with 1086.C gp120, while Cathepsin A co-purified with TV1 gp120. Cathepsin-induced degradation was also reported for other gp120s [The primary amino acid sequences deduced from corresponding cDNA sequences are shown in r 1086.C . Identitr gp120s . Since cO-linked glycosylation on gp120s has been reported previously in the literature [O-linked glycosylation and to characterize the O-glycan(s), a Product Ion Discovery (PID) base approach was used with a Q-TOF MS, which was set to search for de-N-glycosylated peptides that generated the signature sugar peaks upon Collision Induced Dissociation (CID) and target those peptides for sequencing. Three peptides  in TV1.C gp120 and two peptides  in 1086.C gp120 were found to bear O-linked glycans. Since 1086.C peptide 444YKVVEIKPLGVAPTEAKR461 and TV1.C peptide 465VVEIKPLGIAPTKAK479 each contain only one serine or threonine residue, the O-linked glycan could only be on T457 and T476, respectively. Either S1 or T4 in 1086.C peptide 1SWVTVYYGVPVWK13 could be the potential site of O-linked glycosylation. CID from the Q-TOF MS was not able to differentiate the two sites since O-linked glycosidic bonds were labile under CID condition and completely fell off before the peptide backbone was fragmented. Electron Transfer Dissociation, a mild fragmentation technique that preserves the labile glycosidic bonds, was used to specifically target the precursor ion and pinpointed T4 as the O-linked glycosylation site , which fit the N-linked glycosylation consensus motif . To map the exact sites of modification, an approach that combined LC-MS/MS analysis and endoglycosidase treatment was used. Two endoglysosidases, Endo F3 and Endo H, which respectively cleave between the two core GlcNAc on complex N-glycans and high mannose/hybrid glycans leaving only one GlcNAc still attached to the Asn residue, were used. The reasons to use such treatment are two-fold: one is to reduce the complexity of the N-glycans and make MS/MS data easier to interpret; the other is to differentiate sites with complex or high mannose/hybrid glycans. Examples of the site mapping result are shown in N-glycosylation schemes in gp120s  glycans were the main species detected in the gp120 molecules (Glycosylation profiles of the gp120s were characterized by combining fluorescence labeling of the released olecules B. The de402MWQGVGQATYAPPIAGNITCR422 in TV1.C gp120 (17TTLFCASDAK26 in 1086.C gp120 (17TTLFCASDAK26 and 389AIYAPPIEGEITCNSNITGLLLLR412) in 1086.C gp120. Clearly, the un-bonded Cys residues were also subject to inter-molecular disulfide bonding. Overall, disulfide bonding patterns delineated from the extensive LC-MS/MS studies were shown in both expected and alternative disulfide bonding were detected and are shown in TV1.C and 1086.C gp120s each contains 18 cysteine residues that form intra-molecular disulfide bonds and stabilize the tertiary structure. Correct disulfide bonding is critical in maintaining the structural integrity. Heterogeneity has been reported in the literature for several gp140 proteins that were recombinantly produced ,20. It wExtensive characterization using an assay of analytical and biochemical techniques covered the physicochemical properties of recombinant gp120s expressed in CHO cells, which are representative of the protein antigens used in the HVTN 100 clinical trials. The data confirmed the sequence integrity of the molecules and characterized their biophysical immunogenicity, glycosylation patterns, and disulfide bonding properties. These data will set the benchmark for comparison to ensure key structural elements are preserved as the antigens progress through clinical studies and also lay the physiochemical groundwork for interpretation of future clinical trial data.