MET mRNA expression status using RNA in situ hybridization (ISH) technique in primary and metastatic lesions of 535 surgically resected gastric carcinoma (GC) cases. We compared the results with those of immunohistochemistry and silver in situ hybridization, and examined the association with clinicopathologic characteristics and prognosis. Among 535 primary GCs, 391 (73.1%) were scored 0, 87 (16.3%) were scored 1, 38 (7.1%) were scored 2, 12 (2.2%) were scored 3 and 7 (1.3%) were scored 4 by RNA ISH. High MET mRNA expression (score ≥3) was associated with lymph node metastasis (P = .014), distant metastasis (P = .001), and higher TNM stage (P<.001). MET mRNA expression was correlated with protein expression  and gene copy number . The patients showing high-MET mRNA in primary or metastatic lesions had shorter overall survival than those showing low-MET mRNA . The patients showing positive conversion of MET mRNA status in metastatic lymph node had shorter overall survival than those with no conversion (P = .011). Multivariate analysis demonstrated that high MET mRNA expression in metastatic lymph node was an independent prognostic factor for overall survival (P = .007). Therefore, this study suggests that MET mRNA expression assessed by RNA ISH could be useful as a potential marker to identify MET oncogene-addicted GC.We investigated  MET is a proto-oncogene located on the 7q31 locus and encodes an RTK for hepatocyte growth factor (HGF) During the past decade, receptor tyrosine kinase (RTK) pathways have proven to be attractive drug targets for anticancer therapy MET gene amplification (GA) or protein overexpression has been associated with aggressive tumor characteristics and/or worse clinical outcome MET-amplified or -overexpressed GC showed response to treatment with several inhibitors of the HGF/MET signaling pathway in preclinical studies MET gene copy number (GCN) gain ranged 10% to 21.2% in studies using quantitative real-time polymerase chain reaction (qPCR) MET GA ranged 2% to 3.9% in studies using fluorescence in situ hybridization (FISH) For gastric carcinoma (GC), aberrant MET activation has been thought to be related to a gene dosage effect HER2 mRNA expression evaluated by RNA ISH was well correlated with protein overexpression and GA evaluated by IHC and FISH in 211 GC cases MET GCN and protein expression in a previous study MET mRNA expression using RNA ISH method, and compared the results with those of IHC and SISH in a large series of GC. In addition, clinicopathologic parameters and clinical outcomes of GC patients according to MET mRNA expression status were evaluated.In this study, we performed an RNA in situ hybridization (ISH) assay using paired DNA oligonucleotide probes and preamplifier-amplifier-label probes for visualization th edition. Clinical outcomes were followed up from the date of surgery until death or 60 months.We collected archival tissue samples of GC patients who consecutively underwent gastrectomy at Seoul National University Hospital, Seoul, Korea, from January 2004 through December 2005. Finally, 535 samples of primary GC and 199 samples of synchronous regional metastatic lymph node (LN) from 535 patients were available for this study. The clinicopathologic characteristics of the patients were examined by reviewing medical charts and pathologic records . TNM staAll tissue samples were fixed in 10% buffered formalin for 24–48 hours and then embedded in paraffin. Core tissues (2 mm in diameter) were taken using a trephine apparatus . For the primary GCs, the invasion front of each primary tumor was selected. Metastatic LNs were subjected to the tissue array but the cases with micrometastasis were excluded. Total 22 tissue microarray blocks which contained up to 60 cores were constructed.All human specimens were obtained during therapeutic surgery. The participants did not provide the written consent to participate in this study. The retrospective study was performed using the samples over the shelves after the pathologic diagnosis, and all of the samples were anonymized before the study. Our IRB  approved this retrospective study under the condition of the anonymization (Reference: H-1006-035-320).MET mRNA, the RNAscope FFPE 2.0 assay kit  was used according to the manufacturer’s instructions. Briefly, 2- to 3-µm thick FFPE tissue sections were deparaffinized, heated, treated by protease, and hybridized with probe at 40°C for 2 hours . After washing and amplification, 3, 3′-diaminobenzidine was added for detection of target RNA. Nuclei were counterstained with hematoxylin. Positive staining was indicated by brown punctate dots in the nucleus and/or cytoplasm. MET mRNA expression levels were categorized into 5 grades according to the manufacturer’s scoring guideline: score 0, no staining or <1 dot per cell; score 1, 1–3 dots per cell (visible at 20–40×); score 2, 4–10 dots per cell and no or very few dot clusters (visible at 20–40×); score 3, >10 dots per cell and <10% positive cells have dot clusters (visible at 20×); score 4, >10 dots per cell and >10% positive cells have dot clusters (visible at 20×) (UBC (ubiquitin C) and dapB  were used as the positive and negative control, respectively. Samples were considered adequate when the UBC mRNA signals were easily visible under a 10x objective lens and the dapB signal was not visible.For in situ detection of  at 20×) . The proImmunohistochemical staining for MET was performed with anti-total MET (SP44) rabbit monoclonal primary antibodies . An automatic immunostainer  was used according to the manufacturer’s instructions. MET immunostaining was scored with the HercepTest scoring guidelines for GC : score 0, no membrane staining or membrane staining in <10% of tumor cells; score 1, faint/barely perceptible partial membrane staining in >10% of tumor cells; score 2, weak to moderate staining of the entire membrane in >10% of tumor cells; score 3, strong staining of the entire membrane in >10% of tumor cells .MET gene status was classified into 6 groups using the University of Colorado Cancer Center criteria for epidermal growth factor receptor gene Dual-color SISH assay was performed with INFORM MET DNA probe and INFORM Chromosome 7 probe  on a Ventana BenchMark XT following the manufacturer’s protocols. Signals were enumerated in 40 tumor nuclei per core, and 2 test or Fisher’s exact test was used to test the association between MET status and clinicopathologic factors. The Student’s t-test was used to compare means of continuous variables. The Spearman correlation test was used to assess the relationship between RNA ISH results and IHC or SISH results. The Kaplan-Meier method was used to estimate overall survival (OS), and OS differences between the groups with different MET status were compared by using the log-rank test. Multivariate survival analysis was performed using the Cox proportional hazards ratio model. Data analysis was conducted by using SPSS version 20.0 , and the results were considered significant when P<.05.The χMET mRNA group, and total 19 cases (3.5%) belonged to high-MET mRNA.Of 535 primary tumors, 391 (73.1%), 87 (16.3%), 38 (7.1%), 12 (2.2%) and 7 (1.3%) showed RNA ISH score 0, 1, 2, 3, and 4, respectively. When we compared the results of RNA ISH with clinicopathologic data including survival, and analyzed results of RNA ISH with IHC and SISH data, the groups of score 3 and 4 showed distinct features. Therefore, we regarded the score 3 and 4 as high-MET mRNA was associated with older age (P = .002), larger tumor size (P = .006), LN metastasis (P = .014), lymphatic invasion (P<.001), increased number of metastatic lymph nodes (P<.001), distant metastasis (P = .001), and higher TNM stage (P<.001) when compared to low-MET mRNA  showed concordant MET mRNA status and 13 (6.5%) did not. Of these 13 discordant cases, negative conversion was found in 50% (7/14) of high-MET mRNA primary tumors, and positive conversion was found in 3.2% (6/185) of low-MET mRNA primary tumors , 45 (22.6%), 22 (11.1%), 4 (2.0%) and 9 (4.5%) showed RNA ISH score 0, 1, 2, 3, and 4, respectively. Therefore, 13 cases (6.5%) were high-y tumors .P = .005), larger tumor size (P = .009), invasion depth (P = .05), LN metastasis (P = .018), lymphatic invasion (P = .026), increased number of metastatic lymph nodes (P<.001), distant metastasis (P = .007), and higher TNM stage (P = .001). However, MET overexpression did not show any association with gender, tumor location, and Lauren classification , 171 (32%), 113 (21.1%) and 15 (2.8%) primary tumors were scored 0, 1, 2 and 3, respectively. IHC score 3 showed distinct clinicopathologic features, and this MET overexpression group was significantly associated with older age , 92 (46.2%), 46 (23.1%) and 15 (7.5%) showed IHC score 0, 1, 2 and 3, respectively. Among 199 pairs of primary and metastatic lesions, 187 (94.0%) showed concordant MET protein status and 12 (6.0%) did not. Of these 12 discordant cases, negative conversion was found in 36.4% (4/11) of primary tumors with MET overexpression, and positive conversion was found in 4.3% (8/188) of primary tumors without MET overexpression.MET GA was observed in 2.6% (14/535) of primary tumors. MET GA showed significant association with larger tumor size (P = .038), lymphatic invasion (P = .009), increased number of metastatic lymph nodes (P<.001), distant metastasis (P = .005), and higher TNM stage (P = .002). However, MET GA did not show any association with gender, tumor location, Lauren classification, invasion depth and LN metastasis . All 7 cases with RNA ISH score 4 showed MET protein overexpression. Among 12 cases with RNA ISH score 3, 5 cases (41.7%) showed IHC score 3. The cases with RNA ISH score 2 exhibited variable IHC scores. The cases with RNA ISH score 0 or 1 did not show MET protein overexpression except for 1 case. Among 10 cases showing discrepancy , 8 showed IHC score 2 or RNA ISH score 2. In 199 metastatic LNs, there was a good positive correlation between MET mRNA and protein expression  (The correlation between  P<.001) .MET mRNA expression and MET GCN  (MET GA and only one case showed HP (14.3%). The 12 cases with RNA ISH score 3 showed GA (50%) or polysomy (50%) by SISH. The cases with RNA ISH score 2 showed various SISH patterns including GA (5.3%). None of the cases with RNA ISH score 0 or 1 showed GA.In addition, there was a positive correlation between  P<.001) . Among tMET GA (54.5%) or polysomy (45.5%) by SISH, and disomy or trisomy was never observed. In terms of SISH, among the 14 cases showing GA, 11 cases (78.6%) exhibited high expression of both mRNA and protein. Among the 58 cases showing HP, however, only 7 cases (12.1%) exhibited high expression of either mRNA or protein. Among the 111 cases showing LP, only 3 cases (2.7%) exhibited high mRNA expression. All 352 cases showing disomy or trisomy exhibited negative results by both RNA ISH and IHC.MET mRNA in primary tumors or metastatic LNs was significantly associated with poor OS . In the primary tumors with MET overexpression, negative conversion showed a trend of better OS than no conversion, but it did not reach statistical significance (P = .132). In addition, MET GA in primary tumors was also significantly associated with poor OS , Lauren classification , and TNM stage (I–II vs. III–IV). The hazard ratio was 2.27 (P = .007) (P = .067). In addition, MET GA, high-MET mRNA or protein overexpression in primary tumor was not a statistically significant prognostic factor by multivariate analysis (data not shown).In multivariate analysis, high- = .007) . HoweverMET mRNA expression was significantly associated with adverse clinicopathologic features and poor prognosis in a large series of GC patients using RNA ISH method. In addition, RNA ISH results were well correlated with those of SISH and IHC. The previous studies evaluating MET mRNA levels in GC used the Northern blot assay In the present study, we demonstrated that high HER2 mRNA was well correlated with protein overexpression and GA by comparing the results of 4 different in situ-based methodologies  in 211 GC cases Recently, we reported that high levels of MET GA, high MET mRNA and protein overexpression evaluated by SISH, RNA ISH and IHC were highly concordant, and high MET status at the DNA, mRNA, and protein were significantly associated with poor prognosis. These findings support that MET overexpression is mainly due to increased MET GCN and this mechanism contributes to aggressive behavior of MET oncogene-addicted GC. Nevertheless, there were some cases showing inconsistency among the MET GCN, mRNA and protein levels. We speculate that technical problems  and intratumoral heterogeneity of MET status may be the main causes of this discrepancy. However, some biological mechanisms can also be related to this discrepancy. For example, MET overexpression without GA can occur through transcriptional activation via HGF-dependent autocrine/paracrine loops or other signaling pathways MET GA may not increase the gene product. Asaoka et al. reported that a few GC cell lines harboring MET GA expressed the protein as same level as other cell lines without GA, but their tyrosine residues at the kinase domain were more phosphorylated We demonstrated that MET plays a role in metastatic progression of cancer. Several studies showed that MET GA or overexpression was associated with LN metastasis MET activating mutations were selected during metastatic spread of head and neck squamous cell carcinomas by comparing the gene sequence between primary tumor and metastatic lymph node MET mRNA status was significantly associated with poor prognosis by univariate and multivariate analysis. Therefore, these results suggest that the evaluation of MET status in metastatic lesions may be important to predict prognosis and to identify additional candidates for MET-targeted therapy.It is well known that MET, this advantage can be useful because the HGF-producing cells can be visualized in a tissue section using the HGF probe. However, vulnerable mRNA stability during the tissue processing and higher cost than that of IHC are the disadvantages of RNA ISH method. We hope that further technical improvement will resolve these limitations.In situ-based RNA analysis has several advantages over the ‘grind and bind’ analysis such as RT-PCR MET mRNA status evaluated by RNA ISH is well correlated with protein and GCN assessed by IHC and SISH, respectively, and MET GA is highly concordant with high expression of either mRNA or protein. In survival analysis, high expression of MET mRNA in primary or metastatic lesions, and positive conversion of MET mRNA status are significantly associated with poor prognosis. In addition, MET mRNA status in metastatic LNs is an independent prognostic factor by multivariate analysis. Our findings indicate that MET mRNA can be an alternative marker to identify the MET oncogene-addicted GC.In this retrospective study, Table S1(DOCX)Click here for additional data file.Table S2(DOCX)Click here for additional data file.Table S3(DOCX)Click here for additional data file.


MET alterations including amplifications and nucleotide variations have been associated with resistance to therapy and aggressive clinical behavior.MET nucleotide variation or amplification status were reviewed retrospectively (n=178). Categorical and continuous clinical and molecular characteristics were compared using Fisher's exact and Wilcoxon rank-sum tests, respectively. Univariate and multivariate survival were assessed via Kaplan-Meier and Cox regression analysis, respectively.The medical records of patients presenting to the University of Texas MD Anderson Cancer Center Phase I Clinic with relapsed or metastatic ovarian cancers and known MET amplification occurred in 4 (3.5%) of 113 patients, whereas nonsynonomous nucleotide variations were present in 9 (7.4%) of 122 patients. No patients exhibited concomitant amplification and variation. MET variations were observed only in white women with high-grade ovarian tumors, whereas amplifications were observed in both black and white women with high-grade serous ovarian primary tumors. No patients (n=4) exhibiting a MET alteration achieved an objective response when treated on a c-Met inhibitor phase I trial. In addition, ovarian cancer patients treated with a c-Met inhibitor with multikinase activity trended towards a longer time-to-failure compared with those treated with a c-Met-specific inhibitor .MET alterations occur in a minority of patients with ovarian cancer. c-Met inhibitors with multikinase activity may exhibit less activity in ovarian cancer than c-Met specific drugs. These findings warrant further investigation. MET alterations, including amplification and nucleotide variations, have been described and are associated with resistance to therapy and aggressive clinical behavior[The c-Met tyrosine kinase receptor (TKR), upon activation by its cognate antigen, hepatocyte growth factor (HGF), generates proliferation, migration, and survival signals in numerous cancers, 2. This behavior, 4, 7. TMET variations and amplifications in this disease[Ovarian, primary peritoneal, and fallopian tube cancers have similar ontological origins and clinical presentations. Their aggressive metastatic behavior and generally poor prognosis has prompted interest in developing therapies with TKR inhibitors, including those targeting EGFR and c-Met, 9. Howes disease-12. We, MET variations and amplification, respectively. Fifty-seven patients were tested for both variation and amplification. MET nucleotide variations were detected in 9 patients (7.4%): 6 with N375S and 3 with T1010I nonsynonomous variations. MET amplification was detected in 4 patients (3.5%), amplification gene copy numbers (in relation to CEP7) were 2.12, 2.27, 2.55, and 2.78. No patients exhibited concomitant MET nucleotide variation and amplification.One-hundred-and-seventy-eight patients met study inclusion criteria, of whom 122 and 113 were tested for MET variation or amplification status . MET variations were detected only in white women with high-grade primary ovarian tumors (Tables MET variations was predominately serous (74%), with one patient each having carcinosarcoma and clear cell carcinoma . Concomitant mutations included KRAS, BRCA1, ARID1A, and TP53 were identified in 1, 1, 1, and 2 patients, respectively. In addition, one patient had concomitant PTEN loss and another had weak PTEN staining  was compared with that of patients known to be negative for any aberrations (n=50). There was no significant different in median survival in patients exhibiting MET alterations  when adjusting for histology (serous vs. other), age (≥60 vs. <60), and number of prior therapies (>3 vs. ≤3).OS in patients with a MET nucleotide variation and one with MET amplification  exhibited a partial response (PR) or stable disease (SD) lasting ≥6 months. Of these, 2 of 18 patients (11%) achieved a best response of PR  patients tested[MET variations to be 1 and 0, respectively [MET aberrations. Of note, we did not detect any patients with concomitant MET amplification and variation.The literature delineating the prevalence of ts tested. An earlts tested. Two othectively , 11. TheMET nucleotide variations identified here and even whether these specific variations represent somatic mutations or germline polymorphisms have yet to be fully elucidated[MET amplification has been most extensively correlated with tumor invasion and aggressive metastatic behavior in gastrointestinal malignancies[MET variations, the N375S nonsynonmous variation occurs in the extracellular semaphorin domain, whereas the activating T1010I variation occurs in the juxtamembrane domain[MET polymorphisms may enhance c-Met TKR activity and even confer inhibitor resistance[MET alteration achieved an objective response on a c-Met phase I inhibitor trial. Another possible explanation for the lack of an observed objective response are that most patients in this study were enrolled on a Phase I dose escalation trials and may not have received an efficacious study drug dose. Few preclinical data have tested the efficacy of these inhibitors in MET altered patients [The biologic activity of the lucidated, 18. Theignancies, 20. Witesistance, 21. Thipatients -24 and tTP53 mutations exhibited objective responses to c-Met inhibitors and were both treated using a multikinase inhibitor. As such, observed responses may be due to effects on targets other than c-Met, including VEGFR2. This hypothesis is supported by recent evidence from our group showing that TP53 mutations predict responses to anti-VEGFR2 therapies[A subset of all ovarian cancer patients achieved objective responses. Patients treated with a multikinase inhibitor trended toward a longer TTF compared with those treated with c-Met-specific inhibitors . A possible explanation is that activity against VEGFR2, a target with known clinical efficacy in ovarian cancer, may inctherapies. In addiMET alteration versus those without, although in multivariate analysis patients with MET alterations trending towards a worse OS . However it is possible that separate analyses of MET variations and amplifications may yield different results. Due to the limited incidence, we did not observe any significant differences in the characteristics of patients with MET alterations. In addition, a number of concomitant mutations were identified in patients harboring MET variations, whereas there were none in patients with MET amplification other than PTEN loss.We observed no difference in survival in patients with a MET variations and amplifications observed in this study did not provide sufficient statistical power to stringently assess demographic and patient characteristic differences. MET variation testing was also done with some heterogeneity. Although the majority of patients were tested at MD Anderson core laboratories, a minority was tested with outside platforms. In addition, mutations and amplifications were often grouped within a single stratum. With regard to amplifications, the relatively low gene copy numbers found in this study (range 2.12-2.78) may be inadequate to exhibit biological differences. Finally, the few number of patients, especially MET altered patients, treated on c-Met inhibitor phase I trials makes generalizing these observations into a larger patient populations difficult. However, despite these limitations, to our knowledge this is one of the first studies to substantially compare the demographic and clinical characteristics of ovarian cancer patients with MET altered patients and their response to treatment with c-Met inhibitors. The findings of this analysis provide insight into the clinical characteristics associated with MET alterations and, if validated in the prospective setting, may lead to improved strategies utilizing c-Met therapies in this prognostically poor patient population.Outside of limitations inherent to all retrospective reviews, the low frequency of MET nucleotide variations and/or amplification (n=178). This study was approved by the MD Anderson Institutional Review Board and patient confidentiality was maintained following Health Insurance Portability and Accountability Act guidelines.We retrospectively reviewed the medical records of all consecutive patients presenting to our Phase I Clinic starting in May 2010 to November 2012 (n=3607). Patients presented for treatment of recurrent or metastatic disease, usually after multiple prior treatments (range 0-13). Eligibility criteria for inclusion in this study were a histologic diagnosis of ovarian, fallopian tube, or primary peritoneal carcinoma and testing for MET nucleotide variation and amplification status were tested using archival formalin-fixed, paraffin-embedded tissue blocks in addition to biologic material from fine needle aspiration biopsies or excised primary or metastatic tumors from diagnostic and/or therapeutic procedures. Histology was centrally reviewed in the MD Anderson Department of Pathology. MET nucleotide variations were assessed in several Clinical Laboratory Improvement Amendment-certified laboratories using a single test or as part of a gene panel. The majority of patients (n=160) were analyzed at the MD Anderson core laboratories. Of the remaining samples, 15 were tested at Knight Diagnostics , 2 at the Baylor College of Medicine , and 1 using a Foundation One platform  as previously described[described.MET amplification status was analyzed via fluorescence in situ hybridization (FISH) following institutional protocols at MD Anderson. Copy numbers were expressed as a gene copy number in relation to CEP7, a gene located near the centrosome of the same chromosome. Gene copy numbers of 2-fold or greater were considered amplified.All patients were enrolled when possible in a Phase I clinical trial(s) judged to be clinically appropriate by the patient's attending physician. Because confidentiality agreements with the providing pharmaceutical company stipulate that c-Met inhibitor identities cannot be disclosed, c-Met agents were classified as being in one of three categories: c-Met-specific inhibitor , c-Met inhibitor with multikinase activity , and c-Met inhibitor with multikinase kinase activity in combination with a VEGFR2 kinase inhibitor . All c-Met inhibitors with multikinase activity also targeted the VEGFR2 receptor, a molecule associated with known clinical efficacy in ovarian cancer. PatientClinical assessments were performed according to the specific requirements of individual protocols, typically once prior to the initiation of treatment and then at least every treatment cycle. Treatment responses were primarily assessed using computed tomography scans, magnetic resonance imaging, and/or positron emission tomography conducted prior to therapy and every 2 cycles thereafter (6-8 weeks). Radiographs were read in the Department of Radiology at MD Anderson and reviewed by physicians in our Phase I Clinical Trials Program. Objective responses were determined according to RECSIT 1.0 or 1.1 criteria, as specified by individual protocols, and the best responses achieved were recorded.MET nucleotide variation and amplification status. Time to treatment failure (TTF) was defined as the time from the initiation of therapy to its cessation for any reason. Categorical and continuous variables, including TTF and stratification by MET alteration were compared utilizing Fisher's exact and Wilcoxon rank-sum tests, respectively. Overall survival (OS) was assessed starting from the date of the first appointment in the Phase I Clinic using Kaplan-Meier analysis with comparisons via the partial likelihood ratio test. Multivariate and univariate hazard ratios (HR) were calculated via Cox regression. Wald p-values were reported for multivariate analyses. All tests were two-sided when appropriate and considered significant at p<0.05. Statistical analyses were performed using SAS version 9.3 .Patient demographics and tumor molecular and histologic characteristics were summarized in relation to


The Bloom syndrome helicase BLM and topoisomerase-IIβ-binding protein 1 (TopBP1) are key regulators of genome stability. It was recently proposed that BLM phosphorylation on Ser338 mediates its interaction with TopBP1, to protect BLM from ubiquitylation and degradation . Here, w  •Contrary to the findings of Wang et al. (2013), TopBP1 does not maintain BLM levels•BLM phosphorylation on Ser304, not Ser338, is required for TopBP1 binding•Disrupting BLM-TopBP1 binding causes chromosome instability•Crucial residues mediating TopBP1-MDC1 and BLM-TOP3A/RMI interactions identified Wang et al. (2013) proposed that the BLM helicase and TopBP1 interact via phosphorylated Ser338 of BLM, and that TopBP1 shields BLM from degradation. In contrast, Blackford et al. reveal that the BLM-TopBP1 interaction involves Ser304 of BLM, not Ser338, and that TopBP1 has no role in stabilizing BLM. TopBP1 is an essential protein with key roles in DNA replication and DNA damage responses . It has Bloom syndrome is a rare autosomal recessive disorder caused by mutations in the gene encoding the BLM helicase, and is characterized by growth retardation, immunodeficiency, hypersensitivity to sunlight, and cancer predisposition . Cells fWe identified BLM as a TopBP1-interacting protein and found that BLM binding requires BRCT domain 5 of TopBP1, in agreement with a recent report . HoweverWe identified BLM as a candidate TopBP1 interactor by mass spectrometric analyses of TopBP1-associated proteins see A. ConsisTo gain insight into the function of the BLM-TopBP1 interaction, we needed to map their reciprocal binding sites. Usually, one BRCT in a tandem unit contains a conserved lysine residue that directly contacts the phosphate group of a modified protein ligand . AccordiWe next assessed whether any of our TopBP1 BRCT mutants were compromised in their ability to interact with BLM. Strikingly, only BRCT5 mutants lost the ability to bind BLM D and 1E,To establish whether BLM itself or one of its binding partners was mediating BLM-TopBP1 binding, we took advantage of the fact that the N-terminal 132 residues of BLM are required for its interaction with the dissolvasome . ImportaTo determine the region of BLM that binds TopBP1, we examined binding of TopBP1 to a series of GFP-tagged BLM truncation mutants A. These To investigate whether the TopBP1-BLM interaction was direct, we used biotinylated nonphosphorylated peptides or Ser304-phosphorylated BLM peptides in interaction studies. Importantly, only the phosphopeptide retrieved TopBP1 from nuclear extracts H, indicaOur conclusion that Ser304 is the critical residue of BLM that interacts with TopBP1 BRCT domain 5 differed from that of a recent report describing a role for Ser338 in this binding . We initWe considered the possibility that the contradictions between our results and those of Wang et al. might be due to the different methodologies used. We therefore incubated recombinant GST-tagged TopBP1-BRCT5 purified from bacteria with lysates from human cells transfected with plasmids expressing S/Flag/streptavidin-binding peptide (SFB) triple-tagged BLM as described in the previous study . NotablyNext, we examined the relative abilities of peptides encompassing phosphorylated or nonphosphorylated Ser304 or Ser338 of BLM to interact with GST-BRCT5. While the phospho-S304 peptide readily bound the TopBP1 fusion protein, neither the phospho-S338 peptide nor the nonphosphorylated peptides were able to interact with it detectably C. Taken BLM−/− chicken DT40 cells as a complementation system by stably transfecting these with vectors encoding wild-type BLM or BLM-S251A  mutated to alanine (“K3A”) were hypersensitive to ionizing radiation, showed increased phosphorylation of CHK1 and RPA, and had a reduced frequency of random plasmid integration . NotablyIn this study, we identified and characterized the interaction between the Bloom syndrome helicase BLM and TopBP1, and investigated the consequences for cells when binding is disrupted. We found that BLM interacts directly with BRCT domain 5 of TopBP1 via a phosphorylated N-terminal residue (Ser304 in human BLM). This finding was surprising, given a recent report suggesting that Ser338 was important for TopBP1 binding . HoweverBLM−/− clones stably expressing BLM-S251A . We also tested whether we could recapitulate another claim by Wang et al., that TopBP1 stabilizes BLM, but we were unable to do so. In our hands, BLM stability was normal in absence of TopBP1 using various methods, including when we used identical siRNA sequences to deplete TopBP1 in the same cell lines used by Wang et al. In addition, mutant BLM that cannot interact with TopBP1 was not less stable in our studies. Taken together, these data strongly suggest that TopBP1 binding does not protect BLM from proteasomal degradation, and that the BLM-TopBP1 interaction therefore maintains genome stability via another mechanism.Significantly, while Wang et al. showed that TopBP1-BRCT5 mutations lead to increased SCEs as we did, they did not show this phenotype for cells expressing BLM-S338A. By contrast, we clearly observed increased SCEs in multiple Our data cannot rule out an indirect role for TopBP1 in maintaining BLM stability, because they leave open the formal possibility that our TopBP1 siRNA depletions were not as efficient as those of Wang et al. Crucially, however, under experimental conditions where we observed increased SCEs in TopBP1-depleted cells, there was no effect on endogenous BLM protein stability. Thus, even if TopBP1 has some indirect role in maintaining BLM stability that we could not detect due to incomplete TopBP1 depletion (a possibility we consider unlikely), this would still not explain the chromosomal instability phenotypes we see in TopBP1-depleted cells or cells expressing BLM or TopBP1 binding mutants.It was reported that ubiquitylation of lysines 38, 39, and/or 40 in BLM leads to its degradation, that this is prevented by TopBP1 binding, and that cells expressing BLM with these sites mutated (“K3A”) show signs of increased DNA-end resection in G1 . These tA number of issues arise from our work that will be worthwhile to address in future studies. In particular, it remains to be determined which kinase phosphorylates BLM on Ser304 to promote its interaction with TopBP1, and how it is regulated. Finally, given that cells expressing mutant BLM that cannot bind TopBP1 display increased SCEs and chromosomal aberrations, our data may have important implications for understanding the increased cancer susceptibility and pathologies observed in Bloom syndrome patients.293FT, HeLa, U2OS, and DT40 cells were grown as described previously . Wild-tySee the See the hr703 E1B mutant adenovirus was used rather than wild-type virus, because one of the E1B gene products induces BLM degradation , supplemented with Complete EDTA-free protease inhibitor cocktail (Roche) and 25 U/ml Benzonase (Novagen). After nuclease digestion, NaCl and EDTA concentrations were adjusted to 200 mM and 2 mM, respectively, and lysates were cleared by centrifugation. Where appropriate, antibodies were added to a final concentration of 1 μg/mg lysate and incubated for 2 hr at 4°C. Lysates were then incubated with 10 μl of either GFP-Trap agarose beads (ChromoTek), anti-Flag M2 affinity gel (Sigma-Aldrich), or protein G Sepharose  for 2 hr with end-to-end mixing at 4°C. Immunoglobulin-antigen complexes were washed extensively before elution in 2× SDS sample buffer for SDS-PAGE.For preparation of lysates for immunoprecipitations (IPs), cells were washed twice in phosphate-buffered saline (PBS) and lysed in IP buffer  proteins were purified as described (Glutathione escribed . BiotinyPull-downs were performed as described .Cells were prepared for analyses of DNA fibers, SCEs, and chromosomal aberrations as described .A.N.B. carried out the majority of experimental work, with substantial contributions by J.N. and W.N.; R.A.S. performed DNA fiber experiments, Y.G. contributed stable cell lines, and the paper was written by A.N.B. with editing by S.P.J. and W.N.


Complicated intra-abdominal infection (cIAI) is infection that extends beyond the hollow viscus of origin into the peritoneal space, and is associated with either abscess formation or peritonitis. There are few studies that have assessed the actual costs and outcomes associated with failure of initial antibiotic therapy for cIAI. The aims of this study were to evaluate risk factors and impact on costs and outcomes of failure of initial antibiotic therapy for community-onset cIAI.A retrospective study was performed at eleven tertiary-care hospitals. Hospitalized adults with community-onset cIAI who underwent an appropriate source control procedure between August 2008 and September 2011 were included. Failure of initial antibiotic therapy was defined as a change of antibiotics due to a lack of improvement of the clinical symptoms and signs associated with cIAI in the first week.A total of 514 patients hospitalized for community-onset cIAI were included in the analysis. The mean age of the patients was 53.3 ± 17.6 years, 72 patients (14%) had health care-associated infection, and 48 (9%) experienced failure of initial antibiotic therapy. Failure of initial antibiotic therapy was associated with increased costs and morbidity. After adjustment for covariates, patients with unsuccessful initial therapy received an additional 2.9 days of parenteral antibiotic therapy, were hospitalized for an additional 5.3 days, and incurred $3,287 in additional inpatient charges. Independent risk factors for failure of initial antibiotic therapy were health care-associated infection, solid cancer, and APACHE II ≥13.To improve outcomes and costs in patients with community-onset cIAI, rapid assessment of health care-associated risk factors and severity of disease, selection of an appropriate antibiotic regimen accordingly, and early infection source control should be performed. Complicated intra-abdominal infection (cIAI) is an infection that extends beyond the hollow viscus of origin into the peritoneal space, causing peritonitis or abscess formation. These infections typically require both surgical or percutaneous intervention and intravenous antimicrobial therapy, and are associated with substantial morbidity and mortality. Despite improvements in the management of cIAIs, mortality rates are still high, ranging from 2% in appendicitis to as high as 30–35% in patients with peritonitis, depending on the anatomic origin, severity of infection, and comorbidity –3. cIAIsInitial antibiotic therapy for cIAIs is usually empirical and sometimes problematic due to increasing antibiotic resistance. Whereas empirical therapy with broad-spectrum antibiotics might be more effective, its benefits must be weighed against its higher cost and the potential risk of increasing antibiotic resistance. In contrast, although empirical therapy with narrow-spectrum antibiotics may be less costly, it may be associated with a higher rate of clinical failure, thereby increasing total hospital costs. In addition to antibiotic administration, adequate source control is crucial for successful therapy of cIAIs. There are few studies that have assessed the actual costs and outcomes associated with failure of initial antibiotic therapy for cIAIs –8. In thThis study was reviewed and approved by the Institutional Review Boards of Asan Medical Center (IRB number: 2011–0719), Gyeongsang National University Hospital (2011–082), CheongJu St. Mary's Hospital (IRB-53), Chung-Ang University Hospital (C2011161), Kyung Hee University Hospital (1126–04), Soon Chun Hyang University Bucheon Hospital (2011–85), Ulsan Unversity Hospital (2011–092), Soon Chun Hyang University Cheonan Hospital (2011–105), GangNeung Asan Hospital (2011–038), Soon Chun Hyang University Seoul Hospital (2011–109), and Dongguk University Ilsan Hospital (2011–73). Written patient consent was waived by all sites in view of the retrospective and observational nature of the study. The complete protection of patients’ personal data was guaranteed according to the South Korea’s Bioethics and Safety Act.th edition codes. Then, manual reviews of their medical records were conducted to confirm whether study criteria were met.A retrospective study was performed at eleven tertiary-care hospitals in South Korea. Hospitalized adults (≥18 years old) with community-onset cIAI who underwent an appropriate source control procedure such as laparotomy, laparoscopy, or percutaneous drainage of an intra-abdominal abscess between August 2008 and September 2011 were included. cIAIs included conditions such as intra-abdominal abscess related to previous intra-abdominal operations, secondary bacterial peritonitis, appendicitis complicated by perforation and/or a periappendiceal abscess, perforated diverticulitis complicated by abscess formation or fecal contamination, complicated cholecystitis with evidence of perforation, empyema, or gangrene, perforation of a gastric or duodenal ulcer with symptoms exceeding 24 hours in duration, and perforation of the large or small intestine with abscess or fecal contamination . ComputeStaphylococcus aureus (MRSA) infection or colonization; and [Community-onset infection was classified into community-acquired infection and health care-associated infection. Community-onset health care–associated infection was defined when at least one of the following health care risk factors was present:  presenceion; and  a historion; and ,10.Demographic characteristics, laboratory results, underlying diseases or conditions, site of infection, microbiology and antibiogram results, patient management , clinical outcomes, and direct medical costs from the perspectives of the Korean National Health Insurance Corporation were recorded. Failure of initial antibiotic therapy  was defined as a change of antibiotics due to a lack of improvement of the clinical symptoms and signs associated with cIAI within the first week of admission. A change to narrower spectrum antibiotics in patients with clinical improvement was not considered as failure of initial antibiotic therapy. To clearly compare patients with unsuccessful initial antibiotic therapy and those with successful initial antibiotic therapy, patients who underwent additional operative intervention or died within the first week of admission were excluded in the analysis.The primary outcomes were frequency of failure of initial antibiotic therapy, total length of hospital stay, total parenteral antibiotic days, overall medical costs, and in-hospital mortality. The overall medical costs of hospitalization were calculated using standardized reimbursement rates set by the Korean National Health Insurance System. This was possible because South Korea has a national health insurance system with universal coverage of the population. The secondary outcomes were the assessment of risk factors associated with failure of initial antibiotic therapy and the epidemiology of the microorganisms isolated in community-onset cIAIs.2 test or Fisher’s exact test, as appropriate. Normally and non-normally distributed continuous variables were compared using Student’s t-test and the Mann-Whitney U test, respectively. Significant variables with P values less than 0.05 in the univariate analysis were included in the multiple logistic regression model to identify independent risk factors associated with failure of initial antibiotic therapy.Comparisons between patients with unsuccessful initial antibiotic therapy and those with successful initial antibiotic therapy were performed. Categorical variables were compared using the χThe Republic of Korea has universal health coverage of its population, and the national health insurance system pays health care providers on a fee-for-service basis. The overall costs of antibiotic therapy and hospitalization in patients with cIAI were calculated using the standardized reimbursement rates set by the Korean National Health Insurance System. To investigate the factors that affected parenteral antibiotic days, length of hospitalization, and overall costs, univariate linear regression analyses were carried out and the independent covariates were indentified through multiple linear regression analysis. Because the distribution of these variables was skewed, a natural log transformation was performed for the analysis. An analysis of covariance model was used to assess the adjusted mean differences in parenteral antibiotic days, hospital length of stay, and overall costs between the patient groups of successful initial therapy and unsuccessful initial therapy. A two-tailed P value less than 0.05 was considered statistically significant. All statistical analyses were performed using SPSS software, version 18.0 .A total of 539 consecutive patients hospitalized for community-onset cIAI were included. Of these patients, 25 patients who underwent additional operative intervention or died within the first week of admission were excluded and a total of 514 patients were finally included in the analysis. The mean age of the patients was 53.3 ± 17.6 years, 61% (312/514) were male, and 14% (72/514) had health care-associated infection. The most common intraabdominal diagnosis was complicated appendicitis (52%), followed by perforation of the intestine (17%), and perforated duodenal ulcer (16%). Half of the patients had generalized peritonitis. Among the patients included in the study, 180 had documented information on the pathogen . Baseline and clinical characteristics were similar between patients with positive culture and patients without positive culture.Initial antibiotic therapy was successful in 466 (86%) patients and unsuccessful in 48 (9%) patients. Escherichia coli was the most frequently identified pathogen (42%), followed by streptococcus species (15%), Enterococcus species (12%), and Klebsiella pneumoniae (8%). When compared between patients with unsuccessful and successful initial antibiotic therapy, there was no significant difference in the distribution of pathogens and antibiotic susceptibility patterns. Streptococcus species were marginally more likely to be isolated in community-acquired infection than in health care-associated infection (P = 0.084), whereas Enterococcus species and S. aureus were more likely to be isolated in health care-associated infection .Bacterial organisms isolated from cultures of blood and surgical samples obtained during or within 48 hours of surgery/intervention are shown in Enterobacteriaceae isolates causing community-acquired infection was 6% (7/111) and that among Enterobacteriaceae isolates causing health care-associated infection was 21% (6/29). Carbapenem resistant Enterobacteriaceae strain was not isolated. Eighty-five percent (29/34) of Streptococcus species isolates were susceptible to penicillin, and 85% (22/26) of Enterococcus species isolates were susceptible to ampicillin.The extended-spectrum cephalosporin resistance rate among The most commonly used empirical antibiotic regimen was third generation cephalosporin plus metronidazole (52%) . The patPatients with unsuccessful initial antibiotic therapy had poor clinical outcomes and incurred higher medical costs, compared with patients with successful initial antibiotic therapy . FailureP values <0.05 in Among significant univariate variables with We found that 9% of patients admitted from the community with cIAI experienced failure of initial antibiotic therapy in South Korea. Failure of initial antibiotic therapy was associated with poor clinical outcomes and increased costs. After adjustment for covariates, failure of initial antibiotic therapy was significantly associated with longer duration of antibiotic therapy , increased length of hospital stay , and higher medical costs . Independent risk factors for the failure of initial antibiotic therapy were health care-associated infection, solid cancer, and APACHE II ≥13.In the previous studies with a similar operational definition of the failure of initial antibiotic therapy in cIAIs, the failure rate of initial antibiotic therapy was 21–27% ,6. In coThere are a few studies examining the impact of initial antibiotic therapy for cIAIs on outcomes and medical costs –8. InappEnterococcus species, and immunocompromised patients [Enterococcus species and MRSA accounted for about 33% of community-onset health care-associated cIAIs and were significantly more likely to be isolated in community-onset health care-associated cIAIs than in community-acquired cIAIs. Therefore, anti-enterococcal and anti-MRSA coverage should be considered in patients with severe community-onset heath care-associated cIAI to prevent failure of initial antibiotic therapy.In the practice guidelines for cIAIs, empirical anti-enterococcal therapy is recommended for patients with health care-associated infection, particularly those with postoperative infection, those who have previously received antibiotics selecting for patients ,12–15. Epatients . In the Our study has several limitations. Microbial pathogens were isolated in only 35% of patients with cIAI, and most infections were monomicrobial. Therefore, the analysis of the appropriateness of selected initial antibiotics against isolated pathogens was difficult in the present study, because this could be done in a limited number of patients and was not thought to be reliable enough. In the real clinical situation, many surgeons do not routinely obtain operative specimens for bacterial culture in cIAIs. Also in the practice guidelines for cIAIs, routine cultures from lower-risk patients with community-acquired cIAI are considered optional ,16,17. TIn conclusion, failure of initial antibiotic therapy in community-onset cIAIs was significantly associated with longer duration of antibiotic therapy, increased length of hospital stay, and higher medical costs, even after adjustment for potential confounders. Independent risk factors for failure of initial antibiotic therapy were health care-associated infection, solid cancer, and APACHE II ≥13. Therefore, to improve outcomes and costs in patients with community-onset cIAI, rapid assessment of health care-associated risk factors and severity of disease, selection of an appropriate antibiotic regimen accordingly, and early infection source control should be performed.S1 Dataset(XLSX)Click here for additional data file.


IgG4 is the least abundant IgG subclass in human serum, representing less than 5 % of all IgG. Increases in IgG4 occur following chronic exposure to antigen and are generally associated with states of immune tolerance. In line with this, IgG4 is regarded as an anti-inflammatory antibody with a limited ability to elicit effective immune responses. Furthermore, IgG4 attenuates allergic responses by inhibiting the activity of IgE. The mechanism by which IgG4 inhibits IgE-mediated hypersensitivity has been investigated using a variety of model systems leading to two proposed mechanisms. First by sequestering antigen, IgG4 can function as a blocking antibody, preventing cross-linking of receptor bound IgE. Second IgG4 has been proposed to co-stimulate the inhibitory IgG receptor FcγRIIb, which can negatively regulate FcεRI signaling and in turn inhibit effector cell activation. Recent advances in our understanding of the structural features of human IgG4 have shed light on the unique functional and immunologic properties of IgG4. The aim of this review is to evaluate our current understanding of IgG4 biology and reassess the mechanisms by which IgG4 functions to inhibit IgE-mediated allergic responses. The identification of four distinct subclasses of human IgG was first described during the 1960s, when they were designated as IgG1, IgG2, IgG3 and IgG4, based on their relative concentrations in human serum [IgG antibodies interact with immune cells through binding to Fcγ receptors expressed on cell surfaces •. With tIgG4 binds to all of the Fcγ receptors with the exception of FcγRIIIb Table , which cThe crystal structure of human IgG4 Fc was first solved in 1997 in complex with an IgM rheumatoid factor . More reCysteine residues in the hinge region of IgG4 result in intra-heavy chain disulphide bonds, as opposed to the inter-heavy chain bonds present in the other IgG subclasses. In addition, a key amino acid substitution in the Cγ3:Cγ3 interface weakens the domain interactions. Under reducing conditions, the combined effect allows dissociation of the two heavy chains of human IgG4 into half-molecules . Re-asso− B cells [+ B cells express equivalent amounts of IL-10 to their IgG1+ counterparts [+ B cells themselves are not the major source of this cytokine. Circulating IgG4+ B cells from healthy individuals have a similar but distinct phenotype to IgG1+ B cells: they lack surface IgD but express CD27, consistent with the phenotype of class-switched memory B cells [+ B cells compared to IgG1+ B cells from the same individuals. CD21 forms part of the B cell receptor-signaling complex along with CD19 and CD81 and also has an important role in internalization of immune complexes [+ versus IgG1+ B cells. CD23 expression is up-regulated by IL-4, an important switch factor for IgG4, again highlighting the close association between IgG4 and IgE.Naïve B cells express IgM as a monomeric membrane-bound B cell receptor (BCR). Activation of naïve B cells through the BCR can lead to rearrangement of the immunoglobulin heavy chain locus through class switch recombination. This results in the expression of a different ‘switched’ isotype , dependent on additional signals provided by cytokines. Class switch recombination to IgG4 depends on the production of the Th2 cytokines IL-4 and IL-13, along with ligation of CD40 , 16. The B cells , IgG4+ Bterparts ••. This  B cells ••. Expreomplexes . Hence, omplexes •• reportAn important feature of IgG4 production is the association with high-dose chronic antigen exposure. The production of allergen-specific IgG4 is linked to the ‘modified Th2 hypothesis’, whereby an allergen-driven Th2 response in which IgG4 dominates and IgE is absent results in protection from immediate hypersensitivity . This waAIT is an effective treatment for IgE-mediated allergy and induces long-term clinical tolerance, associated with increases in IL-10-producing T regulatory cells and reductions in basophil reactivity . The obsDefinitive evidence that IgG is able to inhibit IgE activity was provided by Van Neerven and colleagues who reported that AIT-induced IgG could inhibit IgE-facilitated allergen presentation (IgE-FAP) by B cells to T cells in vitro . Later sIn addition to inhibiting CD23-dependent IgE activity, IgG4 can also block the effects of IgE signaling through FcεR1, and in turn inhibit immediate hypersensitivity. For example, IgG4 purified from the serum of an immunotherapy-treated individual inhibited IgE-mediated basophil degranulation, which depends on cross-linking of high-affinity IgE receptor (FcεRI) -bound IgE . The abiUsing a bi-specific antibody, Kepley and colleagues demonstrated that cross-linking of FcγRIIb and FcεRI inhibits IgE-mediated basophil activation . The rolKA 2 × 105 M−1) and when IgG4 antibodies were aggregated as F(ab′)2 complexes, binding to FcγRIIb was detected on cell surfaces [The potential for interaction of allergen-specific IgG4 and FcγRIIb to result in effective inhibition of FcεRI signaling remains uncertain. Using surface plasmon resonance, Bruhns and colleagues found that IgG4 bound to FcγRIIb with moderate affinity (surfaces . Howeversurfaces , likely surfaces . This masurfaces , cord-blsurfaces  and synosurfaces  all exprsurfaces  or intessurfaces . TherefoIgG4 is closely associated to the production of IgE and therefore has relevance to the study of allergic disease. Absolute levels of IgG4 often fail to correlate with clinical tolerance, although absolute levels of IgE are similarly poorly predictive of disease severity. Nonetheless, the biological activity of IgG4, in particular the potent inhibition of IgE-mediated basophil/mast cell activation and antigen presentation, suggest that this unique subclass is indeed relevant to disease expression. IgG4 has a long association with tolerance to aeroallergens, both in studies of allergen immunotherapy and the so-called modified Th2 response. An accumulating body of literature also supports a potential wider role for IgG4 in oral immunotherapy studies of food allergy  and also


Background: With the influx of rural migrants into urban areas, the spread of HIV has increased significantly in Shaanxi Province (China). Migrant workers are at high risk of HIV infection due to social conditions and hardships . Objective: We explored the efficacy of a HIV/AIDS prevention and control program for rural migrants in Shaanxi Province, administered at both rural and urban sites. Methods: Guidance concerning HIV/AIDS prevention was given to the experimental group (266 migrants) for 1 year by the center of disease control, community health agencies and family planning department. The intervention was conducted according to the HIV/AIDS Prevention Management Manual for Rural Migrants. A control group of migrants only received general population intervention. The impact of the intervention was evaluated by administering HIV/AIDS knowledge, attitudes and sexual behavior (KAB) questionnaires after 6 and 12 months. Results: In the experimental group; 6 months of intervention achieved improvements in HIV/AIDS related knowledge. After 12 months; HIV/AIDS-related knowledge reached near maximal scores. Attitude and most behaviors scores were significantly improved. Moreover; the experimental group showed significant differences in HIV-AIDS knowledge; attitude and most behavior compared with the control group. Conclusions: The systematic long-term cross-site HIV/AIDS prevention in both rural and urban areas is a highly effective method to improve HIV/AIDS KAB among rural migrants. In 2010, the total number of migrants in China reached 221 million, of which about 72% were rural migrants. It is estimated that more than 3–5 million people will migrate from the countryside to cities and towns in the next 30 years in China . These retc.) ,9,10. Thetc.) ,12. Theretc.) ,14,15, bTo control the rate of HIV/AIDS infection in the general population, effective behavioral intervention programs are provided to migrants . Among tThe objective of the present study was to measure the efficacy of a HIV/AIDS prevention and control program for rural migrants in the Shaanxi Province. Participants were divided into an experimental (EG) and a control group (CG). We analyzed their HIV/AIDS-related knowledge, attitude and behavior (KAB) before, during and after the intervention at both rural and urban sites. We hypothesized that our coordinated intervention at their home village and place of work greatly improve participants’ HIV/AIDS-related KAB. Results of the present study might also be applicable to migrant populations elsewhere in the world.The design of our intervention is shown in We chose one of the poorest counties in China: Lantian County in Shaanxi Province . Most ofInclusion criteria were: (1) 18–49 years old; (2) household registration in rural Lantian; (3) working in a fixed location in downtown Xi’an for at least three months; (4) normal development and language; and (5) willing to participate in this study. Rural migrants with similar geographical or blood relationships tend to work together. Therefore, 293 migrants in three villages of Lantian County  who work in five fixed communities in Xi’an were assigned to the experimental group (EG). The remaining 300 migrants, from other villages of similar background , were assigned to the control group (CG). The protocol was reviewed and approved by the Human Research Ethics Committee of the Xi’an Jiaotong University College of Medicine, and all participants provided written informed consent.The education was tailored to each individual on the basis of the assessment of one’s knowledge about HIV and the capacity to accept the knowledge, and the professional intervention was implemented by the entire intervention group for each individual. The intervention was carried out at least once a month to ensure that the knowledge and methods were taught step by step. Each intervention was based on the examination of the contents of the previous intervention. The content was not repeated if it was mastered, and misunderstood content was corrected; meanwhile, new content was taught. The intervention team included the CDC staff, family planning workers, community health workers and assistants, all with their specifics tasks. The CDC staff was mainly there to train and guide the other members of the intervention team, to provide HIV/AIDS-related knowledge and skills, and to perform HIV testing. In China, family planning workers are in charge of the birth system; their tasks were to perform physical examinations and to assist the community health workers in carrying out HIV/AIDS-related knowledge publicity, and in teaching condom use methods. Community health workers were the implementers of the HIV/AIDS-related knowledge education, and performed counseling psychology, basic physical examinations, education on sexual partner’s communication, and condom use teaching. The other team members were assistants, and were in charge of following up migration population, evaluating intervention’s effect, and assisting the community health workers to carry out health education. The 12-month intervention program includes four aspects . (1) TheThe PMMRM is divided into three parts. (1) Basic information, including the name, age, home address, telephone numbers. The contact information for village health clinics and urban community health centers are also included; (2) The intervention procedure, divided into assessment, survey, implementation, and evaluation. The assessment framework is guided by the KAB theory, including the KAB and its influencing factors, physical assessment (focusing on the reproductive system) and laboratory examination results. The questions based on physical assessment also include the knowledge, attitude, behavior and physical characteristics. The signatures from investigators and surveyed subjects were required to confirm the time, location, content, methods, and names of investigators and migrants in order to supervise the intervention group; (3) The appendix, which contains the basic skills for preventing HIV/AIDS, using texts and pictures. The project meeting was held in the village during the Spring Festival 2009, when migrants returned home. The participants included: co-instructors, rural migrants and their family members. During the meeting, the research purpose, significance and methods were introduced. The participants provided written informed consent, and the PMMRM was distributed. Intervening staff recorded the date at which migrants left for Xi’an, their employer’s address, and their contact information. Before the migrants left, the staff of the migration source area reminded them to take the PMMRM, and informed the intervening staff of the migration destination area. Within 1 week of the migrants arriving in Xi’an, the city’s intervening staff contacted them. Subjects in the EG groups met the intervening staff once a month.Participants in the CG received no specific HIV/AIDS education, except what they may have learned from general education provided to the general population on billboards, pamphlets or other promotional material.The effects of the intervention were measured by the questionnaire of KAB on HIV/AIDS for migrants ,26,27. BThe questionnaires included demographic data , and four statements about HIV/AIDS . All items had three possible responses: “True, Don’t know and False”. Positive statements were scored as “True = 2, Don’t know = 1 and False = 0”, and negative statements are scored as “True = 0, Don’t know = 1 and False = 2”. The total score ranges from 0 to 50. The higher the score, the better the knowledge level of HIV/AIDS is. Attitude: The HIV/AIDS attitude questionnaire consists of 3 dimensions  and 26 questions with a 4-point scale ranging from “agree” to “disagree”. There are 11 positive items scored as “agree = 3, undecided = 2, indifferent = 1, disagree = 0” and 15 negative items reverse-scored. The total possible score ranges from 0 to 78. Higher score shows more positive attitude. Sexual behavior: the behavior questionnaire consists of 16 items, in which 8 are related to sexual behavior, 4 to selling blood, and 4 to drug abuse.The English version of the questionnaire was back-translated and discussed repeatedly by linguists. Before use, the questionnaire was assessed thrice by three experts, who majored in epidemiology, communicable diseases and nursing, respectively, to ensure the format and content validity of the questionnaire. Furthermore, 50 subjects were pre-surveyed. The Cronbach’s alpha coefficient of the knowledge questionnaire was 0.79, 0.78 for the attitude questionnaire, and 0.81 for the whole questionnaire.In the present study, sample size determination was based on the rate of change in high-risk sexual behaviors after intervention. It was reported that the high-risk sexual behavior of rural migrants changes from 0.3% to 18.2% after comprehensive intervention . Thus, bP-values < 0.05 were considered statistically significant.Statistical analysis was performed using SPSS 16.0 for windows . All variables were initially analyzed descriptively, including means and standard deviation for the quantitative data, and frequencies and percentages for the categorical variables. The difference in knowledge or attitude between the EG and CG was assessed by independent sample t-test. The chi-square test was used to detect the difference of behavior on HIV/AIDS. p > 0.05). Most rural migrants were males (53.2% and 56.0% in EG and CG), married (91.8% and 92.3% in EG and CG), and had completed junior middle education level (68.3% and 66.3% in EG and CG)  .p > 0.05). Aside from the relatively high score on transmission route knowledge, the total score and the other dimension scores were low. The score for misconceptions about transmission route knowledge was especially low. There were significant differences between the EG average knowledge score before and after 6 or 12 months of intervention (p < 0.05). During the intervention, the total score and each dimension score gradually increased, and the scores after 12 months were close to the maximum value (p < 0.05). In the CG, total score, basic knowledge score and transmission route score after 12 months were significantly higher than those before the intervention (p < 0.05), but there was no significant increase in the scores for the categories “misconceptions about transmission route” and “prevention-treatment” after the intervention period (p > 0.05).um value . The totp > 0.05). After 6 months, the EG total score and sub-attitude scores relating to behavior changes were significantly increased (p < 0.05). However, the scores of attitude to AIDS and infected patients did not change (p > 0.05). After 12 months, the total score and each sub-score of the EG were significantly higher than those before intervention or those of the CG (p < 0.05); the scores also gradually increased with prolonged intervention time (p > 0.05).ion time . In the Before the intervention, only one participant sold blood or accepted voluntary HIV counseling and testing (VCT). Therefore, the comparison of HIV/AIDS behavior focused on sex-related behaviors. Excluding the subjects who had no sexual activity during the last 3 months, there were 274 (pre-intervention), 280 (post-6 months intervention), and 266 (post-12 months intervention) subjects in the EG, and 286 (pre-test) and 260 (post-test) in the CG in whom sex-related behaviors were compared.p > 0.05). After 6 months, there were significant changes in only 2 items in the EG  (p < 0.05). After 12 months, we found that most behaviors  were significantly improved (p < 0.05), and that only one item  did not improve significantly (p > 0.05). For each item within the CG, there was no significant difference between pre- and post- intervention (p > 0.05).We explored the efficacy of a HIV/AIDS prevention and control program for rural migrants in the Shaanxi Province by analyzing the HIV/AIDS-related KAB variations resulting from interventions at both rural and urban sites. Comparison between the EG and CG highlighted that the intervention achieved a significant improvement of participants’ HIV/AIDS-related KAB.There are many studies on HIV/AIDS prevention in rural migrants, but most interventions are cross-sectional and one-sided ,30. In tvs. high-risk), to his literacy, to his technology accessibility (compact disc vs. brochure) and to his availability. In addition, studies showed that HIV/AIDS prevention knowledge from different sources will produce different results, and that people are most willing to accept the prevention knowledge from medical professionals [Secondly, the independent AIDS-PMMRM was provided for each migrant. Based on systematic assessment, each intervention was implemented in accordance with migrant’s physical and psychological characteristics and lifestyle, to ensure that each participant receives the appropriate educational content. This was done by tailoring the intervention to the participants according to his sexual behaviors  were not improved. These results indicate that the HIV/AIDS propaganda administered in urban and rural areas have a certain impact, but the breadth and depth of the effects are insufficient as a consequence of complex factors, such as time, place, method, educators, The level of HIV/AIDS knowledge directly affects the attitude to AIDS, and participants with a lower cognitive level have more serious discriminatory attitudes towards those suffering from HIV/AIDS ,34. MoreAfter 6 months of intervention, the participants recognized that condoms are a simple and efficient mean to prevent HIV/AIDS, and they learned some skills for communicating with their sexual partners. The reason why the detection of STDs increases is that more migrants are tested. After 12 months, the rates of condom use within the EG were significantly increased. Also, more migrants used condoms for the purpose of disease protection. The incidence of participation in commercial sex also decreased, but the number of sexual partners did not change. We concluded that participants’ understanding of sexual health and the extent to which they trust their sexual partners influenced their sexual behavior. This conclusion is in accord with previously published research . CommercAfter 12 months, the frequency of VCT in the EG were significantly increased. Regular tracking and information collection showed that those who participated in VCT were mainly those who have sold blood or transfused blood, while drug users and people with dangerous sexual behavior still rarely participated in VCT. This means that participation in VCT may be related to the routes of infection. If the route of infection involves what is traditionally considered to be "immoral behavior", infected individuals are less willing to participate in VCT, fearing discrimination and apathy.In the present study, we selected rural migrants aged 18–49 because: (1) migrants aged 18–49 represent 69.9% of all migrants in China [The present study suffers from some limitations. First, the home villages of the participants were relatively close to their working place. Considering that most people in the Lantian County worked in Xi’an city and also in order to facilitate the survey, the Lantian County and Xi’an city were selected as survey locations in this study. Xi’an is the capital city of Shaanxi Province while Lantian County is in the territory of Shaanxi Province. Previous studies have demonstrated that rural migrants working in places farther from home have the higher incidence of high-risk behavior compared with those working closer to home ,14,15. TWe observed that the cross-center HIV/AIDS prevention and management model could improve rural migrants’ HIV/AIDS-related knowledge, attitude and behavior . Further studies would further illuminate the extent of this improvement, examine how it may be maintained, and study the impact of this intervention on the HIV acquisition rate of participants. In addition, this model could be used elsewhere in the world.


Third generation cephalosporin were the most commonly prescribed antibiotics (28.7% of prescriptions). Reasons for antibiotic use included treatment of community—(27%) and healthcare-associated infections (11%) and surgical (39%) and medical (23%) prophylaxis. Among surgical prophylaxis recipients, only 28% of evaluable cases received the first dose within two hours before incision and only 25% of cases received surgical prophylaxis for <24 h. The prevalence of antibiotic use in Egyptian hospitals was high with obvious targets for antimicrobial stewardship activities including provision of antibiotic prescription guidelines and optimization of surgical and medical prophylaxis practices.Inappropriate antibiotic use leads to increased risk of antibiotic resistance and other adverse outcomes. The objectives of the study were to determine the prevalence and characteristics of antibiotic use in Egyptian hospitals to identify opportunities for quality improvement. A point prevalence survey was conducted in 18 hospitals in March 2011. A total of 3408 patients were included and 59% received at least one antibiotic, with the most significant use among persons <12 years and intensive care unit patients ( Excessive and inappropriate use of antibiotics is highly associated with the emergence of antibiotic resistance , which pi.e., participant or non-participant) in an HAI surveillance pilot program. Hospitals were then randomly selected from each group for inclusion in the study.Hospitals included in the study were selected from among 37 Egyptian hospitals that were participating in a larger project to establish surveillance programs for healthcare-associated infections (HAIs) and antimicrobial resistance. For the antibiotic prevalence survey, a 50% sample was selected from among the 37 participating hospitals. Hospitals were stratified into two groups based on their participation status  Project . Data weInformation on patient diagnosis group (body site for which antibiotics were being administered), and the indication of antibiotic use  were obtained from treating physicians. The medical records were examined for the presence of documentation of the indication of antibiotic use.Additional information was collected from the records of patients receiving antibiotics for surgical prophylaxis. These data included timing of the first dose in relation to the timing of the surgical incision and the duration of prophylactic antibiotic administration as of the day of the survey.Each participating hospital identified four persons with medical background to collect the survey data who were familiar with the hospital departments, wards and staff. They were either nurses, doctors or pharmacists. A specific day for data collection was pre-assigned for each hospital ward. All patients on a given hospital ward at 8:00 am on the day that the survey was scheduled were included in the survey. Data were collected on paper forms at each participating facility, reviewed for completeness by the director of the infection prevention and control team, and submitted to project coordinators at U.S. Naval Medical Research Unit No.3 (NAMRU3) in Cairo, where the data were entered into an electronic database for analysis. The study protocol was approved by the NAMRU3 Institutional Review Board as Non-research Protocol No. 1111—(DOD# namru3nr.2011.0011).t-test was used to compare continuous variables. Standard methods were used to calculate 95% confidence intervals (CIs) for the proportion of patients who received antibiotics in each hospital.Statistical analyses were conducted using STATA 11 . Differences in rates between groups were compared using the Chi-square test or Fisher’s exact test; The study was completed during the period March 13–27, 2011. The 18 participating hospitals included five Ministry of Health and 13 university hospitals. Hospitals were classified as general hospitals (11), obstetrics and gynaecology (3), pediatric (2), general surgery (1) and orthopedic (1). The participating hospitals had a total of 7204 beds . Fifty-six intensive care units  were present in the 18 hospitals. Six of the hospitals (33%) reported that antibiotic use guidelines were available, but did not present the guidelines when asked for them. On the days of the survey, there were 3408 patients in the participating hospitals. Within the individual hospitals, the number of patients ranged from 23–447. A total of 2017 patients (59%) were receiving one or more antibiotics. The prevalence of antibiotic use in the participating hospitals ranged from 32.9%–91.7% . The 201The prevalence of antibiotic use was significantly more common among males, younger age groups, and intensive care unit patients . The mosThe most common anatomical sites involved in the prophylactic use of antibiotics were the gynecologic tract (23.9%), gastrointestinal tract (16.3%), and skin, bone, and joints (14.8%) . Among pOut of 1572 patients in the surgery wards, 802 patients (51%) received antibiotics for surgical prophylaxis. Among these 802 patients, 702 (87.5%) had information in their medical charts regarding the start time of the prophylactic antibiotic(s). The duration of antibiotic prophylaxis, however, could be determined for only 333 patients (41.5%) . Out of To our knowledge, this is the first large scale assessment of antibiotic use practices outside of North America and Europe. In this project, we applied the same survey method for the assessment of the prevalence of and indications for antibiotic use as that used by the European Surveillance of Antimicrobial Consumption (ESAC) Project  to charaThis study identified that 59% of patients in the participating Egyptian hospitals were receiving one or more antibiotic agents at the time of survey completion. This is substantially higher than the prevalence of antibiotic use reported in similar studies performed in Europe and the US ,7,8. Alti.e., more than 24 h). Recent European studies have also demonstrated that prolonged administration of antibiotics for surgical prophylaxis is common. In the 2009 ESAC Survey, surgical antibiotic prophylaxis was administered for >24 h in 53% of patients [Evaluation of the specific indications for antibiotic use revealed several specific opportunities for improvement. Surgical prophylaxis was the most common indication for antibiotic administration reported, accounting for 38.4% of all antibiotic prescriptions. A review of the details of surgical prophylaxis prescribing revealed that the selection, timing, and duration of administration were frequently inconsistent with the evidence-based practices recommended in Europe and the US . For exapatients  whereas We also identified that a large proportion of patients receiving antibiotics for “medical prophylaxis” had no justification of a medically-accepted indication for prophylaxis. This group of patients accounted for 14% of all antibiotic use and 60% of patients who were receiving antibiotic therapy for the purpose of medical prophylaxis. Discussions with clinicians suggested that it was relatively common practice in some of the participating hospitals for patients to be given antibiotics during their hospital stay to reduce the risk of acquiring healthcare-associated infection. This suggests that efforts to strengthen basic infection prevention practices would eventually improve clinicians’ confidence in infection prevention and control program which could result in substantial reductions in the use of antibiotic agents and the associated complications among hospitalized patients. Although this study provides novel data that will be useful for quality improvement initiatives in individual hospitals and for the Egyptian healthcare system in general, a few limitations should be noted. First, data were collected from only 5% of the hospitals in Egypt. The data may therefore not be representative of antibiotic use in all hospitals throughout the country. In addition, the data were collected from a convenience sample rather than a random sample of Egyptian hospitals. The distribution of hospital types among those that participated in the survey is quite different from the overall distribution of hospital types in Egypt. We sampled approximately 30% of the university hospitals but less than 2.5% of the Ministry of Health hospitals. Finally, although all persons involved in data collection had completed a formal training program prior to participating in the survey, there was no central validation of the submitted data.In conclusion, this is the first study to quantify and characterize antibiotic use practices in hospitals in Egypt. The information gained from this point prevalence survey has helped to prioritize limited resources by allowing identification of several specific opportunities to improve antibiotic use practices that may result in improved patient outcomes and lower healthcare costs within Egyptian hospitals. Although the data and their implications have largely been presented and discussed here in aggregate form up to the national level, data from the individual participating hospitals may prove to be even more useful for identifying more specific opportunities for quality improvement within these hospitals. We intend to repeat the survey in order to assess changes in the prevalence and characteristics of antibiotic use in Egyptian hospitals over time, as we thought that the point prevalence survey implemented was an efficient and simple method for assessing antibiotic use in Egypt.


Staphylococcus aureus is a leading cause of life-threatening infections worldwide. The MIC of an antibiotic against S. aureus, as well as other microbes, is determined by the affinity of the antibiotic for its target in addition to a complex interplay of many other cellular factors. Identifying nontarget factors impacting resistance to multiple antibiotics could inform the design of new compounds and lead to more-effective antimicrobial strategies. We examined large collections of transposon insertion mutants in S. aureus using transposon sequencing (Tn-Seq) to detect transposon mutants with reduced fitness in the presence of six clinically important antibiotics—ciprofloxacin, daptomycin, gentamicin, linezolid, oxacillin, and vancomycin. This approach allowed us to assess the relative fitness of many mutants simultaneously within these libraries. We identified pathways/genes previously known to be involved in resistance to individual antibiotics, including graRS and vraFG (graRS/vraFG), mprF, and fmtA, validating the approach, and found several to be important across multiple classes of antibiotics. We also identified two new, previously uncharacterized genes, SAOUHSC_01025 and SAOUHSC_01050, encoding polytopic membrane proteins, as important in limiting the effectiveness of multiple antibiotics. Machine learning identified similarities in the fitness profiles of graXRS/vraFG, SAOUHSC_01025, and SAOUHSC_01050 mutants upon antibiotic treatment, connecting these genes of unknown function to modulation of crucial cell envelope properties. Therapeutic strategies that combine a known antibiotic with a compound that targets these or other intrinsic resistance factors may be of value for enhancing the activity of existing antibiotics for treating otherwise-resistant S. aureus strains. Staphylococcus aureus, a leading cause of community- and hospital-acquired infections, would inform the design of new drugs as well as the identification of compounds that enhance the activity of existing drugs. To identify factors that limit the activity of antibiotics against S. aureus, we used Tn-Seq to simultaneously assess fitness of transposon mutants in every nonessential gene in the presence of six clinically important antibiotics. This work provides an efficient approach for identifying promising targets for drugs that can enhance susceptibility or restore sensitivity to existing antibiotics.Bacterial resistance to every major class of antibiotics has emerged, and we are entering a “post-antibiotic era” where relatively minor infections can lead to serious complications or even death. The utility of an antibiotic for a specific pathogen is limited by both intrinsic and acquired factors. Identifying the repertoire of intrinsic resistance factors of an antibiotic for  Staphylococcus aureus is a Gram-positive pathogen with a remarkable ability to withstand antibiotics and evade the human immune system. Many factors, both intrinsic and acquired, have been shown to contribute to its ability to survive specific antibiotic stress. For example, methicillin-resistant S. aureus (MRSA) strains have acquired the mobile staphylococcal cassette chromosome mec element (SCCmec), encoding a transpeptidase, PBP2A, which is naturally resistant to β-lactams, enabling the organism to make cross-linked peptidoglycan when the native transpeptidases are inactivated by the β-lactams   –15 to exTn-Seq involves creating large transposon libraries, sequencing the transposon insertion sites with next-generation sequencing, and mapping the sequence reads to a reference genome , 15. ThiStaphylococcus aureus. We have performed Tn-Seq analysis using transposon libraries treated with six different antibiotics to identify genes with significantly fewer mapped reads than were seen with an untreated control. These genes, which we refer to as intrinsic resistance factors, render the bacteria more sensitive to the antibiotic tested when inactivated. The six antibiotics used in this study, ciprofloxacin, linezolid, gentamicin, oxacillin, vancomycin, and daptomycin, were chosen as clinically relevant representatives of antibiotics that target major pathways: DNA synthesis, protein synthesis, cell wall synthesis, and membrane stability (–Tn-Seq has been used previously to identify antibiotic resistance factors for other organisms –18 but h–tability  and B (1tability –25. In aS. aureus (MSSA) strain HG003 were used in initial screens for mutants exhibiting either enhanced resistance or enhanced susceptibility to an antibiotic (in vivo), the libraries were treated with six antibiotics at concentrations below the MIC of the antibiotics and grown in different media for various numbers of generations .Two different transposon libraries constructed in methicillin-susceptible tibiotic , 26. Thetibiotic . To idenU test, the experimental condition (antibiotic treatment) was normalized to that of the untreated control using simulation-based resampling to minimize differences between the two conditions  for ciprofloxacin to 55-fold for oxacillin. The top genes contributing to susceptibility/resistance for each antibiotic included genes with fewer as well as more reads mapping to them in the treated sample than in the control. The former represent intrinsic resistance factors or impediments to antibiotic inhibition of S. aureus. The latter are also of interest as they provide information on how antibiotic resistance can arise via gene inactivation.Because antibiotics with differing mechanisms of action exert different degrees of selective pressure on the bacterial population at fractional MIC levels, it was necessary to tailor the data analysis approach in a way that enabled comparison across different antibiotic treatments. We therefore adjusted the cutoff value for the fold change in the number of mapped reads/gene for the treated samples relative to the controls to obtain similar numbers of hits for each antibiotic. Only genes corresponding to a Of the genes implicated in antibiotic resistance via the analysis described above, 80 were unique see , 31; dissigB was among the hits identified with oxacillin treatment. Reads mapping to this gene, and to other components involved in the alternative sigma factor pathway, i.e., rsbV and rsbW, were significantly depleted after growth with oxacillin   system that regulates the cell wall stress stimulon  . It has xacillin , 33. Reasistance –36, werestimulon –41, weresistance , 43, wasmprF, ndh, fmtA, components of the graRS and vraFG (graRS/vraFG) multicomponent sensing system, and two genes of unknown function, SAOUHSC_01025 and SAOUHSC_01050.While no inactivated gene altered susceptibility to all six antibiotics, we did identify 21 genes that were hits with two or more antibiotics, and 8 of these were hits with more than two antibiotics  is the only one that, when inactivated, promotes resistance to some antibiotics while promoting sensitization to others. Ndh is a component of the electron transport chain. The electron transport chain creates a membrane potential, which is required for penetration of gentamicin through the cell membrane , a phenotype correlated with persistent infections that are resistant to β-lactams as well as aminoglycosides  background . Si. SimprF ptomycin , 60.S. aureus contains many multicomponent sensing systems, and we identified multiple components of three of these systems, agrABCD, vraTSR, and graXRS/vraFG, among the top hits under conditions of selection with at least one antibiotic (Multicomponent sensing systems (MCSs) allow bacteria to sense and respond to their environments. These systems typically include a membrane-anchored extracellular sensory domain fused to an intracellular kinase domain and a separate, cytosolic response regulator, but they can also include additional elements. A stimulus sensed by the sensory domain results in a change in the phosphorylation state of the response regulator, which in turn modulates the expression of downstream targets . S. aureAgrABCD is involved in quorum sensing and regulation of virulence factors and autolysin expression . We first tested the K-nearest neighbors algorithm by using graX, graS, vraF, or vraG as the query gene and then searched across all nonessential genes for those with the most similar fitness profiles for all six antibiotic selections. For each of these genes, at least two of the other components of the graXRS/vraFG MCS system were among the five genes that most closely paralleled their contribution to fitness in these tests. This result was expected because the individual components of the graXRS/vraFG system would be expected to exhibit similar fitness profiles  most similar to those of profiles . We thenand cvfC . In addiSAOUHSC_01025 (SAOUHSC_01025::Tn) and SAOUHSC_01050 (SAOUHSC_01050::Tn) against a panel of 12 antibiotics with a greater range of different targets. In addition to the six antibiotics originally used, we added moenomycin, targocil, bacitracin, fosfomycin, mupirocin, and rifampin  cation-adjusted broth and incubated for 15 h at 37°C with shaking at 200 rpm. A 10-µl aliquot (106 CFU) of this input culture was then inoculated into a final volume of 200 µl in a 96-well plate broth microdilution format and incubated at 37°C for 8 h, representing approximately 5.5 generations (5 × 107 CFU/200 µl). The 0.5×, 0.25×, and 0.125× MIC wells for the library pool were determined on the basis of the MIC of a small mutant pool (consisting of 10 innocuous transposon mutants). This small pool was used to determine MICs in order to compensate for resistant mutants potentially present in the library pool. The chosen wells  were then subcultured (3 × 105 CFU) into a second iteration of serial dilutions of antibiotics as described above and incubated for 15 h at 37°C, representing approximately 9 generations (2 × 108 CFU/200 µl). The 0.5×, 0.25×, and 0.125× MIC wells were determined based on the small pool, and these wells were transferred to 10 ml of brain heart infusion (BHI) broth and incubated for 4 h at 37°C with shaking at 200 rpm. Biological replicates were conducted for each growth condition. Genomic DNA was harvested using a DNeasy blood and tissue kit  following the manufacturer’s instructions. Library 1 was prepared for next-generation sequencing using the shearing method as described previously . For all antibiotics, an untreated control was prepared in the same media as was used for the tested antibiotic. A stock of the complete library was thawed and diluted to an optical density at 600 nm (OD600) of 0.2 and grown to an OD600 of ~0.4 to minimize changes in library composition prior to treatment. The culture was then diluted to 4 × 105 CFU/ml and added to 1 ml of media with 2× the desired concentration of the antibiotic to give a final starting inoculum of 2 × 105 CFU/ml in 2-ml culture volumes. A 107 CFU/2 ml starting inoculum was used for vancomycin- and daptomycin-treated samples. Samples were grown at 37°C and harvested when they reached stationary phase (~1 × 109 CFU/2 ml). The samples were treated with 2×, 1×, 0.5×, and 0.25× the MIC of the antibiotic. Then, we identified antibiotic concentrations that caused the transposon library to reach stationary phase with a few hours of delay compared to the untreated control. These antibiotic concentrations were prepared for sequencing following the protocol described by Santiago et al. . These were processed for further analysis. This percentage of decrease was chosen so that we could identify genes with an increase and a decrease in the number of reads mapping to them. Library 2 contains transposon constructs with outward-facing promoters that can upregulate proximal genes in addition to the traditional construct which can only insert into and inactivate genes. For these experiments, we considered only data from the inactivation constructs. Data for both library 1 and library 2 were analyzed as described previously , with thsampling , 29. DatqoxA, qoxB, ndh, fmtA, SAOUHSC_01025, and SAOUHSC_01050 were validated using transposon mutants from the Nebraska library in the USA300 LAC JE2 background (dltA and graR deletion mutants were tested in methicillin-susceptible S. aureus (MRSA) strain Newman (2+ (25 mg/liter) and Mg2+ (12.5 mg/liter) and the six antibiotics at concentrations below the MIC. Overnight cultures of mutants were diluted 1:100 in fresh TSB and grown to an OD600 of 1. They were then serially diluted 10-fold, spotted onto an agar plate, and incubated at 37°C overnight. The concentration of the antibiotic at which growth of the WT was severely inhibited, showing growth in only the highest 1 or 2 dilutions on agar plates under these conditions, was determined. This was considered to be the MIC under these conditions. The spot dilution assays were then set up using three different antibiotic concentrations. The concentration closest to the MIC at which the WT was at most 3 logs more depleted than the control was used to calculate fitness. This concentration was used so that reduced fitness of any mutants could be observed. The exception to this was the use of the MIC for gentamicin to evaluate the resistance of inactivation mutants in the oxidative phosphorylation pathway  Schematic of the oxidative phosphorylation pathway is depicted here. Numbers of reads due to transposon insertions were greatly increased under conditions of exposure to gentamicin for the 11 genes illustrated. Inactivation of the oxidative phosphorylation pathway is a known mechanism of resistance to gentamicin, which depends on the membrane potential for cell entry. (B) Results representative of the fold change in the number of reads/gene for a subset of genes involved in oxidative phosphorylation that were tested to determine if inactivation confers resistance to gentamicin. (C) Fitness comparison of the WT to mutant strains in which the indicated genes were inactivated. Spot dilutions of WT and mutant strains were plated on gentamicin, and fitness was calculated as the ratio of the highest dilution that allowed growth of the WT to highest dilution that allowed growth of the mutant . Download Figure S1, PDF file, 0.2 MBFigure S2 SAOUHSC_01025::Tn and SAOUHSC_01050::Tn are particularly sensitive to antibiotics that damage the cell envelope. (A) Data representative of results of analysis of the targeted pathways of the additional antibiotics tested against SAOUHSC_01025::Tn and SAOUHSC_01050::Tn mutants are shown. The abbreviations used here are as follows: mup, mupirocin; lin, linezolid; rif, rifampin; cip, ciprofloxacin; gen, gentamicin; van, vancomycin; bac, bacitracin; tar, targocil; fos, fosfomycin; moe, moenomycin A; dap, daptomycin. (B) A summary of the fitness of these mutants relative to that of the WT was assessed by spot dilution against the various antibiotics tested. (C) Spot dilution assay plates for these mutants and all antibiotics tested are shown here. The results obtained with the first six antibiotics are reproduced from Figure S2, PDF file, 0.3 MBTable S1 A total of 80 unique genes were identified as important for fitness by treatment of pooled transposon libraries with six antibiotics. The top 20 genes with the greatest fold change in numbers of mapped reads are shown for each antibiotic. Fold change in the number of mapped reads is indicated by colored rectangles. Orange rectangles indicate genes for which the numbers of reads due to transposon insertions were substantially lower than in the control, whereas blue rectangles indicate genes for which the numbers of reads due to transposon insertions were substantially higher than in the control. Gray rectangles indicate that they were not identified as a hit using that antibiotic treatment.Table S1, PDF file, 0.3 MB