A possible correlation between MET receptor expression, MET gene alterations and the two most frequent occurring EGFR gene mutations was also investigated.To compare the efficacy of silver MET and IGF1Rgenes by SISH in paraffin-embedded, formalin-fixed material. Correlations were made with the immunohistochemical expression of two frequent EGFR mutations and clinicopathological variables. Univariate and multivariate survival analyses was used to evaluate the prognostic efficacy of the tested markers.Stage I to IIIA tumors from 326 patients with NSCLC were immunohistochemically tested for protein expression of MET and IGF-1. Their cytoplasmic expression was compared with the gene copy number of the MET gene to chromosome 7 ratio was a significant positive prognostic marker (p = 0.005), probably only due to the highly negative prognostic significance of chromosome 7 polysomy (p = 0.002). High IGF1R gene copy number was a negative prognostic marker for all NSCLC patients (p = 0.037). In the multivariate analysis, polysomy of chromosome 7 in tumor cells correlated significantly and independently with a poor prognosis (p = 0.011). In patients with adenocarcinoma, a high cytoplasmic MET expression was an independent negative prognostic marker (p = 0.013). In males a high IGF1R gene copy number to chromosome 15 count ratio was significantly and independently correlated to a poor prognosis (p = 0.018).In univariate analyses, high cytoplasmic MET expression showed a significant negative prognostic effect in adenocarcinoma patients (p = 0.026). IGF1R gene copy number to chromosome 15 count ratio is an independent predictor of inferior survival in male patients with primary NSCLC.MET protein expression provides superior prognostic information compared with SISH. Polysomy of chromosome 7 is an independent negative prognostic factor in NSCLC patients. This finding has an important implication while examining genes located on chromosome 7 by means of SISH. High  Lung cancer is the leading cause of cancer-related mortality in men and the second among women worldwide. With annMET  is activated by its ligand HGF and exerts broad biological effects associated with malignancy including cell proliferation, cell scattering and migration, induction of cell polarity, and angiogenesis. MET is rMET has recently also gained ground as an important target in the treatment of malignancy. Using the MET inhibitor onartuzumab plus erlotinib, an improved progression-free survival (PFS) and a better overall survival (OS) was seen in immunohistochemically MET-positive NSCLC patients, while a worse outcome was observed in MET-negative patients treated with onartuzumab. However,in situ hybridization (SISH). Due to observations reporting MET activation and treatment resistance following anti EGFR treatment,[EGFR; E746-A750deletion mutation of exon 19 (EGFRdel) and the single L858R deletion mutation of exon 21 (EGFRmut). In the light of studies indicating IGF1R as a putative coactivator of MET  in patients with NSCLC.Herein, we conducted a study investigating the prevalence and the prognostic role of MET in NSCLC. We compared the expression of MET as detected by immunohistochemistry (IHC) with its gene amplification by means of silver reatment,, 13 we ar of MET , we invein-situ disease regarding the new lung cancer classification resulting in a total of 326 patients eligible for this study.[This retrospective study utilized primary tumor tissue from patients diagnosed with NSCLC stage I–IIIA; the tissue was surgically resected at the University Hospital of Northern Norway and Nordland Central Hospital between 1990 and 2004. Three hundred seventy-one patients were registered from the hospitals’ databases. The following exclusion criteria were employed: (1) radiotherapy or chemotherapy prior to surgery, (2) other malignancy within 5 years before the NSCLC diagnosis and (3) inadequate paraffin-embedded tissue blocks. Thirty-six patients fell into these three categories  and were excluded from the study. Adjuvant chemotherapy had not yet been introduced as a therapeutic option in Norway during this time span (1990–2004). In total, 335 patients with complete medical records and adequate paraffin embedded tissue blocks were included in this study. The tumors were subtyped and histologically graded according to the recent World Health Organization (WHO) guidelines. The patiis study. The RegiTwo experienced pathologists (S.A.S. and K.A.S.) investigated all the lung cancer specimens thoroughly. Tissue microarray (TMA) blocks were constructed using a tissue-array instrument  as previously described .IGF1R gene and Chromosome 15 probe . MET gene and chromosome 7 probe . IGF-I . The detailed methodology for immunohistochemistry and silver in situ hybridization has been previously published[The applied antibodies have been previously subjected to in-house validation by the manufacturer for IHC analysis of paraffin-embedded material. The antibodies used in this study were as follows: Phospho-MET Receptor . EGF Receptor . EGF Receptor.published.MET and IGF1R genes resulted in signals as black dots on the corresponding chromosomes for both genes, while centromeres of chromosome 7 and 15 were stained as red dots. Regarding SISH scoring, uniform guidelines exist and were strictly followed for the interpretation of gene and chromosome signals.[HER2sish is reported in breast cancer as the ratio of the average number of HER2 gene copies to the average number of chromosome 17 copies (HER2:chr17) per cell. Because no clear guidelines have been established for measuring MET or IGF1Rgene amplification in NSCLC, we also sought to determine whether the absolute number of MET and IGF1Rgene copies detected by SISH  would add prognostic significance beyond that established by the gene copy number to chromosome count ratio. From each tumor, four cores were eligible for scoring. In each core, we counted genes and centromere signals in 20 cells at least in two cores, where one core was taken from the central part of the tumor and the other core was taken at the advancing edge of tumor. An overall average was taken for both gene and centromere count. The other two cores included for the most stromal tissue surrounding epithelial cells of NSCLC. Heterogeneity was not observed while scoring immunostains. Regarding SISH, in cases of heterogeneity hot spots with the highest gene or centromere count were scored. The number of gene copies was assessed according to the manufacturer’s protocols for INFORMHER2 DNA. Briefly, a discrete dot was counted as a single copy of MET, IGF1R, chromosome 7 or chromosome 15. Some nuclei showed multiple discrete copies. Clusters of dots representing many copies of the targetgenes were also apparent; a small cluster of multiple signals was counted as six copies and a large cluster was counted as 12 copies.The tissue cores were scored by light microscopy to determine the degree of cytoplasmic and nuclear expression. Examples of various markers’ expressions are shown . Stainin signals.Even thouStaining for MET, IGF-1, EGFRmut and EGFRdel resulted in a homogenous cytoplasmic staining . Due to MET SISH and EGFRdel) between the pathologists, with an intra-class correlation coefficient of 0.91 (P < 0.001) for MET SISH and 0.93 (P < 0.001) for EGFRdel.All of the anonymized samples were semi quantitatively and independently scored by two experienced pathologists (S.A.S. and E.R). In the event of disagreement, the slides were re-examined and a consensus was reached by the observers. When assessing one variable for a given core, the observers were blinded to the scores of the other variables and the outcome. The reproducibility of the IHC and SISH evaluation in randomly selected specimens is high. The IHC and SISH scores from each observer were compared for interobserver reliability using a 2-way random effects model with absolute agreement definition, yielding an intra-class correlation coefficient (reliability coefficient) and Cohen’s kappa. There was an excellent scoring agreement for two tested markers . DSS was determined from the date of surgery until the time of lung cancer death. Statistical significance between the survival curves was assessed utilizing the log-rank test. Cut-offs were chosen by a minimal p-value approach, with regard to the association between markers and survival endpoints. The survival curves were terminated at 120 months as fewer than 10% of patients were at risk after this point. Statistically significant variables from the univariate analysis were included in the multivariate analysis, applying the Cox proportional hazards model. The data were run in a backward stepwise Cox regression with a probability for stepwise entry and a removal set at 0.05 and 0.10. The significance level was set at a P-value less than 0.05.The statistical analyses presented in this study were performed using the statistical package IBM SPSS, version 24 .Chi-square and Fisher’s exact test were used to examine the correlation among different molecular markers and clinicopathological factors. The We retrospectively examined a non-selected group of NSCLC patients, which was examined in previous works by our research group.Clinical,in situ hybridization analysis of the MET gene copy number on chromosome 7, and 237 cases were eligible for the IGF1R gene copy number analysis on chromosome 15.Of 326 cases, all were immunohistochemically evaluable for the expression of MET and IGF-1, while 295 were evaluable for the silver EGFRgene mutations, 313 cases were eligible for EGFRmut, while 318 cases were eligible for the detection of the EGFRdel mutation.For the immunohistochemical analysis of MET, IGF-1, EGFRmut and EGFRdel showed a homogenous cytoplasmic staining pattern.We found high (moderate to strong) cytoplasmic expression of MET and IGF-1 in 82.8% and 6.1% of valid tumor samples, respectively. The reported prevalence of phosphorylated MET in our material is consistent with results demonstrated by other investigators. –21. HoweMET gene to chromosome 7 ratio higher than 1 was observed in 7.1% of valid tumors, while an IGF1R gene to chromosome 15 ratio higher than 1 was observed in 21.6% of valid tumors. In the male cohort population, 6.1% of valid tumors showed an IGF1R gene to chromosome 15 ratio higher than 1, p = 0.015, while this was observed in 15.5% of all females with NSCLC, p = 0.021. An EGFRdel mutated protein indicating a gene mutation was found in 6% of all valid patients  while 5.4% showed an EGFRmut mutated protein, indicating a mutated gene . Prevalence of investigated factors is shown . We sought to determine if different cut-off points for the MET gene/chromosome 7 ratio would provide an additional prognostic significance. A MET gene/chromosome 7 ratio >1.5 (p = 0.21) did not show a prognostic significance. The same applies for a MET gene/chromosome 7 ratio >2 (p = 0.43). However, the polysomy of chromosome 7 emerged as a highly specific (p = 0.002) negative prognosticator for all patients.IGF1Rgene copy number was a highly negative prognostic marker for all NSCLC patients (p = 0.037), but was even more significant in males (p = 0.015) than in females (p = 0.021).Finally, a high IGF-1 expression showed a trend as a negative prognostic marker in males (p = 0.053) but not for the whole cohort population. There was no significant correlation between DSS and tumor epithelial cell expression of the EGFR gene mutations EGFRmut (p = 0.628) and EGFRdel (p = 0.498).High cytoplasmic MET expression showed a significant negative prognostic effect only in patients with adenocarcinoma (p = 0.026), but not for the whole cohort . For the whole cohort, the histologic subtypes did not reach a statistically prognostic significance, neither for the group of adenocarcinoma (p = 0.165), nor for the group of squamous cell carcinoma (p = 0.7) or for the large cell carcinoma group (p = 0.466).Polysomy of chromosome 7 in tumor cells correlated significantly and independently with a poor prognosis . In patients with adenocarcinoma, a high cytoplasmic MET expression was an independent negative prognostic factor . In males with NSCLC a high MET gene copy number gain in NSCLC. In addition to IHC we used SISH to investigate the METgene. While, in the adenocarcinoma patient population, independent of other clinicopathological variables, a high cytoplasmic MET expression was a significant negative prognosticator, as determined by IHC, a similar correlation was not found in patients with MET gene copy number gain, i.e. the absolute MET gene copy number in tumor cells did not affect the prognosis. Surprisingly, investigating the ratio of MET gene to chromosome 7, we found a trend of better survival in patients with a higher ratio in the whole cohort in the univariate analysis, but not in the multivariate analysis. However, a high MET gene to chromosome 7 ratio appeared to be a rather non-frequent event. We observed a MET gene to chromosome 7 ratio higher than 1 in 7.1% of our non-selected patients. This is in agreement with recent published data by Noonan et al[MET gene to chromosome 7 ratio of 1.8 or higher only in 4.5% of adenocarcinoma patients. The rather confusing positive prognostic effect of a high MET gene to chromosome 7 ratio led us to investigate a possible role of chromosome 7 polysomy in NSCLC patients. Interestingly, we found a high chromosome 7 count, higher than three chromosome copies as a highly independent negative prognostic factor for the whole cohort population. We assumed that a dysfunction in the transcriptional or posttranscriptional controlling mechanisms could partly explain the discrepancies between the IHC and SISH results; still we wanted to explore other genes located on chromosome 7. Chromosome 7 is known to harbor genes whose alteration my play an important role in multiple diseases as cystic fibrosis[EGFR, MET, and BRAF emerge as of special interest in NSCLC. Both MET and EGFR gene amplification are described to have a critical predictive role in NSCLC. [BRAF appears to have a role as a predictive marker in patients with advanced melanoma disease [MET gene copy number count (GCNC) and two of the most observed EGFR gene mutations in NSCLC; EGFRmut and EGFRdel [EGFR L858R specific antibody showed a sensitivity of 95.2% and a specificity of 98.8%. They further found that the EGFR exon 19 mutant specific antibody would detect 100% of 15-bp (base pair) deletions with a high specificity, however, a significant lower sensitivity of about 48.6% in non-15-bp exon 19 deletions was observed. According to the COSMIC database, Non 15-bp exon 19 deletions account for about 35% of exon 19 deletions[The chief aim of our study was to investigate the prognostic role of MET expression and the nan et al, where–u fibrosis, but als fibrosiswith over fibrosis Neverthen NSCLC. BRAF appe disease , 32, wit disease  There ar disease –36 propo disease , 37 Thes disease , 38, 39I EGFRdel , 41in un EGFRdel , 43 Addi EGFRdel –46 have  EGFRdel  reportedMET GCNC and any of the examined EGFR gene mutations. Further, none of the investigated EGFR mutations had a prognostic significance, despite conflicting results by other groups[We did not find a statistic correlation between MET expression, er groups, 49, 50.MET-null mutant mice embryos, with malformation of liver, placenta,[in vivo. Furthermore, dysregulation of the HGF-MET pathway has been demonstrated in malignancies of epithelial cell origin, represented by carcinomas of the lung, mamma, hepatic cells, pancreas ovaries, papillary renal carcinoma, papillary thyroid carcinoma, and carcinomas of the colorectal system.[MET gene mutations, MET amplification, chromosomal rearrangement, MET transcriptional upregulation or changes in the autocrine or paracrine signaling. Several studies have investigated the prognostic role of the MET receptor and the MET gene alteration in NSCLC. While some groups investigated a rather small group of patients [MET gene copy number gain either independent of the histologic type[The essential role of the HGF-MET cellular pathway has been further established following observations on placenta, melanocyplacenta, and testplacenta, Meanwhilplacenta,. There iplacenta, that METl system.Dysregulapatients –57, otheogic type–63 or onogic type or squamogic type.MET GCNC in 140 NSCLC patients, Dziadziuszko et al[MET high (GCNC) occur in a low proportion of primary NSCLCs and are associated with a good prognosis. Awad et al [MET exon 14 alterations. Similar results were also recently published by Lu et al[Exploring both the MET protein expression and the zko et al concludezko et al observedad et al reported ad et al  show tha Lu et al.MET GCNC, and the IGF-1 axis.Recent studies have proposed a ligand-independent MET activation., 72 UsinMET GCNC , the IGF1R GCNC (p = 0.64) or IGF-1 expression (p = 0.31).MET receptor expression did not show a significant correlation with the IGF1R GCNC to chromosome 15, this ratio immerged as a highly significant and independent negative prognostic indicator for disease specific survival in males, whereas it did not show prognostic influence in females. Gender differences in cancer tumorigenesis and survival are most likely associated with different sex hormone effects on various genes. While there are promising preliminary results among breast cancer and NSCLC patients treated with the IGF1R inhibitor Dalotuzumab,[IGF1R GCNC.[However, investigating the prognostic significance of IGF-1 expression in epithelial NSCLC showed a trend towards worse survival. Furthermore, investigating the ratio of otuzumab, accordinotuzumab,There areotuzumab, suggestiotuzumab, and Capuotuzumab,reported otuzumab,–81 have F1R GCNC. However,IGF1R and by immunohistochemistry for MET have a relevant practical implementation. While IGF1R SISH analyses seems to give additional information about the subgroup of NSCLC patients, who most likely would benefit of an anti-IGF1R therapy regimen, the MET SISH analyses seem to be biased when investigating the ratio of gene copy number to chromosome count due to the highly negative prognostic significance of chromosome 7 polysomy, higher than 3 chromosome copies. Our results would most probably apply to any SISH analysis investigating a gene to chromosome ratio located on chromosome 7. Even thought our findings would apply to small subgroups of patients, still finding therapeutic aid to these subgroups would be regarded as a significant step towards personalized NSCLC treatment. Until this, every effort should be made to find more specific and even more personalized potential molecular targets whose status in tumor samples might impact therapeutic responses.Our current prognostic findings as detected by SISH for S1 TableData set with scoring results for all the markers included in the study.(SAV)Click here for additional data file.


The activation of c-Met has been associated with both primary and acquired resistance to EGFR-TKI therapy in NSCLC patients. Thus, c-Met status during EGFR-TKI therapy should receive much attention.P = 0.002). Patients with a baseline soluble c-Met level >766 ng/ml showed inferior median progression-free survival  after EGFR-TKI treatment. Multivariate Cox proportional hazards model analyses demonstrated that the soluble c-Met level was an independent prognostic factor for PFS after EGFR-TKI treatment . In the validation cohort, patients with soluble c-Met levels >766 ng/ml were also determined to have significant short median PFS after EGFR-TKI treatment .Forty-nine patients were selected as training cohort and 52 cases as validation cohort. With disease progression, IHC results showed that 37 (75.5%) of the patients were tissue c-Met-negative, and 12 (24.5%) were tissue c-Met-positive. There was a statistically significant difference in the dynamic change in soluble c-Met between the tissue c-Met-negative and c-Met-positive groups  was used to assess the expression of c-Met in the resistant tissue. Plasma c-Met levels were assayed in duplicate using a human soluble c-Met quantitative enzyme-linked immunosorbent assay (ELISA) kit.Quantitatively determining the soluble c-Met level in plasma by ELISA might provide a non-invasive and sensitive method to predict EGFR-TKI prognosis. Activating mutations in epidermal growth factor receptor (EGFR), particularly the exon 19 del and exon 21 L858R mutations, are associated with sensitivity to EGFR tyrosine kinase inhibitor (EGFR-TKI) therapy . Howeverin situ hybridization (FISH) assays that determine gene amplification [Clinically, c-Met status is most often determined in tissue by immunohistochemistry (IHC), which evaluates protein expression, or by fluorescence fication . Howevervia an enzyme-linked immunosorbent assay (ELISA) in 198 advanced NSCLC patients. We found a statistically significant correlation: patients whose tumor tissue showed c-Met positivity also tended to have elevated soluble c-Met levels in plasma. A plasma c-Met level of 766 ng/ml showed moderate specificity and sensitivity in predicting tissue c-Met protein expression. A high level of soluble c-Met was associated with a poor prognosis (detailed data not shown).The c-Met is a transmembrane protein consisting of an α- and a β-subunit linked together by a disulfide bond. Extracellular fragments of c-Met protein may be shed from the cell surface through a proteolytic process, facilitating the generation of soluble truncated c-Met protein, which can be easily measured in human blood –19. A siThe role of soluble c-Met during EGFR-TKI therapy is unclear. Therefore, the purpose of this study was to examine the dynamic change in the plasma soluble c-Met level in advanced NSCLC patients receiving EGFR-TKI treatment using a human soluble c-Met quantitative ELISA kit. We evaluated the potential usefulness of determining soluble c-Met levels to predict the prognosis of EGFR-TKI treatment.Forty-nine patients were selected as training cohort and 52 cases as validation cohort for prognosis analysis. In the training cohort, most of these patients had adenocarcinoma histology , with progression-free survival (PFS) after EGFR-TKI > 6 months . In the validation cohort, 98.1% (51/52) of these patients had adenocarcinoma histology. The clinicopathological features are summarized in Table P = 0.042; Figure P = 0.008; Table With disease progression (PD), all patients in the training cohort underwent rebiopsy after resistance to EGFR-TKI therapy. Tissue c-Met protein expression was evaluated by IHC according to H score criteria. Of the 49 patients, 37 (75.5%) were tissue c-Met-negative, and 12 (24.5%) were tissue c-Met-positive. We observed a positive correlation between the soluble c-Met level with PD and tissue c-Met status in resistant tumors. We observed no other correlations between plasma c-Met levels and the clinicopathological parameters, including age, sex, histological type, EGFR mutation type, and smoking history. The average plasma c-Met concentration was 601.55 ± 120.94 ng/ml in the tissue c-Met-negative group, which was significantly lower than 986.42 ± 578.36 ng/ml in the tissue c-Met-positive group (P = 0.001). To identify the effect of soluble c-Met dynamic change on the resistance to EGFR-TKI, we divided the 49 patients into two groups according to c-Met status in the tumor tissue with PD. In tissue c-Met-positive patients, we found significantly elevated soluble c-Met levels, and there was a statistically significant difference in the dynamic change in the soluble c-Met level between the tissue c-Met-positive and c-Met-negative groups . The average plasma c-Met was 619.83 ± 166.92 ng/ml  before EGFR-TKI treatment, decreased to 535.07 ± 135.37 ng/ml (95% CI: 496.19 - 573.95) at the time of the best tumor response, and then increased to 695.80 ± 339.98 ng/ml (95% CI: 598.15 - 793.46) with PD  had elevated pre-EGFR-TKI soluble c-Met levels (> 766 ng/ml), and 6  had elevated soluble c-Met levels with PD. In the tissue c-Met-negative cohort, only 3 patients  had elevated soluble c-Met levels pre-EGFR-TKI treatment, and 4 patients  had elevated soluble c-Met with PD.P= 0.051).Soluble c-Met levels > 766 ng/ml before EGFR-TKI treatment were associated with a positive c-Met status of the resistant tumor in 4 of 12 patients (33.3%). Plasma c-Met levels < 766 ng/ml before EGFR-TKI treatment were associated with a negative c-Met status of the resistant tumor in 34 of 37 patients . Patients with soluble c-Met levels > 766 ng/ml were also determined to have inferior overall survival (OS) after EGFR-TKI treatment ; however, this difference was not significant. Multivariate Cox proportional hazards model analyses demonstrated that the soluble c-Met level was an independent prognostic factor for PFS after EGFR-TKI treatment . The above results suggest that soluble c-Met levels pre-EGFR-TKI treatment have potential clinical value as a predictive biomarker for PFS after EGFR-TKI treatment . Among the 49 patients, 7 (14.3%) had baseline soluble c-Met levels > 766 ng/ml. Patients with soluble c-Met levels > 766 ng/ml showed inferior median PFS after EGFR-TKI treatment .The soluble c-Met level in pre-EGFR-TKI plasma test was further assessed in an independent prospectively collected validation cohort of 52 patients Figure . Among tHowever, there was no significant correlation between plasma c-Met levels at the time of the best response or PD and survival after EGFR-TKI therapy .To the best of our knowledge, the present study was the first to discuss the relationship between EGFR-TKI treatment and variations in the soluble c-Met level during certain time points. c-Met is a promising therapeutic target in NSCLC. The expression of c-Met in primary and resistant tumors is essential for EGFR-TKI treatment in advanced NSCLC patients , 12. HowP = 0.002). During EGFR-TKI treatment, we observed that the plasma c-Met level decreased at the time of the best tumor response and increased with PD, particularly in EGFR-TKI-resistant patients with c-Met positivity. Our results suggest that the c-Met signaling pathway might play a role in the response to EGFR-TKI treatment. Treating NSCLC with EGFR-TKI would enrich c-Met-positive tumor cells, with the overexpression and overactivation of c-Met consequently triggering the activation of Her3, thus activating downstream signal transduction molecules such as Akt and Erk, independent of EGFR kinase activity [In our study, we measured the dynamic change in soluble c-Met in plasma during EGFR-TKI treatment. To identify the effect of soluble c-Met levels on EGFR-TKI resistance, we divided the 49 patients into two groups according to c-Met status in the resistant tumor tissue. The average plasma c-Met levels in the tissue c-Met-negative group were 602.72 ± 135.59 ng/ml (95% CI: 403.42 - 873.86) before EGFR-TKI treatment, 531.47 ± 128.85 ng/ml (95% CI: 333.44 - 755.92) at the time of the best tumor response, and 601.55 ± 120.94 ng/ml (95% CI: 386.24 - 881.84) with PD. The plasma c-Met levels in the tissue c-Met-positive group were 672.57 ± 239.58 ng/ml (95% CI: 318.91 - 1232.67) before EGFR-TKI treatment, 546.15 ± 159.54 ng/ml (95% CI: 360.33 - 971.51) at the time of the best tumor response, and 986.42 ± 578.36 ng/ml (95% CI: 483.36 - 2208.06) with PD. There was a statistically significant difference in the dynamic change in the soluble c-Met level between the tissue c-Met-positive and c-Met-negative groups . Multivariate Cox proportional hazards model analyses demonstrated that the soluble c-Met level was an independent prognostic factor for PFS after EGFR-TKI . However, there was no significant correlation between plasma c-Met levels at the time of the best response or PD and PFS after EGFR-TKI therapy. This might be because c-Met-activated tumor cells could presumably proliferate despite EGFR-TKI therapy, and initial disease control was followed by a relatively short PFS compared with tumors without c-Met activation. Taken together, our findings highlight the role of soluble c-Met quantitative analysis in predicting PFS after EGFR-TKI, and might support early administration of a combination of c-Met and EGFR inhibitor therapies. Based on these considerations, our findings form the basis for further studies that may lead to an improvement in current treatment strategies.In advanced NSCLC, the relationship between soluble c-Met levels and prognosis after EGFR-TKI treatment remains unclear. Several studies have detected the coexistence of c-Met dysregulation and EGFR gene mutations in tumor tissues of NSCLC patients, and demonstrated that the PFS of patients with both EGFR mutations and c-Met gene amplification was significantly shorter than in patients with EGFR mutations alone , 23. By vs. 14.5 months, P < 0.001). It provided a highly significant proof of principle.Owing to the small sample size, we further assessed the prognosis of soluble c-Met level in an independent validation cohort of 52 patients Table . In the One potential drawback of our study was that c-Met status was not determined before EGFR-TKI treatment because the tissue samples were not available at this time point. Therefore, we could not determine whether a soluble c-Met level > 766 ng/ml before EGFR-TKI treatment indicated that tissue c-Met protein was expressed. Owing to the small sample size, more studies are warranted to confirm these results in the future.In conclusion, our results demonstrate that the measurement of soluble c-Met using ELISA is feasible and noninvasive with potential clinical applications. Stratifying EGFR mutation NSCLC patients based on the dynamic change of soluble c-Met during EGFR-TKI treatment might allow for individualized treatment. Moreover, tumors with a high soluble c-Met level before EGFR-TKI therapy could exhibit a shorter PFS after EGFR-TKI. Dynamic monitoring of the change in c-Met level might help determine EGFR-TKI prognosis.Between February 2014 and May 2015, we retrospectively analyzed patients with advanced NSCLC  at the Guangdong Lung Cancer Institute who were resistant to EGFR-TKI treatment. The inclusion criteria were as follows: 1) patients being screened for the c-Met inhibitor trial and 2) sufficient plasma samples available for the analysis of soluble c-Met level before EGFR-TKI treatment, at the time of the best response, and PD. We collected plasma and tissue samples with PD, but subsequent treatment was not initiated. The interval time between PD after EGFR-TKI and plasma extract preparation was less than 21 days. PFS after EGFR-TKI was defined as the time interval between the beginning of EGFR-TKI treatment and PD or death. OS was defined as the time interval between the beginning of EGFR-TKI treatment and death. The clinical data were reviewed. The objective tumor response was evaluated by RECIST 1.1 at 8-week intervals, or as clinically indicated, until PD, death, or loss to follow-up. This study was approved by the local Institutional Review Board. All patients provided written informed consent before the collection and use of their tumor specimens and blood samples.The expression of c-Met protein in formalin-fixed paraffin-embedded tissues collected with PD was determined by IHC analysis using an anti-total c-Met rabbit monoclonal primary antibody . IHC staining was carried out according to the manufacturer's protocol using the BenchMark XT platform from Ventana and the ultraView detection kit. Tissue c-Met expression levels were evaluated by IHC according to H score criteria. The expression of c-Met in tumors was defined as follows: no c-Met expression (scored as IHC = 0), weak c-Met expression (scored as IHC = 1+), moderate c-Met expression (scored as IHC = 2+), and strong c-Met expression (scored as IHC = 3+). The fraction of positive cells at each intensity level was estimated as a percentage (0-100%). The H score was the cross-product of the intensity and fraction score, ranging from 0 to 300. Positivity was defined as having ≥50% of tumor cells positive for membranous and/or c-Met immunostaining with strong intensity (IHC ≥3+).The plasma soluble c-Met protein concentration was measured using a human soluble c-Met quantitative ELISA kit according to the manufacturer's instructions .Blood samples were obtained from a consecutive prospective series of advanced NSCLC patients with EGFR mutation treated with EGFR TKI at the Guangdong Lung Cancer Institute from February 2013 to May 2014. Samples were assessed by ELISA blinded to clinicopathologic characteristics.2 and/or t-test. To evaluate the duration of response, Kaplan-Meier estimation was used, and comparisons were made using the log-rank test. Multivariate analysis of categorical variables was performed using logistic regression. A two-sided P value less than 0.05 was considered to be statistically significant.All analyses were performed using SPSS 16.0. Soluble c-Met changes over time were analyzed using repeated measures ANOVA. The associations between c-Met and clinical parameters were evaluated using the χ


Defects in coordinated ribosomal RNA (rRNA) transcription in the nucleolus cause cellular and organismal growth deficiencies. Bloom’s syndrome, an autosomal recessive human disorder caused by mutated recQ-like helicase BLM, presents with growth defects suggestive of underlying defects in rRNA transcription. Our previous studies showed that BLM facilitates rRNA transcription and interacts with RNA polymerase I and topoisomerase I (TOP1) in the nucleolus. The mechanisms regulating localization of BLM to the nucleolus are unknown. In this study, we identify the TOP1-interaction region of BLM by co-immunoprecipitation of in vitro transcribed and translated BLM segments and show that this region includes the highly conserved nuclear localization sequence (NLS) of BLM. Biochemical and nucleolar co-localization studies using site-specific mutants show that two serines within the NLS (S1342 and S1345) are critical for nucleolar localization of BLM but do not affect the functional interaction of BLM with TOP1. Mutagenesis of both serines to aspartic acid (phospho-mimetic), but not alanine (phospho-dead), results in approximately 80% reduction in nucleolar localization of BLM while retaining the biochemical functions and nuclear localization of BLM. Our studies suggest a role for this region in regulating nucleolar localization of BLM via modification of the two serines within the NLS. Transcription of rDNA genes is facilitated primarily by the RNA polymerase I complex in the nucleolus, with the 18S, 5.8S and 28S rRNAs transcribed as a single pre-rRNA transcript that is subsequently processed prior to the assembly of ribosomes [Ribosomal RNA (rRNA) transcription plays a central role in ribosome biogenesis with rRNA an integral part of ribosomal subunits ,3,4,5,6.ibosomes ,8,9. Theibosomes ,8,9,10. ibosomes .rDNA metabolism is associated with nucleolar dysfunction, cellular and organismal growth defects, and cellular transformation; it is often suggested as a primary cause of cellular senescence [rDNA metabolism correlates with rDNA repeat instability and compromised DNA damage responses throughout the genome.Ribosome biogenesis is critical for protein translation, cellular growth and proliferation. The group of disorders termed ribosomopathies, characterized by defects in the ribosome biogenesis pathway and including mutations in RNA polymerase I transcription complex components, results in severe growth impairment ,10,12,13nescence ,16,17,18Blm knockout mouse model results in severe anemia during embryogenesis [rDNA repeat instability, inter- and intra-chromosomal recombination, recombination of acrocentric chromosome arms where rDNA is found, chromosomal breakage and telomeric associations [Bloom’s syndrome (BS), an autosomal recessive human disorder caused by mutations of the recQ-like helicase BLM, is associated with several characteristics of nucleolar dysfunction ,22,23,24ogenesis . Cells wciations ,30,31. TrDNA metabolism and stability. BLM localizes to the nucleolus via the C-terminus and to promyelocytic leukemia (PML) nuclear bodies, implicated in DNA repair mechanisms, via the N-terminus [rDNA and at telomeres. BLM directly associates with rDNA sequences, specifically with the 18S-coding and Alu-repeat regions; its localization correlates with genomic stability [rDNA [rDNA repeat numbers in comparison to wild-type cells. Furthermore, our recent studies demonstrate a role for BLM in facilitating rDNA transcription in the nucleolus [rDNA transcription at least two-fold in vivo. BLM is a component of active RNA polymerase I transcription complexes. Disruption of transcription by the RNA polymerase I inhibitor actinomycin D re-distributes both RNA polymerase I components and BLM from the nucleolus into the nucleoplasm. In vitro, BLM facilitates unwinding of RNA:DNA hybrids (R loops) that have the potential to block ongoing rDNA transcription. This activity is enhanced by its association with TOP1, another integral component of the rDNA transcription machinery [rDNA transcription by removing superhelical tension (relaxation) generated by ongoing transcription [BLM ortholog Sgs1 and Srs2, another 3′–5′ helicase, suggesting a conserved role for recQ-like helicases in nucleolar rRNA transcription [Several studies point to a nucleolar role of BLM in terminus ,33. BLM tability ,33. BLM ty [rDNA : BS cellucleolus ,36. BLM cription ,38,39. Ocription .In addition to RNA polymerase I components, BLM interacts with several other proteins that function in nucleolar metabolism, including nucleophosmin, DNA topoisomerase IIα (TOP2A), WRN and BRCA1 ,44,45,462.GM08505 immortalized BS fibroblasts were obtained from Coriell Cell Repository . Cells were cultured in Minimum Essential Medium  supplemented with 10% fetal bovine serum  at 37 °C and 5% COpYES-BLM yeast expression vector was provided by Ian Hickson [BLM deletion and site-specific mutants were generated by an overlapping polymerase chain reaction (PCR) with the pYES-BLM vector as a template using Phusion High-Fidelity DNA Polymerase . 3′ internal primers were used with the 5′ external primer; 5′ internal primers were used with the 3′ external primer. Corresponding products from both reactions were gel-purified  and used as templates in an overlapping PCR with the 5′ external and 3′ external primers in a 1:1 ratio. Primers (5′–3′) used in BLM cloning were: 5′ external primer: CAGCTTTTGGCCTACTTTGGT3′ external primer: TTCCTTTTCGGTTAGAGCGGA3′ 1332-1349 deletion internal primer: CCACTGGAAGCAGTTCTGGTTTTACTTGCAAAGTAGTGG5′ 1332-1349 deletion internal primer: CAAGTAAAACCAGAACTGCTTCCAGTGGTTCCAA3′ S1342A/S1345A internal primer: CTCTTAGCCCTTTGGGCGGCTGG5′ S1342A/S1345A internal primer: CCAGCCGCCCAAAGGGCTAAGAG3′ S1342D/S1345D internal primer: CTCTTATCCCTTTGGTCGGCTGGC5′ S1342D/S1345D internal primer: GCCAGCCGACCAAAGGGATAAGAG3′ S1342A internal primer: CTCTTAGACCTTTGGGCGGCTGG5′ S1342A internal primer: CCAGCCGCCCAAAGGTCTAAGAG3′ S1345A internal primer: CTCTTAGCCCTTTGGGAGGCTGG5′ S1345A internal primer: CCAGCCTCCCAAAGGGCTAAGAG3′ S1342D internal primer: CTCTTAGACCTTTGGTCGGCTGGC5′ S1342D internal primer: GCCAGCCGACCAAAGGTCTAAGAG3′ S1345D internal primer: CTCTTATCCCTTTGGGAGGCTGGC5′ S1345D internal primer: GCCAGCCTCCCAAAGGGATAAGAGThe  Hickson . BLM delpYES-BLM were digested with SalI and XbaI at 37 °C for 1.5 h per manufacturer’s instructions and gel-purified. T4 DNA ligase (NEB) was used in ligation reactions per manufacturer’s instructions. Ligation reactions were transformed into Dh5α-competent cells. Plasmids were purified with QIAprep Spin Miniprep kit (Qiagen) and E.Z.N.A. Endo-Free Plasmid Maxi Kit . The full sequence of each BLM mutant was verified by the OSU Nucleic Acids Shared Resource. Inserts were transferred into the pEGFP-BLM vector, originally generated by cloning BLM cDNA into a mammalian expression vector containing an N-terminal GFP tag, pEGFP-C1 . The pYES-BLM mutants and pEGFP-BLM vector were digested with AflII and AsiSI at 37 °C for 1 h, and the remaining steps were carried out as previously described.All restrictions enzymes were purchased from NEB. Gel-purified PCR products and TOP1 cDNA was purchased from Thermo Fisher Scientific  as pCR4-TOPO and sub-cloned into the pYES yeast expression vector. A SacI restriction site and His tag were added to the 5′ end of the cDNA and a SalI site added to the 3′ end through PCR. Primers (5′–3′) used in TOP1 sub-cloning were: 5′ TOP1 primer: CGTAGAGCTCGGATCCCTAACCATGCACCACCACCACCACCACAGTGGGGACCACCTC3′ TOP1 primer: TACGCTCGAGTCAAAACTCATAGTCTTCATCAGCCGel-purified PCR products were digested sequentially with SalI at 37 °C for 1 h and SacI at 37 °C for 1 h and purified with QIAquick PCR Purification Kit (Qiagen) in between digests. pYES-BLM vectors were digested with SacI and XhoI at 37 °C for 1 h. Remaining steps were carried out as previously described. The full sequence of TOP1 cDNA was verified by the OSU Nucleic Acids Shared Resource.35S-methionine. Peptides generated with the included luciferase control DNA were used as a negative control in subsequent immunoprecipitation reactions. DNA templates containing the indicated amino acids of BLM were generated by PCR using pYES-BLM as a template. The Δ1332–1349 DNA template was generated by PCR using the pYES-BLM-Δ1332–1349 plasmid as a template. The T7 promoter and a poly-A tail were added by PCR. PCR products were separated on agarose gels and purified with a QIAquick gel extraction kit (Qiagen). IVTT products were incubated with full-length recombinant TOP1 followed by α-TOP1  or isotype control  antibodies at 4 °C for a total of 3 h with rotation in buffer containing 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 10% glycerol, 1 mM DTT, 0.1% Tween-20, 0.1 mg/ml BSA and protease inhibitor . Complexes were immunoprecipitated with Dynabeads Protein G (Invitrogen) at 4 °C for 2 h with rotation. Beads were washed 4 times for 5 min at 4 °C with rotation, and products were eluted with SDS-PAGE sample buffer at 95 °C for 5 min and separated by SDS-PAGE. Gels were dried, exposed to storage phosphor screens , imaged with Typhon FLA 7000 (GE) and analyzed using ImageQuant software (GE). The C-terminus of BLM (amino acids 997–1417) was used as a positive control and luciferase as a negative control for each experimental replicate. Peptide fragment pulldowns were quantified, and non-specific pulldown by the IgG control antibody was subtracted. Each fragment was normalized to its input and expressed as % relative IP compared to the positive control (amino acids 997–1417). Experiments were repeated 3 to 9 times per peptide. Data were analyzed with a Student’s t-test .In vitro transcription and translation (IVTT) reactions were performed with the TNT Rabbit Reticulocyte Lysate kit  according to manufacturer’s instructions with BLM  and TOP1 (pYES-TOP1) were expressed in Jel1Saccharomyces cerevisiae. Yeast cells were lysed with a French Press Cell Disrupter (Thermo Fisher Scientific), and cell debris was pelleted by ultracentrifugation at 65,000 g for 1 h at 4 °C. BLM protein was purified from the lysate by FPLC using Ni-NTA Superflow (Qiagen), Heparin-Sepharose 6 Fast Flow  and Q-Sepharose (Sigma-Aldrich) columns with dialysis between each column in 50 mM Tris-HCl (pH 7.5), 200 mM NaCl, 1 mM EDTA, 10% glycerol, 1 mM DTT, protease inhibitor (Sigma-Aldrich) and 100 mM PMSF. The purified protein was dialyzed in 50 mM Tris-HCl (pH 7.5), 200 mM NaCl, 1 mM EDTA, 20% glycerol, 1 mM DTT, protease inhibitor (Sigma-Aldrich) and 100 mM PMSF, snap frozen in liquid N2 and stored at −80 °C. The BLM mutants eluted at similar salt concentrations as wild-type. TOP1 was purified similarly to BLM by FPLC using Ni-NTA Superflow (Qiagen) followed by Heparin-Sepharose 6 Fast Flow (Amersham Biosciences) columns with dialysis between columns. TOP1 eluted at 140–230 mM imidazole from Ni-NTA, and at 610–670 mM NaCl from the Heparin-Sepharose column. Purified TOP1 was dialyzed in 50 mM Tris-HCl (pH 7.5), 200 mM NaCl, 1 mM EDTA, 30% glycerol, 1 mM DTT, protease inhibitor (Sigma-Aldrich) and 100 mM PMSF, snap frozen in liquid N2 and stored at −80 °C. Single-band purity of the resulting proteins was verified by Coomassie blue staining of SDS-PAGE gels. Protein concentration was measured using Qubit Protein Assay Kit (Thermo Fisher Scientific).BLM was purified as previously described with the addition of a heparin-sepharose column ,36,44. B20: CGCTAGCAATATTCTGCAGCDNA20: CGCUAGCAAUAUUCUGCAGCRNA33: GCTGCAGAATATTGCTAGCGGGAATTCGGCGCGDNA Oligonucleotides (DNA20 and RNA20) were end-labeled with 32P using polynucleotide kinase (NEB) according to manufacturer’s instructions and annealed with the DNA33 oligonucleotide by heating to 95 °C for 5 min followed by slow cooling to room temperature (RT), to generate the corresponding DNA:DNA or RNA:DNA substrates. Double-stranded substrate was separated on non-denaturing polyacrylamide gels and gel-purified. Helicase assays were performed with the indicated concentration of BLM protein and with the indicated substrate (DNA:DNA for protein activity determination or RNA:DNA for TOP1 stimulation experiments) as previously described [2, 2 mM ATP, 1 mM DTT and 0.1 mg/ml BSA. Reactions were stopped at the respective times and products separated on 12% non-denaturing polyacrylamide gels, dried, exposed to storage phosphor screens (GE), imaged with Typhon FLA 7000 (GE) and analyzed using ImageQuant software (GE). TOP1 alone did not unwind the RNA:DNA substrate (data not shown) [2, 1 mM ATP, 0.5 mM DTT and 50 μg/mL BSA, using radioactively labeled DNA:DNA substrate prepared by annealing the following oligonucleotides, as described [TTTTTTTTTTTTTTTTTAGGGTTAGGGCATGCACTACGTAGTGCATGCCCTAACCCTAATTTTTTTTTTTTTTT Reactions were terminated in TOP2A stop buffer ; products were resolved on 10% non-denaturing polyacrylamide gels and quantified as described above.DNA oligonucleotides were purchased from Operon , and RNA oligonucleotides were purchased from Invitrogen. Oligonucleotide sequences (5′ to 3′) are: escribed ,36,48. Bt shown) . Unwindiescribed : TTTTTTTBLM protein sequences of different species were extracted from Ensembl and aligned using ClustalW 1.83 through the European Bioinformatics Institute. The output was re-formatted in MView, using default parameters. Identities were normalized by aligned length. Residues were colored by identity and property. BLM amino acids 1332 to 1349 were analyzed with the following programs: KinasePhos2.0 , ScanSitt-test .GM08505 cells were plated on sterile coverslips and transfected with the indicated GFP-tagged BLM plasmids  with Lipofectamine2000 (Thermo Fisher Scientific) according to the manufacturer’s instructions. Cells were fixed with 4% paraformaldehyde for 15 min 24-h post-transfection, washed with Dulbecco’s phosphate-buffered saline (DPBS) 3 times for 5 min, permeabilized with 0.25% Triton-X-100 for 15 min, washed with DPBS 3 times 5 min and blocked with 10% normal goat serum (Sigma-Aldrich) . Anti-nucleophosmin  was used at 1:1000, anti-PML  was used at 1:500 and Alexa-Fluor (Invitrogen) fluorescent goat anti-mouse was used at 1:2000 in DPBS with 1% BSA (Sigma-Aldrich) and 0.1% Tween 20 (Sigma-Aldrich). Coverslips were mounted with VectaShield plus DAPI mounting medium . Slides were imaged on a Zeiss AxioVert 200 M microscope and AxioCam MRm camera or an Olympus FV1000 filter confocal microscope. Nucleolar localization for 100 cells from 3 to 5 blinded experiments was expressed as percent nucleolar localization. PML body localization for 50 cells from 3 blinded experiments was expressed as percent PML body localization. Data were analyzed with a Student’s rDNA transcription, while BLM stimulates TOP1 activity in relaxation of helical tension generated in DNA during ongoing transcription [35S-methionine-labelled BLM fragments generated by in vitro transcription and translation (IVTT). The BLM protein contains a number of domains  substrate with a 13-base 3′ single-strand overhang. Initial unwinding rates for four protein concentrations were graphed to calculate the specific activities of the proteins, expressed in fmol substrate unwound/min/nM protein. BLM-Δ1332–1349 unwound dsDNA with a lower specific activity than BLM-WT  or aspartic acid (phospho-mimic) and tested for their ability to alter nucleolar localization using GFP-tagged constructs  or to inRecombinant mutant proteins BLM-S1342A/S1345A and BLM-S1342D/S1345D were purified and characterized  and unwirDNA or protein-rRNA interactions and protein-small nucleolar RNA interactions [rDNA via its C-terminus; regions overlapping the RQC (amino acids 1118–1164) and HRDC domains (amino acids 1166–1331) mediate these interactions [rDNA binding domains of BLM are intact in these mutants, our results argue that the effect of the phospho-mimetic modification in limiting nucleolar localization is dominant over the effects of any rDNA transactions or TOP1 interactions that might direct BLM to the nucleolus. Unimpaired localization of these mutants to PML bodies suggests that they would retain BLM functions in global DNA repair. The ability of the BLM NLS to direct TOP1 functional interactions as well as modulate nucleolar trafficking adds to the known functions of the BLM NLS.Unlike nuclear localization sequences (NLS), nucleolar localization sequences are not clearly defined. Both are composed of a high proportion of basic amino acids and may either be distinct or overlap within any one protein . Known mractions ,64. BLM ractions ,33. The ractions ,36. The ractions ,57,58,65ractions  suggesterDNA loci are prone to the formation of aberrant structures and damage that pose blocks to ongoing replication and transcription. BLM is an integral part of DNA damage repair pathways that promote resolution of such aberrant structures. In its absence or inappropriate localization, these transcription/replication blocks could elicit a DNA damage response that without BLM would increase recombination between rDNA repeats.Several studies have identified distinct sites within BLM that are modified post-translationally by phosphorylation, ubiquitination and sumoylation in response to cellular stress . These mrDNA metabolism—transcription, replication and recombination—to ensure stability of the rDNA loci. Early replicating rDNA loci  localize at the nucleolar periphery or outside the nucleolus during early S-phase and are subsequently internalized in the nucleolus [rDNA loci occurs during mid- to late-S phase inside the nucleolus. Likewise, during recombinational-repair of double-strand breaks, there is a transient translocation of rDNA loci from the nucleolus to the nucleoplasm accompanied by a coordinated localization of recombination proteins to the nucleoplasm [rDNA transcription by RNA polymerase I is localized solely within the nucleolus. The dynamics of rDNA loci during the replication/transcription processes may preserve rDNA loci stability by preventing collisions of the transcriptional and replication machinery [rDNA loci by BLM. Likewise, restricting nucleolar entry of BLM may facilitate slower rates of rDNA transcription and the ensuing ribosome biogenesis, perhaps favorable under conditions of starvation or stress [Accumulating evidence suggests a sub-nuclear spatial separation of the different aspects of ucleolus . Replicaleoplasm ,87,88,89achinery ,88. Serir stress ,35,36. S


Rational use of antibiotic is the key approach to improve the antibiotic performance and tackling of the antimicrobial resistance. The efficacy of antimicrobials are influenced by many factors: (1) bacterial status  and inoculum size; (2) antimicrobial concentrations [mutant selection window (MSW) and sub-inhibitory concentration]; (3) host factors (serum effect and impact on gut micro-biota). Additional understandings regarding the linkage between antimicrobial usages, bacterial status and host response offers us new insights and encourage the struggle for the designing of antimicrobial treatment regimens that reaching better clinical outcome and minimizing the emergence of resistance at the same time. Currently, antibiotics are the invaluable weapons to fight against infectious diseases. However, the outbreak of antimicrobial resistance, jointly with the shortage of newly developed antimicrobial drugs, brings a great threat to the health of both human and animals . MIC is defined as, the minimum concentration of an antibiotic to inhibit the bacterial growth. MIC is usually determined by exposing a defined amount of bacterial population to a series of increasing antibiotic concentrations in a standardized growth medium for about 16–20 h  from a clinically susceptible bacteria  that are not killed by antibiotics, and heterogeneous response is repeated when they are expose to the same antibiotic Lewis, . The detMDK provide an apparent predictions of the treatment period that is needed to treat an infection by strains with the tolerance or time-dependent persistence, and could thus be combined with PK/PD models to optimize treatment regimens  or in a cell aggregates (biofilm). Biofilms are consortium of bacteria, which are surrounded in a self producing polymer matrix which consist of a polysaccharides, proteins and DNA  but resistant if the inoculum size is increased. Several studies have been shown that high bacterial inocula at the infection sites may decrease the activity of antibiotic  of wild-type bacteria, the mutants were not eliminated when expose to antibiotic concentrations above the MPC  or a low (105 CFU) inoculum of E. coli, the values of time within the mutant selection window (TMSW) <30% appeared to be a good predictor for prevention of a resistance  or a high (109 CFU) inoculum of Klebsiella pneumonia, the results displayed that for the low inoculum, prevention of resistance occurred for an AUC/MIC ratio of 189 h, while for the high inoculums, resistant bacteria were enriched for AUC/MIC ratios up to 756 h , while linezolid exhibited no inoculum effect at low and medium inocula (104 and 106 CFU/ml) but with the high inoculum , inhibiting DNA replication (quinolones) or its transcription (rifampicin), impairing bacterial ribosomes and protein synthesis , interfering with metabolic pathways (sulfonamides and trimethoprim) or disrupting the cytoplasmic membrane  compared to MIC testing . Mutant selection window (MSW) is a collection of concentrations between the MICs of the susceptible and resistant variants. Resistant mutants may be selected under antimicrobial selective pressures in the MSW Drlica, . The uppThe MSW concept aims to the prevention of a resistance. When the pertinent antimicrobial agent is present at a concentration within the MSW, a selection process of resistance will occur. Therefore, dosages ensuring antibiotic concentrations at the infection site above the MPC are suggested. The emergence of resistant bacteria is a dynamic phenomenon over time, the MPC should be established with new PK/PD knowledge  have been reported to be able to induce the SOS response and the RpoS regulon in bacteria  in test medium (Zhanel et al., in vitro tests (Smith et al., in vitro experiments always require to be assured in in vivo settings. For example, telavancin, a glycopeptides, although the protein binding was above 90% and in vitro studies observed a 10-fold decline of the activity in the presence of proteins; it was recently permitted on clinical base efficacy (Hegde et al., in vivo state. The establishment of a methods for a direct quantification of affinity of protein binding or its reversibility in individual models may leads to a better understanding of the results where the free drug hypothesis contradict (Zeitlinger et al., Living systems are a dynamic compared to static The intestinal microbiota protects the hosts against infections and other pathologies by directly inhibiting colonization of invading microbes or by orchestrating appropriate immune responses, which can control innate and adaptive immunity in the gut (Macpherson et al., In food producing animals, the most common route of antibiotic administration is oral. However, oral antibiotic administration may results in a gut microbiota dysbiosis, which remains for a long periods of time, spanning months, and even years (Francino, The antibiotics induced microbiota alterations can affect the basic immune homeostasis, especially if they occur early in life, which is an important period for maturation of the immune system and establishment of immunological tolerance (Francino, Enterobacteriaceae with a fitness advantage (Faber et al., Gut microbiota alterations may also increase the sensitivity to infections, which can stem from a lately invaded pathogens or from the abrupt overgrowth and pathogenic actions of the opportunistic organisms which are already exist in the gut (Young and Schmidt, H. parasuis infection (Macedo et al., On the other hand, some of the antibiotics, except for antimicrobial activity, exhibit immunomodulatory properties. The 14- and 15-member macrolides can interact with mitogen-activated protein kinases, thereby inhibiting NF-κB-mediated inflammatory responses toward various stimuli (Woodhead et al., in vivo, which makes the serum effect to be considered in an antibiotic therapy. Choosing the precise antibiotic is important, as the serum effect is changed between different antibiotics in the same class or one antibiotic against different microorganisms. Since living systems are more dynamic and complex, the results of in vitro tests always necessitate to be assured in in vivo settings.Rational and correct uses of antibiotics are the key approaches in improving antibiotic performance and tackling the antimicrobial resistance. The efficacy of an antibiotic treatment is influenced by many factors. The sensitivity of the specified pathogens is usually combined with pharmacokinetic parameters to investigate the effectiveness of antimicrobial dosage regimens. It should be noted that only the non-protein-bound fraction of an antibiotic is microbiologically active On the other hand, MIC is not informative for some special bacterial status, such as persistent or tolerant bacteria. In contrast to infections caused by planktonic bacteria, biofilm-forming bacteria tend to cause the chronic infections, especially in the respiratory tract, whereby infections persist despite seemingly adequate antibiotic therapy. This is because emergence of persistent or the tolerant bacterial cells is usually happened in biofilms. Recently, several compounds have been identified as an effective against time-dependent persisters (Kim et al., Antibiotic regimens should be optimized not only for the treatment outcome, but also for the minimization of antimicrobial resistance development (Mouton et al., It should not be ignored that antibiotic induced alterations in composition and functions of the microbiota may also create long-lasting harmful effects for the host and increase the bacterial resistance (Francino, With an improved understanding on the interaction of antimicrobials, pathogens and host gut microbiota and immune response, we have reasons to believe that we will develop judicious antibiotic treatment strategies and better control programs to fight against microbial infections.JL and SX wrote and revised the review. SA, FW, YG, CZ, XC, YW, and JC revised the review. GC contributed to the conception of the review and wrote the review.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The genesis of the AIDS policy and AIDS Space in Brazil (1981-1989)” published in the “Revista de Saúde Pública”, 2016;50:43, DOI: http://dx.doi.org/10.1590/s1518-8787.2016050005801; on page 8, 7th paragraph.In the article: “Where it reads:“azithromycin (AZT)”It should read:“zidovudine (AZT)”.


Fentanyl and fentanyl analogues represent a current and emerging threat in the United States as pure illicit narcotics and in mixtures with heroin. Because of their extreme potency, methods to safely and rapidly detect these compounds are of high interest. This work investigates the use of thermal desorption direct analysis in real time mass spectrometry (TD-DART-MS) and ion mobility spectrometry (IMS) as tools for the rapid and sensitive (nanogram to picograms) detection of fentanyl, 16 fentanyl analogues, and five additional opioids. Competitive ionization studies highlight that detection of these compounds in the presence of heroin is readily achievable, down to 0.1% fentanyl by mass with TD-DART-MS. With IMS, detection of nanogram levels of fentanyl in a binary fentanyl and heroin mixture is possible but can be complicated by decreased resolution in certain commercial instrument models. Modifications to the alarm windows can be used to ensure detection of fentanyl in binary mixtures. Additionally, three complex background matrices  are shown to have a minimal effect of the detection of these compounds. Wipe sampling of the exterior of bags of questioned powders is shown to be a safe alternative method for field screening and identification, removing the need to handle potentially lethal amounts of material. Opioid abuse is a growing epidemic that creates unique challenges for law enforcement, first responders, and medical personnel. Fentanyl and fentanyl analogues are many times more potent than morphine, making accidental exposure through the seizure of powders and pills life-threatening. The Drug Enforcement Administration (DEA) issued a warning to law enforcement in June of 2016 to exercise extreme caution when handling possible fentanyl-containing materials .Field testing that requires direct handling of suspected drug powders is discouraged due to safety concerns, as materials must be treated as potential NPF, both in the field and in the lab, until a qualitative analysis can be performed. This extended period of uncertainty about the contents of the suspicious material is undesirable, as it requires extreme vigilance during handling. A rapid, safe screening test for fentanyl and fentanyl analogues that is successful in realistic sample scenarios (i.e. mixtures with heroin) would protect emergency and laboratory personnel and could provide valuable medical treatment information. Current colorimetric techniques require macroscopic amounts of material, making them unsuitable for safe field testing of NPF. Ion mobility spectrometry (IMS) is a field-deployable technology useful for qualitative screening of street drugs and is sensitive to nanogram levels of fentanyl, even in the presence of heroin . The conAnother potential field deployable, and laboratory-based, screening technique is thermal desorption direct analysis in real time mass spectrometry (TD-DART-MS), which has been demonstrated for direct analysis of drugs collected onto wipes without the traditional solution-based sample preparation . TD-DARTThis study provides some of the necessary data needed to support the use of IMS and TD-DART-MS for applications in the law enforcement, emergency, and medical communities. Fentanyl, sixteen different fentanyl analogues, and five opioids  were evaluated for detection by IMS and TD-DART-MS, to determine spectral characteristics and limits of detection. Selected compounds were evaluated for changes in sensitivity and selectivity of detection when combined with heroin and/or excipients, and against background collected from sampling the outside of bags, fingerprints, and dirt. The potential for false negatives or false positives for NPF using IMS was evaluated.* in powdered form or, when possible, as 1 mg mL−1 methanolic solutions. Fentanyl was purchased in powder form as fentanyl citrate  and the remaining opioids were purchased as 1 mg mL−1 methanolic solutions from Cerilliant . Powdered samples were dissolved gravimetrically in methanol , and all samples were further diluted in methanol. Solutions were then pipetted or inkjet printed +, and the reduced mobilities are linearly correlated with molecular weight (0 (differences of less than ±0.002). These pairs are: 1) acetyl and benzyl, 2) p-fluoroisobutyryl and furanyl, 3) butyryl and isobutyryl, 4) isobutyryl and trans-3-methyl, and 5) 4-methoxy and valeryl fentanyl. In addition to the fentanyl analogue overlaps, despropionyl fentanyl and naloxone could not be differentiated on the basis of K0. Since IMS is based on cross-sectional area in addition to molecular mass, the technique was able to differentiate one set of isomeric compounds that the TD-DART-MS could not (o-fluorobutyryl and p-fluoroisobutyryl). Of the 22 compounds listed in 0 with the illicit drugs already active in the instrument library, and no false positive alarms for these drugs were observed.Determination of representative IMS responses were completed by analyzing 1 ng to 100 ng of the compounds deposited individually on meta-aramid wipes. In most cases, the spectra were simple combinations of the RIP and one distinct peak for the compound, as represented by fentanyl and carfentanil in r weight . There a90 values were determined for a small subset of compounds on IMS , as shown in −1, highlighting the possibility of detection by swiping the outside of a bag, or other surface, instead of needing to sample bulk powder.LODThough it is useful to demonstrate low level detection of these compounds, it is more important to evaluate whether or not these compounds can be detected in the presence of other drugs or excipients. Since fentanyl is commonly encountered in the United States as a minor component in a mixture with heroin , it is n−1 deposits of fentanyl. Levels of the “competing compound” (heroin or the excipient) were 0 ng wipe−1 , 1 ng wipe−1, 10 ng wipe−1, 25 ng wipe−1, 50 ng wipe−1, 100 ng wipe−1, and 1000 ng wipe−1 to obtain competing compound: fentanyl ratios of 0:1, 1:1, 10:1, 25:1, 50:1, 100:1, and 1000:1. Once analyzed, the extracted ion chronograms of the fentanyl molecular ion (337.228 m/z) were used to calculate peak areas. The response of fentanyl in the binary mixture relative to pure fentanyl was then calculated by plotting the peak area in the mixture relative to peak area of pure fentanyl . The stability of the fentanyl signal is promising as it shows that fentanyl can be detected in a heroin-fentanyl mixture regardless of the relative percentages of the two compounds. This data also indicates that the proton affinity for fentanyl is likely higher than that of heroin. More interesting than the heroin mixture response is that of fentanyl in the presence of mannitol (−1 of mannitol). This increase may be attributed to either additional proton donation from mannitol, which has a higher propensity to form deprotonated molecular ions than protonated molecular ions [f heroin . No decrmannitol . With thlar ions  or the n−1 heroin and 1 ng wipe−1 of the analogue), and comparing the response to the signal of the pure analogue. A second study was completed to evaluate whether or not the more potent fentanyl analogues and opioids  would have similar responses to fentanyl in the presence of heroin. −1 used as the constant mass of fentanyl and  as the masses of competing compounds. This provided ratios of competing compound: fentanyl of 0:1, 1:1, 2.5:1, 5:1, 10:1, 50:1, and 100:1. The response of fentanyl, measured as the maximum peak height during analysis, showed little suppression in the presence of acetaminophen , reported separation of fentanyl and heroin peaks, with detection of both compounds over a wide range of binary compositions [0 value of heroin. The combination peaks that form from these 3 analogues with heroin cannot be reliably differentiated from each other, or from the fentanyl:heroin combination peak. However, a combination peak can be a reliable indicator of the presence of one of the 4 fentanyl compounds in a mixture with heroin. Carfentanil, benzyl, furanyl, acetyl, and valeryl fentanyl have K0 values that are further away from heroin, and can be resolved for the entire range of studied compositions . For these resolved mixtures, ion suppression could be evaluated by monitoring peak intensity, with carfentanil maintaining its intensity even at the highest additions of heroin  with a slight increase in buprenorphine (approximately 25%) when compared to the pure compounds. Like the fentanyl-heroin binary mixture, these results are promising as they demonstrate that both compounds would be detected in the drug, at least at these lower mass loadings.A similar competitive ionization study was completed with buprenorphine and naloxone, the two active ingredients in the drug suboxoneThe final set of experiments aimed to establish whether or not realistic background matrices would hinder the detection of trace levels of these compounds. In scenarios encountered by first responders or forensic scientists, samples would be collected from materials stored under non-ideal conditions, and therefore would likely contain materials such as phthalates and plasticizers from plastic bags, fingerprint residues, and dirt or dust, in addition to heroin or excipients. To evaluate whether background matrices would complicate detection of these compounds, a binary 10:1 heroin: fentanyl mixture in each of three simulated background matrices was analyzed. Background matrices included artificial fingerprint residue, standard reference material (SRM) sediment to simulate dirt, and retrieval of the materials off of a plastic bag.The first challenge matrix examined was an artificial fingerprint material containing over forty compounds commonly found in fingerprint residue at biologically relevant concentrations . ApproxiAlong with fingerprint residue, it is likely that a surface that is a candidate for bearing trace narcotics will contain dirt or dust. Dirt presents several challenging aspects for trace detection, namely the presence of trace organics, trace inorganics, and particulates which can clog the instrumentation. The effect of dirt on the detection of the binary heroin fentanyl mixture was examined by depositing approximately 50 μL of an aqueous suspension of NIST SRM 1944 (New York/New Jersey Waterway Sediment) onto the wipe along with 100 ng heroin and 10 ng fentanyl followed by direct insertion into the instruments. The aliquot of SRM 1944 was allowed to dry prior to deposition of the heroin and fentanyl. NIST SRM 1944 contains a number of organic and inorganic constituents, as well as particulate with a mean diameter of approximately 150 μm. The final background matrix intended to investigate real-world wipe collection and the effects of phthalates and plasticizers on detection by collecting the fentanyl and heroin deposit off of a plastic bag using a wipe. To do so, a polyethylene bag, approximately 7.5 cm by 11.5 cm, was handled for several minutes to purposefully contaminate the bag with real fingerprint residue. After handling, for TD-DART-MS, 100 ng fentanyl and 1 μg heroin were deposited. The entirety of the bag was then wiped and inserted directly into the instrument. The higher levels of fentanyl and heroin were chosen because typical collection efficiencies of the PTFE-coated wipes are on the order of single percentages, which allowed for the collection of approximately 5 ng fentanyl and 50 ng heroin . Fig. 4eRapid detection of fentanyl and fentanyl analogues with nanogram to sub-nanogram sensitivity is possible using both TD-DART-MS and IMS. Detection of low levels directly wiped off of a surface can be advantageous to law enforcement, first responders, and forensic scientists as it provides an option for screening an item of evidence without having to come in direct contact with the bulk powder. Both techniques detected fentanyl in the presence of heroin down to 0.1% by weight fentanyl. Lower instrumental resolution in the IMS caused the formation of a heroin and fentanyl combination peak which could be reliably traced to the presence of heroin and fentanyl or a fentanyl analogue. Other adulterants provided varying degrees of signal enhancement or suppression; however, with TD-DART-MS, the fentanyl signal was maintained regardless of excipient and amount present. IMS provided similar results, except with procaine, which consumed all available charge at higher than 1:1 ratios of procaine to fentanyl. Complex matrices, including artificial fingerprint residue, dirt simulant, and a plastic bag, did not prevent detection of fentanyl on either system. The 16 fentanyl analogues behaved similarly to fentanyl, and it is expected comparable results would be obtained in the presence of excipients or background matrices.0 values for naloxone and despropionyl fentanyl. This work highlights the ability of TD-DART-MS and IMS to rapidly detect fentanyl and fentanyl analogues, though prior work already has highlighted their capabilities in the detection of more conventional narcotics. Both tools provide potential avenues for rapid on-site or laboratory-based screenings from mobile units to forensics laboratories to emergency rooms.While these techniques are not quantitative, they do constitute qualitative tools to help identify and properly mitigate fentanyl and fentanyl analogues in evidence, on persons, and elsewise. Analysis by these techniques is both rapid (less than 10 s), sensitive, and generally discriminative. DART was able to separate all but one pair of isomeric compounds, while IMS had less selectivity in discriminating similar analogues. However, the only identified potential issue for false alarms is the similar KSupp1


Corrosion of concrete reinforcement members has been recognized as a predominant structural deterioration mechanism for steel reinforced concrete structures. Many corrosion detection techniques have been developed for reinforced concrete structures, but a dependable one is more than desired. Acoustic emission technique and fiber optic sensing have emerged as new tools in the field of structural health monitoring. In this paper, we present the results of an experimental investigation on corrosion monitoring of a steel reinforced mortar block through combined acoustic emission and fiber Bragg grating strain measurement. Constant current was applied to the mortar block in order to induce accelerated corrosion. The monitoring process has two aspects: corrosion initiation and crack propagation. Propagation of cracks can be captured through corresponding acoustic emission whereas the mortar expansion due to the generation of corrosion products will be monitored by fiber Bragg grating strain sensors. The results demonstrate that the acoustic emission sources comes from three different types, namely, evolution of hydrogen bubbles, generation of corrosion products and crack propagation. Their corresponding properties are also discussed. The results also show a good correlation between acoustic emission activity and expansive strain measured on the specimen surface. Reinforced concrete (RC) structures constitute the majority of civil infrastructures of the world. Corrosion of concrete reinforcement members has been recognized as one of the predominant deterioration mechanisms for steel reinforced concrete structures. The two major factors responsible for corrosion of steel reinforcement are carbonation of concrete and chloride penetration. Of the two, chloride induced corrosion is a far more serious problem. Under chloride attack, the passivation of steel within the concrete is broken down and indicates the initiation of reinforcement corrosion. The corrosion products are usually two to six times more voluminous than the original steel consumed , resultiStructural health monitoring for concrete structures has attracted a lot of attention ,4,5. A wConcrete expansion and cracking are among the most significant and obvious physical features related to concrete deterioration due to reinforcement corrosion, and thus monitoring of these two features is critical for evaluating the safe performance of reinforced concrete structures. These two features can be readily detected using acoustic emission (AE) techniques and fiber optic sensing, both of which have recently emerged as new methods to measure and characterize corrosion in concrete structures.Acoustic emission is comprised of high frequency elastic waves or stress waves generated when there is a rapid release of energy within a material that is undergoing deformation ,15,16. IFiber optic sensors carry numerous advantages over traditional sensors, such as small size, immunity to electromagnetic interference, corrosion resistance, and real-time monitoring ,21. TherIn the present study, an approach based on the combination of the AE technique and FBG strain measurements is presented with the aim to monitor the progression of reinforcement expansion and concrete cracking due to reinforcement corrosion in concrete structures. Acoustic emission measurement helps to detect corrosion initiation and crack growth while FBG strain measures the extent of corrosion and identify time of cracking. Such a combined approach has not yet been reported in the literature, and the results of such a study can provide a correlation between acoustic emission events and concrete surface strains. A reinforced mortar specimen was prepared and an accelerated corrosion test was performed in laboratory to verify the proposed method. The rest of the paper is organized as follows: a, b and c are the variables that depend on the alkalinity of the pore water solution of concrete, the oxygen supply and the moisture content.Corrosion of steel reinforcement members leads to the desolvation of metallic iron into corrosion products as a result of the oxidation process. This process gives rise to an increase in volume, which, depending on the level of oxidation, may be up to about six times the original iron volume. The composition of the expansive corrosion products usually exist in the form of According to the corrosion cracking model for reinforced concrete described by Zhao and Jin , the steTo verify the proposed approach for reinforcement corrosion monitoring through a combined AE and FBG detection system, a cylindrical reinforced mortar specimen was prepared and an accelerated corrosion test was conducted under laboratory conditions. Both AE transducers and the FBG sensor were used in the same corrosion specimen so as to investigate the correlation between acoustic emission events and concrete surface strain.The cylindrical reinforced mortar specimen was 80 mm in diameter and 200 mm in height, as shown in Two AE transducers were mounted on the specimen. One transducer was on the top of the steel plate, and the other was on the top surface of the mortar, as shown in The corrosion monitoring systems consists of two separate systems, namely, the AE corrosion monitoring system and the FBG strain monitoring system. The layout of these two systems are depicted in For the FBG strain monitoring system, the wavelength variation due to corrosion expansion was interrogated by an FBG interrogator . The interrogator was connected to a laptop via Ethernet, and the wavelength data was stored in the laptop with a saving frequency of one sample every 100 s.The attenuation rate of acoustic emission signal transmitted in concrete and steel materials are different. To check the sensitivity of each sensor, a pencil lead break test was conducted on the mortar specimen. The test consists of breaking a 0.5 mm diameter pencil lead by pressing it against the specimen surface. The testing points were along the longitudinal direction and the spacing between these points was 40 mm. Thus, there will be six testing points in total.To corrode the reinforced mortar in a reasonable amount of time, an accelerated corrosion test was performed. The specimen was placed in a bucket filled with 3.5% NaCl solution, which was used to simulate seawater. The specimen was immersed in the NaCl solution for three days before a constant current was applied. The constant current was set at 80 mA by a power supply. The anode was connected to the rebar and the cathode was connected to the sacrificial stainless steel plate, as shown in Attenuation of the generated acoustic emissions was considered as having a major influence on the sensitivity of the transducer and accuracy of the collected data. A pencil lead break test was used to produce similar AE events and check the sensitivity of each transducer. The signal amplitude received from both transducers during the pencil lead break test is shown in AE hits from both transducers during accelerated corrosion of the reinforced mortar specimen are shown in Peak frequency is the frequency with the maximum magnitude as determined by a fast Fourier transform (FFT) of the time domain signal. The peak frequency can serve as a good indicator for different AE sources arising from reinforcement corrosion. Other indicators of frequency, like average or central frequency, share the same properties. The cross-plot is an effective technique to analyze the features of an entire AE signal for the purpose of distinguishing signal characteristics. The circumferential strain variation of the reinforced mortar was measured using an FBG strain sensor. Measurement commenced the moment that the current was applied all the way to when the specimen cracked. The strain data over the time span of about 36 days is shown in The above results showed that FBG strain measurement is effective in monitoring reinforcement corrosion induced mortar expansion and cracking. For the case investigated in this study, a single strain curve provides corrosion information from these three aspects. First, the tensile strain around the mortar specimen due to expansive pressure exerted by the formation of corrosion products was continuously monitored. Second, the crack initiation and propagation period, featured by notable jagged variation of strain curve, can be easily recognized. Third, the moment of the first appearance of macrocracks on the mortar surface was detected and the crack width can be estimated from strain value. In future work, more samples will be tested and additional experiments will be designed to validate the efficacy of FBG strain measurement.The presented integrated corrosion monitoring technique was performed under controlled laboratory conditions. As for field application of the integrated corrosion monitoring technique, there will be many practical challenges. Since the reinforced concrete structure is generally large in size, surface mounting AE sensors may reduce the sensitivity and efficacy of the AE sensors. Alternatively, we recently proposed the low-cost, embedded smart aggregate as a type of embedded AE sensor . The embIn this paper, we present an integrated monitoring technique for reinforced concrete corrosion based on the novel coupling of acoustic emission and FBG strain sensing. A reinforced mortar was prepared and the corrosion process was accelerated through the injection of constant current. The setup allowed the study of corrosion characteristics at different stages. Corrosion monitoring can be divided into two aspects: the corrosion initiation and cracking propagation can be captured by an acoustic emission system, whereas the mortar expansion due to corrosion product generation will be monitored by a fiber Bragg grating strain sensor.It was found from the pencil lead break test that the attenuation of AE signal transmitted in mortar material is greater than that in steel material. Analysis from AE hits show that the hit history resembles the phenomenological model of reinforcement corrosion loss in a marine environment. AE signals are generated from three types of sources, including hydrogen bubble evolution, generation and movement of corrosion products, and micro-crack development and crack propagation. These three types of AE sources distinguish themselves in parameters like duration, energy and signal strength. Through continuous monitoring of tensile strain using FBG strain sensors, crack initiation and the propagation period were identified, and the time of concrete cover cracking was detected. Good agreement was obtained between acoustic emission activity and expansive strain measured on the specimen surface. Specifically, a similar trend in AE activity and strain measurement was observed as corrosion progressed. The AE events characterize the corrosion development based on corrosion induced AE events, and FBG strain measurement characterized the corrosion in terms of concrete expansion induced by rebar corrosion. Results have shown that the integration of these two nondestructive techniques is promising for reinforced concrete corrosion monitoring and characterization.