Various helicases and single-stranded DNA (ssDNA) binding proteins are known to destabilize G-quadruplex (GQ) structures, which otherwise result in genomic instability. Bulk biochemical studies have shown that Bloom helicase (BLM) unfolds both intermolecular and intramolecular GQ in the presence of ATP. Using single molecule FRET, we show that binding of RecQ-core of BLM (will be referred to as BLM) to ssDNA in the vicinity of an intramolecular GQ leads to destabilization and unfolding of the GQ in the absence of ATP. We show that the efficiency of BLM-mediated GQ unfolding correlates with the binding stability of BLM to ssDNA overhang, as modulated by the nucleotide state, ionic conditions, overhang length and overhang directionality. In particular, we observed enhanced GQ unfolding by BLM in the presence of non-hydrolysable ATP analogs, which has implications for the underlying mechanism. We also show that increasing GQ stability, via shorter loops or higher ionic strength, reduces BLM-mediated GQ unfolding. Finally, we show that while WRN has similar activity as BLM, RecQ and RECQ5 helicases do not unfold GQ in the absence of ATP at physiological ionic strength. In summary, our study points to a novel and potentially very common mechanism of GQ destabilization mediated by proteins binding to the vicinity of these structures. Escherichia coli RecQ, Saccharomyces cerevisiae Sgs1p, Schizosaccharomyces pombe Rqh1 and human WRN, RECQL1, RECQL4 and RECQL5 helicases (Human Bloom helicase (BLM) is a member of RecQ family ,2, whichelicases –7. RecQ elicases ,9. BLM ielicases  and otheelicases . Howeverelicases . BLM worelicases ,14. Evenelicases .S. cerevisiae Pif1 or human BLM, resulted in increased DNA breaks in PQS and severe retardation of DNA replication (G-quadruplex structures (GQs) are non-canonical DNA structures that form in guanine-rich regions of the genome. GQs are stabilized by Hoogsteen hydrogen bonding between guanosines, stacking of G-tetrad layers, coordination of Hoogsteen bonds by monovalent cations that intercalate between the tetrad layers and by hydration –18. Telolication ,44–48. Ilication . These mlication . In addilication .In vitro studies have demonstrated that both Sgs1 and BLM bind GQ with high affinity (DK ≈5 nM) and can efficiently unfold intermolecular GQs  to measure unfolding of intramolecular GQ by RecQ-core of BLM in the nucleotide-free (nt-free), ATPγS and ADP states under physiological salt concentrations. This truncated BLM construct, lacking the oligomerization region, was used to study the activity of BLM monomers  and will3GGG, as well as a 3′ overhang of 2–15 nt. For the exception, we used a sequence that forms a three layer GQ with 1 nt loops: (GGGT)3GGG. The sequences of these constructs  are given in Table + ions. In order to minimize the interaction of the GQ and BLM with the surface, partial duplex DNA (pdDNA) constructs with an 18 base pair long RNA/DNA heteroduplex stem and a flanking 3′ ssDNA were used. The flanking 3′-ssDNA contains the GQ forming segment and the overhang of 2, 4, 6, 8, 10, 12 or 15 nt 642–1290) was purified and characterized as described , and purified using a similar protocol to core BLM. Core BLM lacks the oligomerization domain, and therefore all experiments in this report are performed with monomeric BLM. BLM protein was stored in storage buffer that contains 50 mM Tris-HCl (pH = 7.5), 200 mM NaCl, 50% (v/v) glycerol and 1 mM dithiothreitol (DTT), and diluted in imaging solution, described in the smFRET Assay section below, to the desired concentration before being added to the microfluidic channel. Purification of E. coli RecQ  is obtained as a result of this protocol. Note that all experiments were carried out with samples that had undergone annealing the same day.To create the sample chamber, double-sided tape was sandwiched between a quartz slide and a glass cover slip. The quartz slide was first drilled, cleaned and coated with polyethylene-glycol (PEG) and biotin-PEG in the ratio of ∼100:1 (m-PEG-5000:biotin-PEG-5000 from Laysan Bio Inc.). Nucleic acid immobilization on the surface was achieved via a neutravidin-biotin linker. DNA and RNA-stems were hybridized by annealing them at 95°C for 5 min., followed by cooling to room temperature over 2–3 h. A stock concentration of 1 μM was prepared for this annealing step, which was diluted to 15 pM in multiple steps before injection into chamber. After 1–2 min of incubation, the chamber was washed to remove excess DNA. A density of roughly 250 molecules/imaging area  is significant for GQ stability, we henceforth refer to KCl as K+. In all assays performed in this study, the Mg++ was kept at 5 mM, regardless of monovalent cation type or concentration. Therefore, only the cation concentration will be mentioned where relevant, but it should be assumed that 5 mM Mg++ is also present in all assays. BLM as well as ATP, ADP and ATPγS were mixed in imaging solution at the desired concentration. The imaging solution was incubated with the DNA sample for 15 min to allow the system to reach steady state. Longer incubation times up to 1 h did not have a significant effect on the smFRET distributions. Long (1000–4000 frames) and short movies (30 frames) were recorded for two different types of analysis.A prism-type total internal reflection microscope built around an Olympus IX-71 microscope was used for all the measurements. Data were acquired at 40 ms time resolution using Andor Ixon EMCCD camera . The imaging solution used in all measurements contained Tris base , 2 mM trolox, 0.8 mg/ml glucose, 0.1 mg/ml bovine serum albumin, 1 mM DTT, 0.1 mg/ml glucose oxidase, 0.02 mg/ml catalase, 5 mM MgClFRET) time traces for each molecule. Such traces were filtered to ensure that only single molecules were selected and the background was subtracted from each of these selected molecules using a custom Matlab code. These traces were used to build population histograms of EFRET. Folded GQ constructs show high EFRET ≈ 0.60–0.80, whereas unfolded and protein-bound unfolded constructs show significantly lower EFRET, which depend on the construct used but is typically <0.40. The histograms were normalized to a percentage scale such that the total number of molecules in the histogram represents 100%. This normalization is necessary for the subtraction analysis which is employed to quantify the effect of BLM or nucleotides (effectors) on the EFRET distribution. In this analysis, a normalized reference histogram, i.e. the histogram representing the state that does not contain the titrated effector, is subtracted from normalized histograms representing various effector concentrations. The relevant reference histogram used for each subtraction analysis is specifically mentioned in the related section describing the data. In general, if BLM is titrated (at a constant nucleotide concentration), the histogram at zero BLM concentration is used as the reference. On the other hand, if a nucleotide is titrated (at constant BLM concentration) then the histogram at zero nucleotide concentration (in the presence of relevant BLM concentration) is used as the reference. For example, for BLM titration experiments in Figure +) before BLM is introduced to the chamber , is used to analyze the results obtained from these subtractions. The independent variable x is the effector concentration (either BLM or nucleotide concentration depending on the measurement), and the dependent variable y is the percent unfolded population with respect to a reference state. In this expression, Keq is the equilibrium constant and α describes the maximum unfolded population under saturating effector concentration. The error bars associated with the data were obtained from standard deviations of multiple data sets obtained for a given condition. The error values for the parameters of the Langmuir binding isotherm fits were obtained as a result of a weighted fit, i.e. the uncertainties in the individual data points were taken into account while fitting the data. Subtraction analysis and fits to data were performed using Origin Pro 8.5.Finally, Langmuir binding isotherm fit, of the form + ions, resulting in a high EFRET. Interaction of BLM with these folded GQ constructs results in various states with lower EFRET , unfolded GQ  and BLM-bound unfolded DNA. EFRET for the folded GQ is determined in the presence of 150 mM K+, the physiological concentration, before BLM is added to the sample chamber. In a few cases either 50 mM K+ or 50 mM Na+ was used instead, and is explicitly stated. In the case of pd-hGQ12T, which is a pdDNA GQ construct with a 12 thymine long overhang formed by hybridizing hGQ12T and RNA-Stem of Table FRET = 0.64 ± 0.07 and EFRET = 0.78 ± 0.05. In human telomeric constructs with shorter overhangs, these multiple peaks were interpreted as signatures of different folding conformations, which are also observed in circular dichroism (CD) and NMR studies . The BLM-bound folded state would have an EFRET between the folded and unfolded states, hence EFRET ≈ 0.55, which is distinctly observed in single molecule traces (data not shown) and is also visible in the steady-state histograms in Figures FRET for the unfolded states should be similar for the two main constructs of this study, pd-hGQ12T and pd-hGQ15T, as they only differ in length by 3 nt. Correspondingly, EFRET for their BLM-bound unfolded states should be similar. Therefore, the EFRET levels measured for pd-polyT35 for unfolded and BLM-bound unfolded states should also be similar to those of pd-hGQ15T, respectively. pd-polyT12 and pd-polyT12–5′ differ only in terms of the directionality of their overhangs but otherwise have the same separation between their donor–acceptor pairs. Therefore, the EFRET values for these constructs should be the same.The coiled DNA is stabilized into a GQ structure by KT Figure . The obs studies . We soug+, 5 mM Mg++, 50 mM Tris-HCl (pH 7.5) and 1 mM DTT. Binding reactions were carried out for 10 min at 22°C and then mixtures were loaded onto a 4–20% polyacrylamide TBE gel and run in 0.5× TBE at 50V for 5 h at 4°C. Gel was imaged using a Molecular Dynamics Typhoon 9400 Multilaser Scanner.BLM binding to pd-hGQ12T construct was measured by EMSA. Native polyacrylamide gel electrophoresis (PAGE) was used for this measurement. The partial duplex construct was formed by annealing the two strands via heating/slow cooling prior to the gel binding assay. Annealed substrates were mixed with varying BLM concentrations (0–500 nM) in 50 mM K+). In this study we used a core BLM polypeptide, BLM(642–1290), that exists only as a monomer both in solution and in the DNA-bound state  and BLM-bound unfolded (EFRET = 0.20 ± 0.08) states identified in Supplementary Figure S1, an equilibrium of both states is represented by the peak at EFRET = 0.30. Figure Keq = 305 ± 16 nM and α = 49 ± 1%, . We have further confirmed BLM-mediated GQ unfolding in the absence of ATP with a CD assay. In this assay, we measured the CD spectrum of DNA before and after adding 1 μM BLM to the environment. The characteristic peaks indicating GQ formation are diminished after adding BLM to the environment, indicating destabilization of GQ . Finally, smFRET measurements were performed on a DNA construct in which donor–acceptor fluorophores are moved directly to the ends of the GQ. This construct eliminates any significant FRET changes that might take place due to binding of BLM to the overhang, and results in lower FRET efficiency only when GQ is unfolded. The schematic of this construct and the smFRET data are shown in Supplementary Figure S2B. Even though this construct resulted in a reduction in the amount of unfolding in 150 mM K+, probably due to the internal fluorophore interfering with BLM-GQ interactions, a significant low FRET population, representing unfolded GQ was observed in 50 mM and 10 mM K+ . As is clear from these different measurements, BLM does not need ATP or any other nucleotide to unfold an intramolecular GQ at physiological pH and ionic strength. This is a novel observation as earlier studies performed on intermolecular GQs (We employed smFRET assay to measure intramolecular GQ unfolding activity of BLM in nucleotide-free (nt-free), ATPγS and ADP states under physiological salt concentrations . No GQ unfolding was observed upon addition of storage buffer without BLM or in the presence of heat-denatured BLM, confirming that activity required native BLM and was not due to GQ-destabilizing elements, such as metal chelators in the buffer . We ruled out the possibility of ATP contamination in purified BLM by testing whether BLM could unwind a forked DNA substrate  at saturating BLMK695M (500 nM) and ATP concentration (1 mM). Despite the lack of helicase activity, BLMK695M showed significant GQ unfolding activity in the absence of ATP . These measurements confirm that BLM does not require ATP hydrolysis to unfold GQ.Finally, to ensure BLM-mediated GQ unfolding does not require ATPase and helicase activity, we performed measurements with a mutant BLM construct that cannot hydrolyze ATP, BLM earlier ,62. The Keq = 4.2 ± 1.1 μM (for ATPγS) and α = 30 ± 4%. The relatively low Keq obtained suggests that the system reaches saturation at very low ATPγS concentration. The α parameter of this fit is a measure of the difference in the efficiency of BLM-mediated GQ unfolding in the ATPγS state relative to the nt-free state .Figure e Figure . In thise Figure , while te Figure . Taking Keq = 1.2 ± 0.1 μM (for ADP) and α = 14 ± 1%. The low Keq suggests that BLM-mediated GQ unfolding is very sensitive to ADP concentration and even a low micromolar concentration of ADP is adequate to reduce the unfolding activity by as much as 14%. Langmuir binding isotherm fit constants for Figure Figure The BLM footprint on ssDNA has been reported as either 7  or 14 nt+ and pH 7.5. However, we observed that BLM not only unfolds the GQ but also unwinds the RNA-DNA heteroduplex stem at significant levels at saturating BLM and ATP concentrations . To avoid complications that would arise due to duplex unwinding, we repeated these measurements with a DNA construct in which the polarity of DNA was reversed, e.g. 5′ is the free end, and the overhang that enables BLM binding is placed between the duplex and GQ . Figure FRET ≈ 0.85. Figure FRET ≈ 0.60 represents the DNA population that is bound by BLM. A subtraction analysis performed on these data with the coiled state in Figure The data presented thus far show that BLM-mediated GQ unfolding is most efficient in the ATPγS state, followed by the nt-free and ADP states. Earlier bulk measurements have shown that the stability of BLM binding to ssDNA in different nucleotide states follows a similar order . This cox = 2, 6, 8, 10, 12 or 15 and pd-hGQ4nt DNA constructs were used for these studies . BLM titration in the nt-free state resulted in α = 55 ± 4% and Keq = 28 ± 9 nM. Note that the Keq at 50 mM K+ is 10.8-fold lower than that at 150 mM K+ . The protein stock used for these measurements was in Na+, therefore, the measurements were performed in Na+ rather than K+. As BLM binding to ssDNA should not depend on the type of monovalent ion, this change is not expected to influence the results. We used a pdDNA with a 15-nt long 3′ ssDNA tail (pd-polyT15). These measurements showed ∼4.7× increase in Keq in 150 mM Na+ compared to 50 mM Na+. As we observed a 10.8× decrease in Keq in 50 mM K+, we conclude that the decrease in GQ stability contributes ∼2.3-fold to the increase in the Keq in 150 mM K+. These results demonstrate that both BLM binding affinity and GQ stability are significant contributors to the change in the equilibrium constant for BLM-mediated GQ unfolding. Finally, we measured BLM-mediated GQ unfolding as a function of ATPγS concentration in 300 nM BLM at 50 mM K+. These measurements resulted in α = 23% and Keq = 0.7 ± 0.1 μM, which is 6-fold lower compared to Keq at 150 mM K+  in contrast to the significant binding (36%) observed for the corresponding 3′ construct  even though the nt-free state still did not show significant BLM binding. Interestingly, BLM-mediated unfolding of pd-hGQ12T-5′ was observed in the nt-free state at 50 mM K+ in the presence of 1 μM BLM . BLM-mediated GQ unfolding was not observed in this construct at 150 mM Kn Figure . The lowy Figure , demonstM Figure , even th+. Changing the loop lengths, number of layers or the overhang sequences could result in changes in conformations . Figure +. Figure + and 150 mM K+. Note that 22 ± 3% and 17 ± 2% GQ were unfolded by 300 nM BLM in 150 mM K+ and 150 mM Na+, respectively. Similarly, 41 ± 6% and 38 ± 2% GQ were unfolded by 1000 nM BLM in 150 mM K+ and 150 mM Na+, respectively. Therefore, BLM unfolds GQ at similar levels, within the uncertainties of the measurements, in Na+ and K+. These results suggest that BLM unfolds different conformations of GQ with similar efficiency.GQs often fold into multiple conformations in Krmations  in addit0 mM Na+ . In ordeE. coli RecQ, human RECQ5 and human WRN, all full-length constructs, were used for these studies. WRN has been shown to unfold GQ in the presence of ATP  while signs of GQ destabilization were observed at 50 mM Na+ in both nt-free and ATPγS states . Therefore, the shift of the FRET peak position upon adding RecQ is most likely due to binding of one or more RecQ proteins to the 12-nt long overhang, rather than unfolding of the GQ. Similarly, RECQ5 did not unfold GQ at 150 mM K+ , but showed GQ unfolding activity at 50 mM Na+ in both nt-free and ATPγS states  of GQ  modulating the binding stability of the protein on the overhang, via changing the nucleotide state or the length of the overhang or (ii) changing the GQ stability, via monovalent ion concentration or changing the loop length in GQ.The correlation between the binding stability of BLM to the overhang and the BLM-mediated GQ unfolding activity highlights the significance of the overhang region in the vicinity of GQ. The function of the overhang region was previously studied in the context of intermolecular GQs and it was shown that BLM unfolds GQ with an overhang as short as 4 nt but cannot unfold GQ that lack an overhang . Our resF) of GQ . These IAnother important issue that should be addressed is whether the truncated BLM construct, RecQ-core of BLM or core-BLM, used in this study is representative of the full-length BLM (wt-BLM) construct that is physiologically relevant. Even though core-BLM has been shown to be a good model system for kinetic studies , its DNAAnother important point highlighted in this study is the influence of the overhang on BLM-mediated GQ unfolding. Various helicases have been shown to possess GQ unfolding activities and the efficiencies of these activities show large variations ,48. Our We demonstrate that BLM-mediated intramolecular GQ unfolding does not require ATP, and can occur in any nucleotide state including the nt-free state. Hence, translocase or helicase activity of BLM is not required for unfolding intramolecular GQ that has a long enough overhang. A limited range of measurements also showed WRN to have a similar GQ unfolding activity. RecQ and RECQ5 were able to unfold GQ only when the ionic concentration was lowered significantly below the physiological conditions, which resulted in a weaker GQ structure. The GQ unfolding activity of these proteins in the absence of ATP are in contrast to prior bulk studies performed on intermolecular GQs which showed GQ unfolding only in the presence of ATP. We also observe that BLM-mediated GQ unfolding is most efficient in the ATPγS state followed by the nt-free state and least efficient in the ADP state. This order correlates with BLM binding stability to the overhang ssDNA in these nucleotide states. We also show that BLM-mediated GQ unfolding becomes more efficient as the overhang length is increased or as the GQ stability is reduced. Finally, we showed that BLM does not show a significant conformation dependence in unfolding GQ. Our study raises a number of questions that challenge our current understanding of helicase-GQ interactions and the mechanism behind the GQ unfolding that results from these interactions. In particular, binding of proteins in the vicinity of GQ, even if the protein is not a translocase or translocation does not take place for different reasons, results in significant destabilization of GQ, which could be a major mechanism for resolving these structures inside the cells.Supplementary Data are available at NAR Online.


Top10%). The increment of 5-year (2010–2014) field weighted citation impact (FWCI) of internationally co-authored papers over the 5-year overall FWCI of the institutions in SciVal® is used as another indicator to eliminate the effect of discipline difference in citation rate. The results show that, for most top institutions, the difference between the citations per paper (CPP) for their publications with and without international co-authorship is positive, with increase of up to 5.0 citations per paper over the period 1996–2003. Yet, for some Asian institutions, by attracting a lot of researchers with international background and making these collaborating “external” authors as internal researchers, these institutions have created a special kind of international collaboration that are not expressed in co-authorship, and the CPP gaps between publications with and without international co-authorship are relatively small (around 0–1 citations per paper increment) for these institutions. The top old institutions have higher CPP than young institutions, and higher annual research expenditures; while young universities have a higher relative CPP increment for the current 5-year period over the previous 5-year period. The Δ%Top10% for international co-authored publications is generally higher than that for all journal publications of the same institution. With the increase of international co-authorship ratio, the mean geographical collaboration distance  of one institution based on the Leiden Ranking data also increases, and young institutions have relatively higher CPP increment over MGCD increment. International co-authorship has a positive contribution to the FWCI of the institution, yet there are untapped potential to enhance the collaboration among young institutions.We investigated the effect of international collaboration  on the impact of publications of young universities (<50 years old), and compared to that of renowned old universities (>100 years old). The following impact indicators are used in this study, they are: (1) the 5-year citations per paper (CPP) data, (2) the international co-authorship rate, (3) the CPP differential between publications with and without international co-authorships, and (4) the difference between the percentage of international co-authored publications falling in the global top 10 % highly cited publications and the percentage of overall publications falling in the global top 10 % highly cited publications (Δ% It is widely presumed that research collaboration; especially international collaboration, has benefits for both the researchers and the organisations involved, and enhances the quality of research  are used as the impact indications. These data are extracted from the Thomson Reuters Web of Science (WoS) database and Essential Science Indicator (ESI) based on papers published from 2004 to 2013. The mean geographical collaboration distance (MGCD) as a function of PP(int collab)  in the Leiden Ranking  field weighted citation impact (FWCI) of internationally co-authored papers over the 5-year overall FWCI of the institutions in SciValTop10%) are the other two indicators of impact. The analysis is based on papers published from 2004 to 2013. Only publications in the Web of Science Core Collection: Citation Index Expanded (SCI-Expanded), Social Sciences Citation Index (SSCI) and Arts & Humanities Citation Index (A&HCI) are used for publication search.The Thomson Reuters Web of Science database is used to extract the publication data, and the international co-authorship rate was calculated from the number of international collaborating publications therein. The 5-year CPP data for All Fields in the Thomson Reuters Essential Science Indicators is used as one of the impact indicators. The 5-year CPP difference between the publications with and without international co-authorships (ΔCPP) are calculated from the publication data, and the difference between the percentages of international collaborated publications falling in the global top 10 % highly cited publications and the percentage of overall publications falling in the global top 10 % highly cited publications  and PP(int collab)  in the Leiden Ranking  of internationally co-authored publications and the 5-year overall FWCI of the collaborating institutions are extracted from SciValFigures Figure Figure Figure For the 16 renowned young and old universities, 57 bilateral collaboration couples with 50 and more co-authored publications are identified, and the FWCI increment data for these collaboration couples are include in the plot. The original data for the plot are provided in Tables Moed  has doneFigure Top10%). It can be seen that for all the selected young and old institutions, this difference is generally positive, means that internationally collaborated publications generally have a higher rate of high citation publications among all publications. Yet, this difference varies from one institution to another institution. For some renown world top universities like Caltech, Stanford University and University of Cambridge, although their overall CPP for their publications is already very high, the percentage of their internationally collaborated publications falling in the global top 10 % highly cited publications is still higher than the percentage of their overall publications falling in the global top 10 %. Further investigation is needed to have an adequate explanation for this phenomenon.Figure Table In this study, the effect of international co-authorship on the impact of publications of selected young universities and well established renowned universities was investigated. The results show that, both young and old institutions benefitted from international co-authorship as evident by the citation impact of their publications. For example, the CPP increased by 4.12 cites for every 10 % increase in international co-authorship rate for MIT, 3.42 cites for University of Oxford, and 3.01 cites for Stanford University. Among young universities, NTU, the increase is 2.24 CPP per 10 % Intl Collab increment, and for Plymouth University it is 3.02 CPP per 10 % Intl Collab increment, and 0.73 CPP per 10 % Intl Collab increment for King Fahd Univ of Petr and Min. Young universities has a higher relative CPP increment for rolling 5-year measurement in the period 2003–2013.The percentage of publications in the ESI global top 10 % highly cited publications for international co-authored publications is generally higher than that for all journal publications of the same institution. Yet, this difference varies from one institution to another institution. For some renowned top universities like Caltech, Stanford University and University Cambridge, although their overall CPP for their publications is already very high, the percentage of their internationally collaborated publications falling in the global top 10 % highly cited publications is still higher than the percentage of their overall publications falling in the global top 10 %.Asian institutions like HKUST, NUS and NTU have attracted a lot of researchers with international background to work in there. As the collaborating “external” authors are internalized in these institutions, a special kind of international collaboration that are not expressed in co-authorship has been created, and the CPP gaps between publications with and without international co-authorship are relatively small (around 0–1 citations per paper increment).The international co-authorship also increases the FWCI of the institution, yet there are untapped potential to enhance the collaboration among young institutions.With the increase of international co-authorship ratio, the mean geographical collaboration distance (MGCD) of one institution also increases. Although the old universities has a higher CPP value in general, young institutions have relatively higher CPP increment over MGCD increment.


The use of antibiotic prophylaxis in inguinal hernia repair is a controversial issue. Accepted randomized controlled trials or registry data with specific analysis of endoscopic repaired patients do not exist.The data presented in this study compared the prospectively collected data from the Herniamed Registry on all patients who had undergone unilateral, bilateral or recurrent repair of inguinal hernias using either endoscopic or open techniques between September 1, 2009, and March 5, 2014. In total, 85,033 patients were enrolled. Of these patients, 48,201 (56.7 %) had an endoscopic and 36,832 (43.3 %) an open repair. The target variables analyzed were impaired wound healing and deep infections with mesh involvement within 30 days after the operation.n = 48,201) did not identify any significant influence of antibiotic prophylaxis on postoperative impaired wound healing, which occurred in 53 cases (p = 0.6431). Nor was it possible to identify any significant impact of antibiotic prophylaxis on the deep infections seen in 27 cases (p = 0.8409). Analysis of the open inguinal hernia repair group revealed that, unlike the laparoscopic/endoscopic group, antibiotic prophylaxis had a significant impact on the postoperative impaired wound healing and deep infection rates. The risk of postoperative impaired wound healing with antibiotic prophylaxis was significantly lower .Analysis of the patient group with endoscopic/laparoscopic inguinal hernia repair (The positive impact of the endoscopic/laparoscopic technique on avoidance of impaired wound healing and deep infections with mesh involvement is already so great that antibiotic prophylaxis has no additional benefit. In contrast, antibiotic prophylaxis should be administered for open inguinal hernia repair. The use of antibiotic prophylaxis in inguinal hernia surgical repair is a controversial issue. Prospective randomized controlled trials (RCTs) have identified postoperative infection rates  of between 0 and 8.9 % in the absence of antibiotic prophylaxis and of between 0 and 8.8 % on administration of antibiotic prophylaxis .Of the 14 RCTs that compared inguinal hernia repair while using antibiotic prophylaxis versus placebo, 13 surgical procedures were performed using open repair and only one with an endoscopic technique , the 10-year OR estimate was given.Since the properties of endoscopic and conventional techniques are presumably different, further analyses are made in these subgroups.n = 85,033 inguinal hernia operations were recorded in the Herniamed Registry in accordance with the inclusion criteria. Antibiotic prophylaxis was administered in n = 60,831 cases (71.54 %) and was not in 24,202 cases (28.46 %). Of the 60,831 cases that had received antibiotic prophylaxis, n = 59,177 (97.27 %) received single-shot antibiotic prophylaxis, n = 253 (0.42 %) for 1 day, n = 607 (0.99 %) for 2–3 days, and n = 794 (1.31 %) received antibiotic treatment for more than 3 days. No details of the antibiotic used were recorded in the registry study. Based on the recommendations of the German Paul-Ehrlich Society, in general ampicillin with a beta-lactamase inhibitor or a group 1 or 2 cephalosporin was administered . Likewise, there were significantly fewer cases of postoperative impaired wound healing among men  as well as for primary hernias . The negative influence of the ASA status was imputed especially to the increased impaired wound healing rates observed for ASA IV status [IV vs I: OR 4.226  p = 0.001] , p = 0.006, was calculated for antibiotic prophylaxis. The risk of postoperative deep infection was significantly lower for the laparoscopic/endoscopic surgical technique . Deep infections also occurred significantly more often among ASA IV patients  , 08; 0.862n = 48,201 endoscopic/laparoscopic and n = 36,832 open inguinal hernia operations, antibiotic prophylaxis significantly reduced impaired wound healing and deep infection rates.In summary, it can be concluded that impaired wound healing and deep infections occurred significantly less often after endoscopic/laparoscopic inguinal hernia surgery compared with open repair. In the total patient collective comprising n = 48,201) did not identify any significant influence exerted by the different variables on postoperative impaired wound healing, which occurred in 53 cases (p = 0.6431). Nor was it possible to identify any significant impact of the variables on the deep infections seen in 27 cases (p = 0.8409) , The present Herniamed Registry study investigated the influence exerted by antibiotic prophylaxis on the occurrence of infectious complications following laparoscopic/endoscopic inguinal hernia operations and compared these findings with those obtained for open inguinal hernia surgery. Besides, other risk factors were identified for onset of impaired wound healing and deep infections with mesh involvement. This also applies for patients with risk factors. For the total patient collective comprising 85,033 patients with 48,201 endoscopic/laparoscopic and 36,832 open inguinal hernia operations, it was revealed that postoperative infectious complications were significantly reduced by use of the endoscopic/laparoscopic technique and antibiotic prophylaxis. Indeed, analysis even revealed that the influence exerted by the endoscopic/laparoscopic technique on onset of postoperative infectious complications had a more preventive effect than that of antibiotic prophylaxis. Hence, the minimally invasive procedure for inguinal hernia repair made a greater contribution to the prevention of impaired wound healing and deep infections with mesh involvement than did antibiotic prophylaxis.If one analyzes the group of 48,201 endoscopic/laparoscopic surgery patients as a separate entity, one notes that the use of antibiotic prophylaxis was unable to reduce further the rates of impaired wound healing and deep infections with mesh involvement. Hence, antibiotic prophylaxis should not be administered for endoscopic/laparoscopic inguinal hernia surgery. Conversely, analysis of the 36,832 open inguinal hernia repair group showed that antibiotic prophylaxis had a significant impact on onset of postoperative impaired wound healing and deep infections with mesh involvement. Accordingly, antibiotic prophylaxis should be administered for open inguinal hernia repair.Of the 14 prospective randomized controlled trials that compared inguinal hernia surgery under antibiotic prophylaxis versus placebo, the endoscopic technique was performed only in one case trial .Nor has the impact of antibiotic prophylaxis on occurrence of infectious complications following endoscopic/laparoscopic inguinal hernia operations been investigated by other studies recorded in the review . As suchHowever, the converse situation applies for open inguinal hernia repair. Multivariable analysis identified that antibiotic prophylaxis had a significant effect on onset of impaired wound healing and deep infections with mesh involvement. That concords with the findings of several meta-analyses –12. Conv


A contemporary reading of Romans 1:27 was disguised as a saying by Paul Benjamin, AD 58 and administered to 275 randomly selected members of a private Christian university community in south western Nigeria in West Africa. Participants were asked to respond to a two-item questionnaire on their perception of the cause of HIV/AIDS either as a judgment from God or consequence of individual lifestyle choices. The apparent consensus drifted in the direction of God as the culprit handing down his judgment to perpetrators of evil who engage in the homosexual lifestyle. The goal of this paper was to examine the implications of a judgmental stance on addressing the psychosocial needs of Persons Living with HIV/AIDS in religious environments. It also explores how service providers in faith-based environments can work around the Judgment versus Consequence tussle in providing non-discriminatory services to persons diagnosed with HIV/AIDS. The Global Health Council  estimateUNAIDS: ‘AIDS in Africa: Three scenarios to 2025’ It has been observed that religious doctrines and moral positions by religious leadership have helped create and support perceptions that those infected have sinned and deserve their punishment, thus increasing the stigma associated with HIV/AIDS The meanings of these biblical passages attributable to Apostle Paul the writer of Romans and 1 Corinthians may be taken for granted by Christians, but on a closer examination may evoke negative reactions about PLWHIV. This study examined the belief about AIDS as reflected in responses to Romans 1:27 and its impact on attitudes and service delivery in a faith-based community. The study will not be making reference to the 1 Corinthians text of the bible as a point of reference in the questionnaire administered to respondents in the study. The biblical passage given to respondents will be restricted to Romans 1:27 to reduce the incidence of confusion on the part of the respondents.‘And the men, instead of having normal sexual relationships with women, burned with lust for each other. Men did shameful things with other men and, as a result, suffered within themselves the penalty they so richly deserved’ (Romans 1:27 New Living Translation).Given the generally increasing awareness that the virus does not discriminate by age, race, gender, ethnicity, sexual orientation or socioeconomic status, it is also a growing fact that certain groups are at particular risk of HIV, including men who have sex with men, injecting drug users and commercial sex workers  were students. Of the students 11% were in high school, 71% were in undergraduate programs, 18% were in the post-graduate program. The majority of the respondents identified themselves as Christian (98.1%) and were single (75.7%).To test the knowledge base of the respondents regarding the opinion if there is any relationship between homosexual lifestyles and HIV/AIDS the following question was asked:‘In your opinion is there a connection between the homosexual and gay lifestyle and HIV/AIDS. [Human Immunodeficiency Virus (HIV) – Acquired Immune Deficiency Syndrome (AIDS).] Please explain.’63% indicated yes and 22% indicated no while 15% stated, I do not know.Between genders, more female respondents stated that there is a connection between the homosexual lifestyle and HIV/AIDS as illustrated in In response to the question, ‘In your opinion, do you believe that HIV/AIDS is God's punishment for the participants of gay/lesbian lifestyle?’ most of the female respondents believed that HIV/AIDS is a punishment from God as shown in Thirty percent of the respondents who stated that HIV/AIDS was a punishment from God on persons involved with homosexual life style were students in post secondary institutions.A female university student between ages 26 and 40 said ‘Yes, it's likely that bacterias can form into HIV after sexual intercourse.’A male staff member also in the age range of 26–40 stated with a certainty of knowledge ‘Yes, because it's been recognized as a primary mode of transmission.’A female Christian student within the age range of 15–25 confessed her limited knowledge by stating, ‘I don't think so. The virus started when a white lady slept with a Gorilla/Monkey.’A male Christian student within the age range of 15–25 responded no to the question stating, ‘No, because AIDS is sexually transmitted between opposite sex.’A female Christian student within the age range of 15–25 responded ‘No, because HIV/AIDS came to being by a man who slept with a dog. I feel it is transferred from man to woman not from man to man.’The second question sought to elicit the opinion of the respondent on their understanding of a biblical statement disguised as a writing of one of the early Church fathers:‘And the men, instead of having normal sexual relationships with women, burned with lust for each other. Men did shameful things with other men and, as a result, suffered within themselves the penalty they so richly deserved’ (Romans 1:27 New Living Translation).In your opinion do you believe from this statement that HIV/AIDS is God's punishment for the participants in the lifestyle? Please explain .Fig. 3.Of 263 respondents, 128 or 49% responded that it was God's judgment, 118 or 45% did not think it was God's judgment and 17 or 6% claimed they did not know the answer.A male Christian student stated that ‘HIV/AIDS is not God's punishment for the participant but it's punishment for disobeying God's word.’I do not think so. the virus started when a white lady slept with a Gorilla/Monkey.No, because it can only be transferred from male to female.Yes, because in order to satisfy their sexual urge, men go after animals and men.Yes, there's a connection because it is a sinful attitude.No, there's no relationship between homosexuals and gay lifestyle with HIV/AIDS even though the lifestyle is not healthy and also not godly.Yes, there is a connection, because originally, HIV was actually gotten from there, by sleeping with dogs, the same sex, etc.These are satanic ways of creating in people's mind bad image about our creator. God ordained man to woman relationship not man to man.Is there a connection between HIV/AIDS and homosexual lifestyle? Sample of respondents statementsAs Agadjanian  has poinet al. (2002) was confirmed in this study that erroneous belief about HIV/AIDS could fuel stigmatization. Some of the respondents stated that HIV/AIDS came as a result of a human having sexual intercourse with an animal. There is therefore an ongoing need to educate young people about the etiology of AIDS/HIV especially in post secondary institutions. Current epidemiological studies have established three modes of transmission of HIV/AIDS, which are sexual intercourse with an infected person; second, through exposure to contaminated blood, blood products or transplanted organs and tissues; and third, from an infected mother to her fetus or infant before, during or shortly after birth  gave them the admonition to ‘go and sin no more’. This admonition was a two-edged statement of empowerment to live a sin-free as well as condemnation-free life. It was also an indication that they remove themselves from situations and circumstances that precipitated and predicated infection or re-infection of disease, in this case with HIV. It is therefore suggested that part of the educational process for the target population be focused on sexual abstinence training and faithfulness to single sexual partner activity.One of the limitations of this study is that it was carried out in a Christian private university. The findings may not be generalizable to other religious settings such as Islam or traditional religion. Also it was restricted to the university community. A follow-up research may be carried out in church-based settings. The small sample size of 300 questionnaires may also be viewed as a limitation. Finally, the instrument utilized was a two-point questionnaire, which may have been simple for respondents to understand. The format of the questionnaire may not have allowed for the capture of more details about behavior among respondents.This study was carried out in a faith-based University in Nigeria to explore the beliefs and perceptions about AIDS/HIV in response to a contemporary reading of Roman 1: 27. A majority of the respondents believed that AIDS/HIV was a punishment from God and erroneous beliefs about causality and modes of transmission were expressed. The study made recommendations for FBOs to provide a forum for accurate education and socialization of congregants to be open to providing services to PLWHA. Religious leaders were encouraged to extend the Christian tenets of caring just as Jesus himself demonstrated to include services to PLWAH.


MET fluorescence in situ hybridization and MET and hepatocyte growth factor immunohistochemistry. Eleven different entities were included, among them the most common and clinically relevant subtypes and tumors with specific translocations or complex genetic changes. MET protein expression was observed in 2.6% of the cases, all of which were either undifferentiated pleomorphic sarcomas or angiosarcomas, showing positivity rates of 14% and 17%, respectively. 6% of the tumors showed hepatocyte growth factor overexpression, mainly seen in undifferentiated pleomorphic sarcomas and angiosarcomas, but also in clear cell sarcomas, malignant peripheral nerve sheath tumors, leiomyosarcomas and gastrointestinal stromal tumors. MET and hepatocyte growth factor overexpression were significantly correlated and may suggest an autocrine activation in these tumors. MET FISH amplification and copy number gain were present in 4% of the tumors (15/413). Two samples, both undifferentiated pleomorphic sarcomas, fulfilled the criteria for high level amplification of MET, one undifferentiated pleomorphic sarcoma reached an intermediate level copy number gain, and 12 samples of different subtypes were categorized as low level copy number gains for MET. Our findings indicate that angiosarcomas and undifferentiated pleomorphic sarcomas rather than other frequent adult-type sarcomas should be enrolled in screening programs for clinical trials with MET inhibitors. The screening methods should include both in situ hybridization and immunohistochemistry.Soft tissue sarcomas are a heterogeneous group of tumors with many different subtypes. In 2014 an estimated 12,020 newly diagnosed cases and 4,740 soft tissue sarcoma related deaths can be expected in the United States. Many soft tissue sarcomas are associated with poor prognosis and therapeutic options are often limited. The evolution of precision medicine has not yet fully reached the clinical treatment of sarcomas since therapeutically tractable genetic changes have not been comprehensively studied so far. We analyzed a total of 484 adult-type malignant mesenchymal tumors by  Soft tissue sarcomas represent a heterogeneous tumor group which consists of over 50 different histologic subtypes . Taken tKIT or PDGFRA  mutations in approximately 90%. For patients with other soft tissue sarcoma entities, however, new effective and reliable molecular based treatments are desirable. Targeting tyrosine kinases has given promising results in many malignancies, but comprehensive studies on soft tissue sarcomas are still lacking.For some sarcoma subtypes tumor specific molecular aberrations such as gene translocations, amplifications or mutations have been identified, however, their exploitation as therapeutic targets has been limited. The best example for molecular targeted therapy in mesenchymal tumors is treatment with tyrosine kinase inhibitors in gastrointestinal stromal tumors (GIST) which carry activating One of those therapeutically tractable tyrosine kinases is MET which is currently subject to many clinical trials with promising results. MET is a transmembrane tyrosine kinase receptor and is also known as hepatocyte growth factor receptor (HGFR) . It is min vitro and in vivo. Analyzed cell lines included clear cell sarcomas [The HGF/MET pathway can therapeutically be targeted in a variety of ways, e.g., blockade of the ligand-receptor interaction using HGF antagonists, HGF neutralizing antibodies or MET antibodies, inhibition of receptor dimerization and inhibition of kinase activity with interruption of downstream signaling using small molecule inhibitors . There asarcomas , rhabdomsarcomas , osteosasarcomas , alveolasarcomas , as wellsarcomas  and malisarcomas . HoweverMET gene amplification, and MET as well as HGF protein overexpression in clinically relevant sarcoma entities. We describe frequencies and patterns of MET gene copy number alterations by fluorescence in situ hybridization (FISH) as well as MET and HGF expression by immunohistochemistry. Our results were obtained by up-to-date biomarker assays and provide first comprehensive epidemiologic data for ongoing and upcoming clinical trials with MET/HGF inhibitors in soft tissue sarcomas and GIST.In this study we aimed at investigating the HGF/MET axis in a comprehensive and well characterized cohort of various adult-type soft tissue sarcomas and GIST. Our goal was to determine the prevalence of the three major mechanisms of MET activation, i.e., MET gene copy numbers.A total of 484 malignant mesenchymal tumors were selected for analysis from the files of the GIST and sarcoma registry Cologne/Bonn, Germany. The samples were collected between 1992 and 2011. All tumors were reviewed by experienced pathologists  based on histology, immunohistochemistry and molecular findings according to the current WHO classification [This study was conducted in accordance to the Declaration of Helsinki and to guidelines of the local Ethics Committees. Cases were derived from our consultation files and originated from various primary sites. Samples were obtained as part of routine clinical care with verbal informed consent from each patient. Patients and their physicians agreed in sending paraffin blocks to us for further analyses. Met studies within this work were carried out anonymously as a retrospective epidemiologic investigation. Therefore, the Ethics Committee of the University Hospital Cologne, Germany, waived the need for a specific approval or written informed consent.For immunohistochemical determination of protein expression antibodies against MET , Rabbit Monoclonal Primary Antibody, Ventana, Tucson, AZ)  and HGF in situ hybridization (FISH) for analysis of MET gene copy numbers was performed with the ZytoLight SPEC MET/CEN7 Dual Color Probe . Hybridization was carried out as previously published [MET/CEN7 probe was applied on the tumor tissue. Denaturation, 75°C for 10 minutes, and hybridization, 37°C overnight, were carried out in the ThermoBrite humidity chamber . After posthybridization washes the slides were counterstained with DAPI and coverslipped. Using a fluorescence microscope  equipped with the appropriate filters, the whole tumor area was evaluated with a 63x or 100x objective. The numbers of MET and centromere 7 signals were counted in 60 nuclei obtained from three different areas with the highest gene count. Average gene copy number and ratio (MET/CEN7) as well as the percentages of tumor cells with ≥4, 5 and 15 gene copies were calculated for each tumor. Tumors were sorted into four categories based on a MET scoring system previously introduced by us in a lung cancer study [high-level amplification was defined asMET/CEN7 ratio ≥2.0 orMET gene copy number per cell of ≥6.0 oraverage MET signals.≥10% of tumor cells containing ≥15 intermediate-level copy number gain being defined asMET signals and≥50% of cells containing ≥5 criteria for high-level amplification are not fulfilledlow-level copy number gain was defined asMET signals and≥40% of tumor cells showing ≥4 criteria for high-level amplification or intermediate-level copy number gain are not fulfillednegative.all other tumors were classified as The fluorescence ublished . Brieflyer study . In brieFISH positivity was defined by categories i-iii, including amplification and copy number gains.For statistical analyses SPSS 21.0 software  was used. All tests were two-sided, with a 95% confidence interval.Immunohistochemical staining results were available for MET in 388/484 and for HGF in 466/484 tumors. Results for both parameters were obtained in 382 tumors.MET: Out of 388 analyzable samples 10 tumors (2.6%) were MET immunopositive (MYC amplification status (p = 0.662). For a subset of angiosarcomas and for clear cell sarcomas we could not obtain valid data on MET immunohistochemistry  (MYC amplification (p = 0.478). The majority of the conventional tissue slides prepared from angiosarcomas and clear cell sarcomas were homogeneously stained with the same immunoscore in all malignant cells.s (6.0%) , Fig. 1.388 tumors were evaluable for MET and HGF immunostains. Out of the 10 MET positive tumors, HGF results were available for 9 samples. MET and HGF immunoscores were highly significantly correlated  with an average MET gene copy number of 5.13/nucleus. MET/CEN7 ratio was 1.95 in this case. The other 12 positive cases were tumors with low level copy number gains . The MET/CEN7 ratio in this subgroup was 0.86–1.55. The percentage of tumor cells with ≥4.0 MET signals ranged from 41.7% to 60%. Low level copy number gain was seen in angiosarcomas , undifferentiated pleomorphic sarcomas , dedifferentiated liposarcomas , pleomorphic liposarcomas , leiomyosarcomas  and myxoid liposarcomas . Out of the 398 FISH negative tumors 194 were disomic  and 204 polysomic for MET . In a subset of angiosarcomas and clear cell sarcomas whole tumor sections were hybridized all of which showed a homogeneous signal distribution. Heterogeneity, as defined by 5–50% of tumor cells fulfilling at least one of the MET FISH positivity criteria .The higher the FNCLCC histologic grade, the more tumors were FISH positive: 6.2% of grade 3 sarcomas , 3.2% (2/63) of grade 2 and none (0/50) of grade 1 tumors were FISH positive. However, the correlation was not significant. Sarcoma entities with complex karyotypes were more often FISH positive (7.3%) than sarcomas with specific translocations or mutations (1.2%) (p = 0.001 Chi square). There was no significant correlation with etiology of angiosarcomas  or MET FISH were available for 341 cases  were negative for both MET IHC and MET FISH.Results for both MET immunohistochemistry and 41 cases . NotablyIn the evolving era of precision medicine previous studies on therapeutic targets and biomarker assays have mainly focused on large tumor entities, such as, for example, breast cancer or lung tumors. These neoplasms and the recent improvements in their therapeutic options are publicly well recognized and, thus, have led to rapid development of novel biomarker assays and enrollment of many patients in clinical trials. Contrarily, rare cancers as sarcomas and orphan diseases were not extensively studied up to now. There is, however, an urgent clinical need to improve the outcome of sarcoma patients. Roughly 5,000 estimated sarcoma related deaths per year in the U.S. indicate that the prognosis has not fundamentally changed over years. One approach to tackle this issue is to include sarcomas in prevalence studies of novel biomarkers and in early clinical trials.MET gene amplification which is discussed as potential predictive marker for anti-MET therapy with tyrosine kinase inhibitors.In this study we have investigated the prevalence of MET alterations in a comprehensive cohort of well characterized adult-type soft tissue sarcomas and gastrointestinal stromal tumors. MET is currently studied in early and late-stage clinical trials. Activation of the MET/HGF axis can occur through diverse mechanisms suggesting several options for therapeutic intervention. Among the most promising approaches are tyrosine kinase inhibitors  as well as anti-MET and anti-HGF antibodies. Therefore, we aimed to investigate the protein expressions of the MET kinase and its ligand HGF which might serve as biomarkers for the corresponding therapeutic antibodies. Furthermore, we investigated MET amplification, MET copy number gain or MET or HGF overexpression. This finding might be relevant for future clinical trials since liposarcomas represent one of the most common sarcoma types in adults. If not further specified “soft tissue sarcomas” are included in clinical trials with MET inhibitors it is conceivable that a considerable number of those patients would suffer from one of the liposarcoma entities which, based on our study, are unlikely to receive benefit from MET-targeted therapy. The same holds true for other clinically relevant adult-type sarcoma entities such as leiomyosarcomas and MPNST as well as for GIST.We have included tissues from the major clinically relevant sarcoma entities which occur predominantly in adults. Based on our results we can clearly show that 94% of these tumors do not have any changes in the MET/HGF axis as measured by IHC and FISH. Notably, none of the more than 150 liposarcoma samples of any type  showed MET amplifications, MET copy number gains or show MET/HGF overexpression. Both entities represent tumors with poor clinical outcome for which targeted therapies have not been established. Interestingly, the high level MET amplifications in our study are restricted to UPS where they are found in 6% of the analyzed cases. MET overexpression was found in 17% of angiosarcomas and 14% of UPS. Notably, MET overexpression—measured by IHC—was not restricted to MET FISH positive cases whereas MET and HGF overexpression were found to be significantly correlated. Therefore, MET overexpression in FISH negative but HGF-positive cases could indicate autocrine or paracrine stimulation of the MET/HGF axis. MET overexpression in HGF-negative tumors that are FISH negative might be due to other genetic alterations of the MET gene such as activating mutations or transcriptional regulation. Processing of MET-RNA, translational and protein regulation might as well result in an overexpression of MET.However, we were able to show that two distinct sarcoma entities out of the eleven tumor types included in our work harbor potentially tractable MET alterations. We demonstrate that a subset of angiosarcomas and undifferentiated pleomorphic sarcomas (UPS) harbor MET gene copy number gain and amplification with IHC status is peculiar. As a matter of fact, tissues collected in a phase II trial evaluating the anti-MET antibody onartuzumab in advanced NSCLC showed that MET amplified cases represented a subpopulation of MET positive cases as defined by IHC [MET harbor mutations preventing transcription or translation into a stable protein which provides suitable epitopes for immunohistochemistry. It might as well be possible that the poor correlation of MET amplification and gene copy number alteration with IHC reflects a specific feature of sarcomas since the similar observation has recently been reported by another group [MET copy number gain and amplification are frequently associated with lack of IHC staining in this disease group.The poor correlation between d by IHC . The lacer group . It willMET amplifications were found. Based on our findings we recommend applying both methodologies, in situ hybridization as well as immunohistochemistry, for further molecular screening procedures in prevalence studies of sarcomas. One major challenge in that context to date is that the criteria for FISH or immunopositivity are not yet consistently defined. So far as we know from previous investigations of MET in various entities it is important to adjust biomarker assays and criteria for “MET positivity” for each tumor type individually. In terms of MET immunohistochemistry we have tested various antibodies  and finally decided to perform our study with the SP44 antibody which revealed the most reliable stainings in a number of test tissues. Similar findings were recently reported by Koeppen et al. [Based on our findings angiosarcomas and undifferentiated pleomorphic sarcomas rather than other frequent adult-type sarcomas should be considered for clinical trials with MET inhibitors. Angiosarcomas of the skin and the deep soft tissues occur in at least three different clinical settings: i) sporadically, ii) associated with chronic lymphedema and iii) induced by radiation. The incidence of post-radiation angiosarcomas is steadily increasing mainly since most breast cancer patients receive post-surgery radiation, and in a considerable proportion of those patients angiosarcomas occur years after treatment . These ln et al. . They coThe immunohistochemical scoring system we worked with was four-tiered, similar to that applied for assay validation  and lateMET copy number changes.We used immunohistochemistry also for the detection of hepatocyte growth factor overexpression, and we applied a scoring system similar to MET IHC in which the strongest staining intensity and the highest proportion of stained cells (which was observed only in non-sarcoma control tissues) was defined as 3+. We are, however, aware of some methodological limitations of this approach. To date, there are currently only few HGF antibodies on the market and none of them has been comprehensively validated for the use in clinical trials. We strongly recommend verifying our data with novel monoclonal antibodies when available or with other methods. Our preliminary data on HGF expression, however, are promising since we could demonstrate a statistically significant correlation between HGF and MET overexpression namely in angiosarcomas and undifferentiated pleomorphic sarcomas. Cases with 2+ HGF staining intensity were also seen in leiomyosarcomas, MPNST and clear cell sarcomas. These entities should, therefore, be included in confirmatory studies on HGF expression. Unfortunately we could not correlate our findings of HGF expression in clear cell sarcomas with MET immunohistochemistry due to limited tissue resources. Based on previous reports clear cell sarcomas do show MET expression to a substantial extent , 42, 43,in situ hybridization for the detection of MET amplifications in these tumors up to now. Based on our findings and on published reports we propose evaluation criteria which are easily applicable in sarcomas. Previously described criteria for MET FISH in different solid tumors varied, sometimes judging carcinoma cases as “positive” which were in fact far away from gene amplification. Therefore, we aimed to create a category of MET FISH positivity limited to truly MET amplified tumors. This “high level” category is defined by a MET/CEN7 ratio ≥2.0 or an average MET gene count per cell ≥6.0 or ≥15 gene copies in ≥10% of tumor cells. These criteria are generally accepted for amplification of many other genes [MET gene copy numbers (“MET negatives”). There is, however, a group of tumors with slightly or moderate increases in gene copy numbers which we categorized as intermediate (≥50% of tumor cells with ≥5 gene copies) or low level cases (≥40% of tumor cells with ≥4 gene copies). Currently we do not know whether these latter categories have any clinical relevance. We have, however, introduced these groupings for two reasons: i) to allow comparison of MET FISH data with previous results obtained by other groups since our criteria for low and intermediate level have been suggested as parameters for MET amplification in earlier reports on various cancers; ii) to provide a level of probability for response to anti-MET treatment. It is currently not known whether sarcoma patients will benefit from such a therapy. It would, however, be wise to include cases from that high level category with homogeneous MET amplification in early clinical trials at first.With more than 400 sarcoma and GIST cases included in our study this is the largest investigation on fluorescence er genes , 45. By MET copy number changes. Based on preliminary data in NSCLC patients with high-level MET amplification seem to be most likely to benefit from MET-targeted therapy [Clinical trials with MET inhibitors could be discussed for sarcoma patients but will require careful planning. Based on our findings first and foremost patients with angiosarcomas or undifferentiated pleomorphic sarcomas could be considered as these entities represent sarcoma types which show first evidence of potential dysregulation of the MET pathway. Strategies to enrich for patients most likely to respond to MET-targeted therapies should be employed and should not only include IHC for MET and HGF but also determination of  therapy . The lowMET gene amplification. We have established biomarker assays for immunohistochemistry and FISH and provide first data on frequency of MET alteration in sarcomas. We could demonstrate that angiosarcomas (either primary or radiation induced) and undifferentiated pleomorphic sarcomas represent those entities with a significant proportion of MET positivity. Therefore, we provide evidence that these sarcoma entities are candidates for upcoming clinical trials with compounds inhibiting the HGF/MET axis.In summary, we investigated a comprehensive series of more than 400 adult-type sarcomas for MET and HGF overexpression as well as for


MET gene amplification and overexpression predicted the sensitivity to MET inhibitors in gastric cancer. However, the identification of drug-resistant tumors has encouraged the pre-emptive elucidation of the possible mechanisms of clinical resistance. The current study assessed a number of patient-derived gastric cancer models with MET amplification and overexpression, including CNGAS028. The tumor tissues were subjected to microarray analysis (using single nucleotide polymorphism 6.0 and human genome U133 arrays) followed by western blotting. The results demonstrated that CNGAS028 xenograft tumors did not respond to treatment with a selective MET inhibitor. Additional analysis indicated that FGFR2 overexpression contributed to the resistance to MET inhibitors. Furthermore, treatment with a combination of fibroblast growth factor receptor 2 and MET inhibitors inhibited the growth of CNGAS028 xenograft tumors in vivo. In conclusion, the current results aid in understanding the mechanism of inherent resistance to selective MET inhibitors as well as provide important information for patient selection and clinical treatment strategies.Gastric cancer is one of the most malignant diseases and one of the leading causes of cancer-associated mortality worldwide. Although advances have been made in surgical techniques, perioperative management and the combined use of surgery with chemotherapy and/or radiotherapy, patients with advanced stage gastric cancer continue to face poor outcomes. Furthermore, it was reported that  Gastric cancer is the second most prevalent cause of cancer-associated mortality worldwide . HoweverMET was initially identified as an oncogene encoding the receptor tyrosine kinase (RTK) for hepatocyte growth factor. The MET gene has been identified on chromosome 7q21-q31, where it encodes a single precursor that is digested and glycosylated post-transcriptionally, resulting in an extracellular α-chain (50-kDa) linked to a transmembrane β-chain (140-kDa) via disulfide bonds. Oncogenic activation of MET suppresses apoptosis and promotes cell survival, proliferation, migration and differentiation, as well as gene transcription and angiogenesis  by Shandong Tumor Hospital Experimental Animal Center  within 1 h of removal from the patients. The samples were cut into 3×3×3-mm sections, soaked in 50% Matrigel™  and subcutaneously implanted into the flank of the nude mice. The tumors were passaged when the tumor volume reached ~300 mm3. Tumor volumes were calculated using the following standard formula: Tumor volume = (length × width2)/2. Written informed consent was obtained from all patients and the study was approved by the ethics committee of Shandong Tumor Hospital Experimental Animal Center.Female athymic BALB/c nude mice (n=200), aged 6–7 weeks, were purchased from Shanghai Laboratory Animal Centre Co., Ltd. . Mice were maintained under super-specific pathogen-free conditions and housed in barrier facilities on a 12 h light/dark cycle, with food and water provided http:www.aroma-project.org/), according to the methods described by Bengtsson et al (The GeneChip® genome-wide human single nucleotide polymorphism (SNP) 6.0 and human genome U133 plus 2.0 arrays  were used to analyze the genomic gene copy number and gene expression levels in all established patient-derived gastric xenograft tumors, respectively. MAS5 software  was used to analyze the U133 results. Gene profiling comparison was performed by calculating the fold change of the copy number and gene expression between these tumors. The data were processed using the aroma.affymetrix R package : Group 1, once-daily dose with vehicle by intravenous (i.v.) tail injection; and groups 2, 3 and 4, once-daily dose with 10, 20 and 30 mg/kg PHA665752 by i.v. tail injection, respectively. PHA665752, a selective MET inhibitor, was purchased from Selleck Chemicals . In a subsequent experiment, the CNGAS028 model was also treated with vehicle, 15 mg/kg PHA665752, the pan-fibroblast growth factor receptor (FGFR2) selective inhibitor NVP-BGJ398  or 30 mg/kg PHA665752 in combination with 15 mg/kg NVP-BGJ398, respectively. All treatments were continued for 21 days and the mice were sacrificed by CO2 inhalation 2 h after the last treatment.Gastric tumors (2-cm diameter) were aseptically resected from established patient-derived gastric cancer xenografts with The tumor tissues were resected 2 h following the final treatment with PHA665752 or/and NVP-BGJ398 on day 21 of the efficacy studies. The tumor tissues were then homogenized and lysed in cell lysis buffer  containing phosphatase inhibitor cocktail and proteinase inhibitor cocktail , and the protein concentrations were determined using the bicinchoninic acid protein assay kit . Subsequently, equal quantities of protein (30 µg) were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis on 8% gels, blotted on polyvinylidene difluoride membranes , then probed with monoclonal phosphorylated (p)-MET , polyclonal p-FGFR2 , monoclonal MET  and monoclonal FGFR2  rabbit anti-human antibodies. Subsequently, the membranes were incubated with goat anti-rabbit horseradish peroxidase-conjugated secondary antibodies  and detected by chemiluminescence. Gel Doc™ XR+  was used to visualize the western blots. All antibodies were purchased from Cell Signaling Technology, Inc. , unless otherwise stated.All data are presented as the mean ± standard deviation for the indicated number of independently performed experiments. Statistical analyses were conducted using GraphPad InStat software . Student's t tests were performed and P<0.05 was considered to indicate a statistically significant difference.MET was highly amplified and expressed in 16.7% (5/30) of the Chinese gastric cancer xenograft models (MET amplification was positively correlated with MET overexpression in the xenograft models.Chinese patient-derived gastric cancer models (n=30) were established from 83 gastric cancer specimens. The established models were termed CNGAS001-030. The CNGAS001, CNGAS002 and CNGAS003 mouse models are indicated in t models . From thMET amplification and overexpression . The results demonstrated that four gastric cancer xenograft models were significantly sensitive to PHA665752 treatment (P<0.05). However, the CNCAS028 model was resistant to PHA665752 ; As indicated in FGFR2 amplification and overexpression as well as PHA665752 resistance, PHA665752 was combined with a selective pan-FGFR2 kinase inhibitor (NVP-BGJ398) to treat the CNCAS028 model  patient-derived gastric cancer xenografts. These results indicated the therapeutic potential of MET inhibitors in gastric cancer. Additional analysis identified that a selective MET inhibitor (PHA665742) was able to significantly inhibit tumor growth in 4/5 gastric cancer models with MET amplification. These results are consistent with a previous study, which demonstrated that MET amplification was associated with the response of the MKN45 gastric cancer cell line to PHA665752 treatment (Gastric and gastroesophageal cancer affect 1 million individuals worldwide every year and are the second most common cause of cancer-associated mortality . Targetereatment .et al (in vitro and recovered tumorigenicity in vivo. In addition, Lee et al (SND1-BRAF fusion gene exhibited resistance to the MET inhibitor PF-04217903 in GTL16 cells via RAS/RAF/ERK signaling pathway activation. By contrast, the current study determined that a MET-amplified CNCAS028 model was resistant to MET inhibitor PHA66575 as a result of FGFR2 gene amplification and overexpression. Inhibition of FGFR2 signaling in this xenograft model recovered its sensitivity to PHA665752.Aberrant RTK expression produces growth and survival signals that are essential for the pathogenesis and progression of various types of cancer. Furthermore, cancer patients treated with targeted inhibitors of key oncogenic kinase drivers, including imatinib, gefitinib and erlotinib, have exhibited promising clinical outcomes . Howeveret al  reportedee et al  reportedMET and FGFR2 amplification. Future studies should be performed to investigate whether similar results could be obtained in an acquired resistant model.In conclusion, MET and FGFR2 coactivation may increase resistance to targeted therapy, possibly due to activation of multiple growth and survival signaling pathways. These findings indicate that combination therapy with MET and FGFR2 inhibitors may be a promising strategy to treat inherent resistance to MET inhibitors in cases of gastric cancer harboring


Mesenchymal epithelial transition factor receptor (Met) is a receptor tyrosine kinase that plays a critical role in promoting cancer cell malignant progression. Met is activated by its ligand hepatocyte growth factor (HGF). HGF-dependent Met activation plays an important role in stimulating epithelial-mesenchymal transition (EMT) in tumor cells, resulting in increased tumor cell proliferation, survival, motility, angiogenesis, invasion, and metastasis. The HGF/Met axis has thus attracted great interest as a potential target in the development of novel cancer therapies. In an effort to suppress tumor cell malignant progression, efforts have been made to develop agents capable of inhibiting inhibit Met-induced EMT, including specific Met tyrosine kinase inhibitors, HGF antagonists that interfere with HGF binding to Met, and antibodies that prevent Met activation and/or dimerization. Tocotrienols, a subgroup within the vitamin E family of compounds, display potent anticancer activity that results, at least in part, from inhibition of HGF-dependent Met activation and signaling. The present review will provide a brief summary of the increasing importance of the HGF/Met axis as an attractive target for cancer chemotherapy and the role of tocotrienols in suppressing Met activation, signaling and HGF-induced EMT in breast cancer cells. Evidence provided suggests that γ-tocotrienol therapy may afford significant benefit in the treatment of breast cancers characterized by Met dysregulation.The online version of this article (doi:10.1186/s40169-014-0030-5) contains supplementary material, which is available to authorized users. Traditional cancer chemotherapeutic agents typically exhibit non-selective activity and often produce low response rates and severe toxic side effects in patients . Thus, tHGF-dependent activation of Met leads to receptor dimerization, tyrosine autophosphorylation, and initiation of down-stream signaling, including activation of MAPK, PI3K/Akt, and STAT pathways, all of which play an important role in cancer cell proliferation and survival ,9],10],[10]9],9],,11],,. EpithelTumors exhibiting rapid growth often face insufficient blood flow to meet their high oxygen demands, but are able to adapt to hypoxic conditions by altering metabolism and phenotypic functions . A typicVarious approaches have been used to inhibit Met activation and signaling, including agents that inhibit tyrosine kinase activity , neutralizing antibodies (anti-HGF antibodies) and competitive antagonists (NK4 and uncleavable HGF) that interfere with HGF binding and activation of Met, and agents that block HGF binding to Met and/or Met receptor dimerization (anti-Met antibodies) ,[25]. ThMet is expressed exclusively in epithelial cells, whereas its natural ligand HGF is produced solely by mesenchymal cells ,4],26],[26]4],4],. MediateDifferent biological effects of Met signaling are directly associated with activation of specific intracellular pathways. HGF-induced cell proliferation is mediated by activation of the MAPK cascade , whereasMet mutations, overexpression and/or dysregulation commonly occur in a wide variety of cancers, characteristics that are associated with promotion of cancer progression, including tumor cell proliferation, survival, motility, EMT, angiogenesis, invasion and metastasis ,10],,, while mSince Met plays an important role in malignant progression, it has emerged as a practical target in cancer chemotherapy, with various strategies currently under development to inhibit Met activation and signaling. Agents that decrease receptor tyrosine kinase activity include low molecular weight molecules that block ATP binding to Met's catalytic, thereby inhibiting receptor autophosphorylation and recruitment of molecules associated with downstream signaling . SeveralAgents that inhibit HGF include NK4, anti-HGF neutralizing antibodies, and an uncleavable HGF agonist. NK4 is a HGF-like ligand that binds to Met without activating the receptor , whereasVitamin E represents a family of compounds that is divided into structurally similar tocopherol and tocotrienol subgroups ,52]. Th. Th52]. Studies using + SA mammary tumor cells maintained in serum-free defined media containing HGF as a mitogen show that HGF induces a dose-responsive increase in growth and corresponding increase in Met autophosphorylation, whereas combined treatment with γ-tocotrienol blocks these effects . In addiSubsequent studies confirm and extend these findings in human breast cancer cell lines . ResultsHGF-dependent Met activation in transformed epithelial cells promotes expression of a malignant mammary epithelial phenotype . Met-indin vitro occur in in vivo.Since combined treatment with low dose of γ-tocotrienol and SU11274 has profound inhibitory effects on tumor cell growth and HGF-dependent Met activation and signaling, additional studies aimed to determine effects of these agents on breast cancer cell epithelial and mesenchymal cell marker expression. Results show that neoplastic mammary epithelial cells grown in serum-free culture medium containing HGF as a mitogen display a strong transition from epithelial to mesenchymal cell phenotype, as evidenced by low epithelial marker and high mesenchymal marker protein expression . Specifiin vitro and in vivo[2-induced hypoxia are shown in Figure Met expression is also responsive to various types of stress, particularly low oxygen tension or hypoxia . Rapidlyd in vivo. AlthougIt has become increasingly evident during the past decade that HGF-dependent Met activation and signaling plays a major role in malignant progression and metastasis. As a result, substantial interest has focused on developing therapeutic strategies that target the HGF/Met axis. A variety of emerging approaches are directed toward one or more of the components within this signaling pathway. Although the majority of research conducted in this area has focused on preclinical studies using cell culture and animal experimental models, a great number of therapeutic agents  developed and refined in preclinical experiments are increasingly the subject of clinical trials. Therapies that disrupt cancer cell proliferation, survival, motility, angiogenesis, invasion and metastasis will provide great benefits in the treatment and cure of cancers that display aberrant Met activity. Recent evidence suggest that γ-tocotrienol may also provide some benefit in the treatment of breast cancers that displays Met dysregulation.The corresponding author was responsible for organizing, writing and preparing the illustrations presented in this article.Dr. Paul W. Sylvester is the Pfizer, Inc. - B. J. Robison Endowed Professor in Pharmacy in the School of Pharmacy at the University of Louisiana at Monroe, Monroe, LA. Dr. Sylvester received his B.S. degree in Biology from Western Michigan University, Kalamazoo, and his Ph.D. in Physiology from Michigan State University in East Lansing, and completed his postdoctoral training in Experimental Therapeutics at Roswell Park Cancer Institute in Buffalo, NY. Dr. Sylvester's research interests include examining the relationship of nutrition and cancer, with particular emphasis on understanding the intracellular mechanisms mediating the anticancer effects of tocotrienols. Dr. Sylvester is a member of the American Association for Cancer Research since 1986.


Since 20 % of gastric cancer patients show high level of c-Met expression, c-Met has been identified as a good candidate for targeted therapy in gastric cancer. Herein, we report our newly synthesized c-Met inhibitor by showing its efficacy both in vitro and in vivo.c-Met signaling has been implicated in oncogenesis especially in cells with Compounds with both triazolopyrazine and pyridoxazine scaffolds were synthesized and tested using HTRF c-Met kinase assay. We performed cytotoxic assay, cellular phosphorylation assay, and cell cycle assay to investigate the cellular inhibitory mechanism of our compounds. We also conducted mouse xenograft assay to see efficacy in vivo.c-met amplified cell lines, c-Met inhibitors reduced the downstream signals including Akt and Erk as well as c-Met activity. In vivo Hs746T xenograft assay showed KRC-00715 reduced the tumor size significantly.KRC-00509 and KRC-00715 were selected as excellent c-Met inhibitors through biochemical assay, and exhibited to be exclusively selective to c-Met by kinase panel assay. Cytotoxic assays using 18 gastric cancer cell lines showed our c-Met inhibitors suppressed specifically the growth of c-Met overexpressed cell lines, not that of c-Met low expressed cell lines, by inducing G1/S arrest. In Our in vitro and in vivo data suggest KRC-00715 is a potent and highly selective c-Met inhibitor which may have therapeutic potential in gastric tumor with c-Met overexpression.The online version of this article (doi:10.1186/s12885-016-2058-y) contains supplementary material, which is available to authorized users. Oncogene addiction, which was first proposed by Dr. Bernard Weinstein, offers a rationale for the recent targeted therapy in cancer biology . Oncogenc-met amplified cell lines, whereas it had no effect on the cell lines without c-met amplification [c-met gene. Moreover, a large number of reports identified c-met amplification even in 10–20 % of gastric cancer [The c-MET receptor tyrosine kinase forms a heterodimer and consists of an extracellular α-chain and a membrane-spanning β-chain , 4. c-MEfication . It strofication , 13. It c cancer –18. It mc cancer .Gastric cancer is the second leading cause of cancer related mortality worldwide with the incidence of 18.9/100,000/year . MoleculVarious approaches have been conducted to inhibit the aberrant c-Met kinase activity, such as c-Met biologics, HGF antagonist peptides, and HGF antibodies as well as small molecule inhibitors –29. HereKRC-00509 and KRC-00715 were synthesized according to the procedures published in patent, KR2012-0022541. All compounds including crizotinib were dissolved in DMSO. Compounds were formulated in 20 % PEG-400, 3 % Tween-80, 77 % distilled water for all in vivo studies. Kinase domain of c-Met was purchased from CarnaBio Science (JAPAN).50 was calculated using GraphPad Prism version 5 for Windows. The curves were fit using a nonlinear regression model with a log (inhibitor) versus response formula.Experiment procedure was followed by the manufactured instruction . The reaction was initiated by ATP addition to a mixture containing the c-Met enzyme, peptide substrates, and inhibitors. After 30 min, EDTA containing solution was added to stop the reaction. EDTA containing solution has Europium conjugated anti-phosphoresidue antibody and SA-XL665 for the detection of the phosphorylated peptide product. After 1 h incubation, fluorescence was measured with 337 nm excitation and dual 665 and 620 nm emission of the Envision reader. ICAll cell lines used in this paper, except Hs746T, were purchased from Korean Cell Line Bank . Hs746T cell line was purchased from ATCC. These are all gastric adenocarcinoma cells. SNU-5, SNU-620, SNU-638, MKN-45, and Hs746T cell lines show high expression of c-Met, whereas others show low level of c-Met. These cell lines were maintained in RPMI 1640 medium supplemented with 10 % FBS  using a humidified incubator with 5 % CO2 at 37 °C.The following antibodies were obtained from Cell Signaling Technology: c-Met , phospho c-Met tyrosine 1234/1235 , phospho-Erk threonine 202/204 , phospho-Akt serine 473 , phospho-tyrosine . Tubulin antibody  was purchased from Sigma-Aldrich. HRP-conjugated anti-mouse , and HRP-conjugated anti-rabbit  antibodies were obtained from Thermo Scientific. For immunoblotting, cells were washed in PBS, lysed in 1 X sample buffer (50 mmol/L Tris–HCl (pH 6.8), 10 % glycerol, 2 % SDS, 3 % β-mercaptoethanol), and boiled for 10 min. Lysates were subjected to SDS-PAGE followed by blotting with the indicated antibodies and detection by Western blotting substrate ECL reagent (Thermo Scientific). Images were quantified using a LAS3000 instrument and Image Lab software.50s were calculated using GraphPad Prism version 5 for Windows. The curves were fit using a nonlinear regression model with a log (inhibitor) versus response formula.For viability experiments, cells were seeded in 96-well plates at 30 % confluency and exposed to chemicals the next day. After 72 h, WST-1 reagent was added and absorbance at 450 nm was measured on a Spectramax spectrophotometer  according to the manufacturer's instructions. ICWe followed the manufacturer’s instruction to NucleoCounter NC-250  to analyze the cell cycle distribution. Briefly, cells treated with vehicle or compounds for 24 h were suspended by lysis buffer supplemented with 10ug/ml DAPI. After 5 min incubation at 37 °C, cells were suspended by stabilization buffer. Cells were loaded into the chambers of the slide and were analyzed by NucleoCounter NC-250.6 in 100 μl) were implanted s.c. into the right flank region of each mouse and allowed to grow to the designated size. Once tumors reached an average volume of 200 mm3, mice were randomized and dosed via oral gavage daily with the indicated doses of compounds for 10 days. Mice were observed daily throughout the treatment period for signs of morbidity/mortality. Tumors were measured twice weekly using calipers, and volume was calculated using the formula: length × width2 × 0.5. Body weight was also assessed twice weekly. Significance differences between the treated versus the control groups (P ≤ 0.001) were determined using one-way ANOVA.Female athymic BALB/c (nu/nu) mice ( 6 weeks old) were obtained from Charles River of Japan. Animals were maintained under clean room conditions in sterile filter top cages and housed on high efficiency particulate air-filtered ventilated racks. Animals received sterile rodent chow and water ad libitum. All of the procedures were conducted in accordance with guidelines approved by the Laboratory Animal Care and Use Committee of Korea Research Institute of Chemical Technology. Hs746T cells . The reaction was initiated by the addition of the MgATP mix. After incubation for 40 min at room temperature, the reaction was stopped by the addition of 3 % phosphoric acid solution. 10 μl of the reaction was then spotted onto a P30 filtermat and washed three times for 5 min in 75 mM phosphoric acid and once in methanol prior to drying and scintillation counting.Kinase panel assay was done by Millipore. Kinases were incubated with peptide together with γ-50s of KRC-00509 and KRC-00715 were 6.3 nM and 9.0 nM, respectively . Tumor volumes were measured about for 10 days. KRC-00715 showed very dramatic result in vivo as shown in Fig. To see if our c-Met inhibitors block the tumor growth in vivo, we used mouse Hs746T xenograft model. After Hs746T cells were implanted on nude mouse, we administered KRC-00509 and KRC-00715 orally at doses of 50 mpk daily when tumor size reaches to certain point (100 mmc-met gene [c-met amplified cancer cell [In this present study, we demonstrated a potent inhibitor of c-Met and its excellent cytotoxic effect on c-Met over-expressed gastric cancer cells. 10–20 % of gastric cancer tissues and 40 % of the scirrhous histological subtype have been known to harbor amplified met gene , 33, 34.cer cell , 35, 36.cer cell . X-ray ccer cell , 38. Tabcer cell –41. If wIn summary, we demonstrate newly synthesized c-Met inhibitor which is orally available. KRC-00715 is highly selective to c-Met, and shows excellent efficacy in vitro and in vivo.


The most notable advance in resuscitating VBNC bacteria is the discovery of resuscitation-promoting factor (Rpf), which is a bacterial cytokines found in both Gram-positive and Gram-negative organisms. VBNC state is a survival strategy adopted by the bacteria, which has important implication in several fields, including environmental monitoring, food technology, and infectious disease management; and hence it is important to investigate the association of bacterial pathogens under VBNC state and the water/foodborne outbreaks. In this review, we describe various aspects of VBNC bacteria, which include their proteomic and genetic profiles under the VBNC state, conditions of resuscitation, methods of detection, antibiotic resistance, and observations on Rpf.Under stress conditions, many species of bacteria enter into starvation mode of metabolism or a physiologically viable but non-culturable (VBNC) state. Several human pathogenic bacteria have been reported to enter into the VBNC state under these conditions. The pathogenic VBNC bacteria cannot be grown using conventional culture media, although they continue to retain their viability and express their virulence. Though there have been debates on the VBNC concept in the past, several molecular studies have shown that not only can the VBNC state be induced under  The viable but non-culturable (VBNC) state is a unique survival strategy adopted by many bacteria in response to adverse environmental conditions. When the VBNC concept was proposed some 30 years ago, many issues related to its importance were raised, since there was no demarcation between dying cells and adaptive strategy made by the bacteria to cope with stressful conditions . The VBNViable but non-culturable pathogenic bacteria are considered as a threat to public health and food safety due to their non-detectability by conventional food and water testing methods. Hence, the identification of VBNC bacteria and understanding their mechanisms of their survival and persistence in the environment are of utmost clinical importance. Several pathogens in this condition may contribute initially to non-apparent infection, which are followed by disease manifestations weeks or months later.Vibrio cholerae transformed into coccoid cells in an aquatic microcosm after incubation for 60 days at 4°C  when they enter into the environments –6. For es at 4°C . In thisV. cholerae, membrane integrity and colonizing ability of VBNC cells were confirmed in the gut of iron-dextran-treated mice  in the functional form and produce biologically active toxin , however, it regained its virulence properties , prolonged the duration of the VBNC state. The viability of E. coli O157:H7 10 months after its induction into the VBNC state by river or chlorinated waters has been demonstrated by establishing the presence of mRNAs of rfbE and fliC genes that encode parts of O-antigen transporter and flagellar filament structural proteins, respectively  O157, resistance to oxidative stress has been generally found in isolates from foods while those of the patients remained susceptible , translation , ATP synthase , gluconeogenesis-related metabolism , and antioxidants  were identified after stimulation and/or maintenance of the cells under VBNC state  or superoxide dismutase (SodA) encoded by the genes katA and sodA, respectively. These genetic changes rendered the bacterial cells highly susceptible to seawater stress at 4°C, thereby preventing their resuscitability , flagellar motor switch protein G (fliG), flagellin subunit protein C (flaC), and iron(III) ABC transporter encoding gene (VC0230) was found to increase  confirming their virulence in this state  poorly in the VBNC state  caused rapid induction of Salmonella enterica serotypes Oranienburg and Typhimurium to the VBNC state  compared to the normal cells  of al cells . ChangesNC state . This file salts .V. parahaemolyticus, ahpC1 (VPA1683) and ahpC2 (VP0580) are located in chromosomes II and I, respectively. These ahpC genes were shown experimentally to protect the pathogens against extrinsic peroxides H2O2 and tert-butyl hydroperoxide (tBuOOH) thereby prolonging the time taken by cells to enter into the VBNC state (dacB), which encodes d-alanyl-d-alanine carboxypeptidase, was more during the cold induction of VBNC. The process was accompanied by the disruption of cell wall synthesis resulting in aberrantly shaped cells (Alkyl hydroperoxide reductase subunit C (AhpC) is the catalytic subunit responsible for the detoxification of reactive oxygen and facilitates the survival of pathogenic bacteria under environmental stresses or during infection. In NC state . It was ed cells .H. pylori and V. fluvialis, the genetic backgrounds of the parent strains remain unchanged even after the cells enter the VBNC sate. This suggests that the genomes of the bacteria do not undergo radical change when they enter the VBNC state, even though the cells undergo morphological and other metabolic changes  is a strong inducer of the VBNC state. Gourmelon et al.  studied E. coli O157:H7, can be resuscitated using its autoinducers such as 2(A1-2) produced during biofilm formation in a serum-based medium  technique with ciprofloxacin has been used to detect VBNC state of this pathogen  and 4,6- diamino-2 phenylindole (DAPI) has also been used in the detection of VBNC and total bacterial cells. In the DAPI counterstaining, cells fluoresce in blue allowing proper contrast, permitting simultaneous enumeration of both total and viable bacteria using a single filter . LIVE/DEVibrio-specific 16S rRNA-directed primers has been performed using the total DNA extracted from the water samples followed by denaturant gradient gel electrophoresis (DGGE) and qPCR for quantification of Vibrio population  was successfully used for the detection and enumeration of H. pylori in the VBNC state in freshwater/seawater samples  agar supplemented with eukaryotic cell extracts, which are thermostable and proteinase-K sensitive  is a technique that can detect individual genes in a single bacterial cell and thus can, unlike the conventional FISH which targets abundant genes, help enumerate the number of bacteria in a sample even if their number is low. A highly sensitive aptamer-based viability sensing technique has been developed by Labib et al.  and the organism changed its morphology mostly into coccoid forms. Expression of vacA gene and levels of 16S rRNA were constant at all test time points , which was first described as a secretory protein of s luteus  and may Mycobacterium spp., Corynebacterium spp., Streptomyces spp., L. monocytogenes, Tomitella biformata, and S. enterica serovar Typhimurium  inhibitors of the muralytic activity of Rpf has been reported, which opens a new approach to the study of cell-wall hydrolyzing enzymes. It has also been observed that peptidoglycan-dependent resuscitation activity can be suppressed by means of an inhibitor (4-benzoyl-2-nitrophenylthiocyanate) specific for Rpf . This coThe resuscitation of VBNC state is not only important in the field of medicine, but also for several industrial processes to improve biodegradation and flocculation . AlthougThe 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.