With the continuous and expanding use of implantable biomaterials in a clinical setting, this study aims to elucidate the influence of sterilization techniques on the material surface and bulk properties of two polyurethane nanocomposite biomaterials. Both solid samples and porous membranes of nondegradable polyhedral oligomeric silsesquioxane poly(carbonate-urea) urethane (POSS-PCU) and a biodegradable poly(caprolactone-urea) urethane (POSS-PCL) were examined. Sterilization techniques included conventional steam sterilization (autoclaving), gamma irradiation, and disinfection via incubating with ethanol (EtOH) for 10 min or 24 h. After treatment, the samples were examined using gel permeation chromatography (GPC), attenuated total reflectance Fourier transform infrared spectroscopy, and tensiometry. Cytotoxicity was evaluated through the culture of endothelial progenitor cells and the efficacy of sterilization method was determined by incubating each sample in tryptone soya broth and fluid thioglycollate medium for cultivation of microorganisms. Although EtOH did not affect the material properties in any form, the samples were found to be nonsterile with microbial growth detected on each of the samples. Gamma irradiation was not only effective in sterilizing both POSS-PCU and POSS-PCL but also led to minor material degradation and displayed a cytotoxic effect on the cultured cells. Autoclaving was found to be the optimal sterilization technique for both solid and porous membranes of the nondegradable POSS-PCU samples as it was successful in sterilizing the samples, displayed no cytotoxic side effects and did not degrade the material. However, the biodegradable POSS-PCL was not able to withstand the harsh environment during autoclaving, resulting in it losing all structural integrity. Biomaterials play a critical role in the healthcare industry with applications ranging from medical implants, prosthetics, and diagnostic devices. However, the ever increasing use of implantable medical devices has led to a significant increase in the number of infections, with at least half of all cases of nosocomial infections associated with medical devices.3in vitro to disinfect the polymer sample. It is particularly useful for disinfecting biodegradable tissue engineering scaffolds, such as glycolic acid-based materials, which, by their very nature, tend to be fragile.6Sterilization can be achieved in a number of ways, including steam and gamma irradiation. Although numerous investigations have characterized microbial activity after sterilization, as a marker of efficacy, the impact of sterilization technique on the material bulk, and surface properties is often ignored, whereas it is often the material bulk and surface properties that determine the success of the implant/device. The sterilization process usually involves physical or chemical treatment, which results in the elimination of organic macromolecules and/or microorganisms. Given the nature of their action, the techniques can also react with the biomaterial. Steam sterilization can lead to hydrolysis, softening, and degradation of the polymer because of the high temperature, pressure, and humidity.in vivo failure observed on prolonged implantation, primarily because of polymer degradation, is a major stumbling block for their continued use.Polyurethanes (PUs) represent a large family of biomaterials commonly found in a clinical setting. They consist of different organic units linked together by urethane bonds [[—]NHC(O)O[—]], which affords them a high degree of versatility. With excellent biocompatibility and mechanical properties, PUs are amenable to a number of applications, including catheters, biomedical implants, and tissue engineering scaffolds.in vitro and in vivo, possess antithrombogenic properties, and sustain cell growth on its surface; a combination of properties making it suitable for a number of biomedical applications, including heart valves, prosthetic grafts, and stents.14In an effort to produce more durable and biostable PUs, our group tethered polyhedral oligomeric silsesquioxane (POSS) nanocages to a poly(carbonate-urea) urethane (PCU) backbone Figure . SiloxanThe purpose of this study was to evaluate the impact of the sterilization technique on the bulk and surface properties of solid samples, referred to as cast sheets, and porous membranes of POSS-PCU and POSS-PCL polymers developed in our laboratory. Porous membranes were fabricated using a phase separation method described previously.Polycarbonate polyol, 2000 mwt  and trans-cyclohexanechloroydrinisobutyl-silsesquioxane (Hybrid Plastics Inc) were placed in a 250-mL reaction flask equipped with mechanical stirrer and nitrogen inlet. The mixture was heated to 135°C to dissolve the POSS cage into the polyol and then cooled to 70°C. Flake 4,4′-methylenebis (phenyl isocyanate) (MDI) were added to the polyol blend and then reacted, under nitrogen, at 75–85°C for 90 min to form a pre-polymer. Dimethylacetamide (DMAc) was added slowly to the pre-polymer to form a solution; the solution was cooled to 40°C. Chain extension of the pre-polymer was performed by the drop wise addition of a mixture of ethylenediamine and diethylamine in DMAc to form a solution of POSS-modified polycarbonate urea-urethane in DMAc.Dry polycaprolactone diol (2000 mwt) and POSS were placed in a 250-mL reaction flask equipped with mechanical stirrer and gas inlet. The mixture was heated to 135°C to dissolve the POSS cage into the polyol and then cooled to 90°C. 4, 4′ methylenebis(cyclohexyl isocyanate)  was added to the polyol blend and then reacted, under nitrogen, at 90°C for 120 min with catalyst (Bismuth neodecanoate) to form a pre-polymer. Dry DMAc was added slowly to the pre-polymer to form a solution; the solution was cooled to 40°C. Chain extension of the pre-polymer was performed by the drop wise addition of a mixture of ethylenediamine and diethylamine in dry DMAc. After completion of the chain extension, 1-butanol in DMAc was added to the polymer solution forming a POSS-PCL solution.Unless otherwise stated, all chemicals and reagents were purchased from Aldrich Limited, Gillingham, UK.The 18% (w/w) solutions of polymer in DMAc were cast onto glass petri dishes and left in an oven at 60°C overnight to evaporate the solvent. The resulting solid sheets of polymer were then sterilized accordingly and used for future experiments.3, 50% (w/w), 40 µm particle size; Bruner Mond, Cheshire, UK] was dispersed into an 18% (w/w) solution of the polymers in DMAc containing 2% Tween 80 surfactant. The mixture was mixed and degassed in one process using a Thinky ARE 250 mixer  resulting in a viscous slurry, which was then cast onto a stainless steel sheet and placed in distilled water for 48 h at room temperature. A sheet of porous polymer was formed via the immersion precipitation process as a result of solvent exchange with distilled water. The polymer sheets were left immersed in distilled water for a period of 48 h to ensure complete removal of solvent and NaHCO3. The membranes were then removed from the steel support, air dried for a further 48 h, and sterilized appropriately for future experiments.Sodium bicarbonate O[—]C), 1400 cm−1 (C[—]C aromatic ring), 1540 cm−1 (N[—]H and CN), 1589 cm−1 (CC aromatic), 1632 cm−1 (NH2), 1736 cm−1 (CO), 3422 cm−1 (N[—]H).ATR-FTIR spectroscopy was used to analyse surface chemical changes on sterilization with the results summarized in Figure −1, attributed to the POSS moiety was detected for the porous sample incubated in EtOH for 24 h. Meanwhile, autoclaving POSS-PCU caused a slight decrease (p < 0.05) in the intensity of the ether peak at 1245 cm−1 for both the cast and porous samples.Ethanol treatment had no significant impact on either of the POSS-PCU or POSS-PCL samples, neither cast nor porous. A slight increase in peak at 1100 cm−1 on both cast and porous samples. There was, however, an increase in the intensity of the peak at 1095 cm−1 for the cast POSS-PCU sample (p < 0.001). Similarly, both POSS-PCL samples suffered a reduction in peak intensity, after exposure to gamma irradiation, at 1730, 1540 1240, and 1165 cm−1. However, the cast sample of POSS-PCL also exhibited an increase in peak intensity at 1100 cm−1.Exposure of POSS-PCU to gamma irradiation led to significant reductions in peak intensity at 1245, 1540, and 1740 cmMw) of 108,500 and a number average molecular weight (Mn) of 42,600, whereas POSS-PCL had an Mw of 62,800 and Mn of 31,200. After treatment with EtOH, there was a negligible impact on either Mn or Mw regardless of incubation time, for any of the four samples.Gel permeation chromatography (GPC) results are summarized in Figure p < 0.05) in Mw but no change in Mn possibly suggesting a degree of cross linking. No changes in molecular weight distributions were detected in autoclaved porous samples of POSS-PCU.Autoclaving the cast sheet of POSS-PCU resulted in a 9.3% increase . Meanwhile, gamma irradiation increased both the Mn and Mw of the porous POSS-PCU by 27.3 and 17.1%, respectively. Both cast and porous samples of POSS-PCL exhibited a significant (p < 0.001) decrease in Mn (11.2 and 7.2%) and Mw (13.4 and 10.4%), respectively, after exposure to gamma irradiation.Exposure to gamma irradiation had a significant impact on all of the samples. The The results of the Alamar Blue assay are presented in Figure The polymeric materials were incubated in TSB and THY to test the efficiency of sterilization with the resultant level of bacterial growth reported in Table Sterilization is defined as the complete removal or destruction of viable organisms. More practically, however, it is better defined as a process capable of delivering a certain probability that the device is free from viable microorganisms. The physical or chemical agents capable of achieving sterilization without adversely affecting the material quality, function and use, are rather limited for healthcare products. Two of the more common sterilization techniques are moist heat (autoclaving) and ionizing irradiation (gamma irradiation). Additional methods capable of inactivating viable organisms exist, but are less effective than terminal sterilization. These include disinfectants such as EtOH incubation.Depending on the chemical nature of the material and the processing method, sterilization techniques have been reported to induce changes in material properties. In this study, we investigated the effects of sterilization on two POSS polyurethane nanocomposites: a nondegradable POSS-PCU with an aromatic hard segment and carbonate-based soft segment, and a biodegradable POSS-PCL based on an aliphatic hard segment and caprolactone soft segment. Furthermore, we investigated the effect of sterilization on porous membranes of POSS-PCU and POSS-PCL generated through phase separation. Phase separation is a popular method to create a porous, 3D environment for tissue engineering applications.−1 possibly indicating a molecular rearrangement after EtOH incubation resulting in the migration of the POSS moiety to the material surface. No further significant changes in ATR-FTIR analysis were found for the EtOH-incubated samples indicated that no changes in the surface chemical composition took place. Furthermore, there were no significant variations in molecular weight or mechanical properties, suggesting that neither surface nor bulk properties of POSS-PCU or POSS-PCL were affected by ethanol irrespective of incubation time. Although ethanol has been shown to be useful in disinfecting lipophilic viruses and Gram-positive, Gram-negative, and acid-fast bacteria; hydrophilic viruses and bacteria spores are resistant to the microbial effects of ethanol making it unsuitable for sterilizing biomedical devices for in vivo applications.The four samples assessed were incubated with 70% ethanol for a period of 10 min and 24 h. The porous POSS-PCU sample incubated for 24 h in EtOH exhibited a slight increase in the peak at 1100 cmThe process of autoclaving requires samples to be exposed to high pressure saturated steam at temperatures of 121°C or more. The combination of high temperature, humidity, and pressure has been shown to be detrimental to PUs with decreases in UTS, hydrolysis, and oxidation being reported in polyether-based PUs.Mw of the cast sample, which may explain the slight increase (9.8%) in elongation at break experienced by the autoclaved POSS-PCU. The high temperatures involved in autoclaving could have induced a nominal degree of cross-linking in the sample, which could be responsible for the slight increases in Mn and elongation at break; however, no evidence was uncovered for cross-linking from the IR spectra.The nondegradable POSS-PCU samples were, however, relatively unaffected by exposure to autoclaving with the cast and porous samples retaining their shape and structure. GPC analysis suggested there was a minor increase (9.3%) in −1. These results are consistent with chain scission of the hard segment. The porous samples have a significantly increased surface area, compared with the cast sample, offering a possible explanation for the difference in behavior between the solid sheets and porous samples. Chain scission may be induced by the moist environment at the surface of the material as opposed to the high temperatures being experienced by the bulk. No significant changes were recorded in the molecular weight profile of the porous POSS-PCU sample further reinforcing the belief that chain scission is a surface effect as opposed to bulk. Autoclaving appears to be an effective sterilization technique as no sign of microbial growth was detected in either of the broths. Furthermore, no cytotoxic side effects were noted during cell culture  in elongation at break alongside a reduction in ATR-FTIR peak intensity at 1245cm.9% in elMixed findings have been reported regarding the effects of gamma irradiation on PUs. Although some investigators have found no change in material properties after exposure to gamma irradiation, an equally significant number of publications indicate severe degradation of material.However, gamma irradiation did lead to POSS-PCU samples displaying cytotoxic side effects on the cultured EPCs Figure . Althoug21Gamma irradiation was found to have an impact on both material appearance and properties. Although there were no changes in the appearance of POSS-PCL samples, POSS-PCU had discolored and became yellow. The difference between the two samples is, again, most likely due to the MDI in the aromatic hard segment of POSS-PCU. Exposure to gamma irradiation may have oxidised the central methylene group of the biphenyl leading to a highly conjugated quinone chromophore Figure .−1 are all consistent with hydrolysis of the hard segment. The reductions in molecular weights for both the cast and porous samples of POSS-PCU would seem to corroborate those findings. However, the Mn for cast samples of POSS-PCU increased suggesting that a degree of cross linking is also taking place. Because Mn is a more sensitive parameter to the lower end of the molecular weight distribution, these results would suggest that chain scission is taking place followed by cross linking of the low molecular weight chains of the cast POSS-PCU. An increase in ATR-FTIR peak intensity at 1095 cm−1 would reinforce these findings because ∼1100 cm−1 is the region where we would expect to find the ether peak as a result of cross linking. However, it is difficult to assign this peak with any certainty as the Si-O-Si of POSS is also expected in this region.Analysis of the surface functional groups of the cast and porous samples of POSS-PCU via ATR-FTIR provided further evidence of material degradation. Reductions in peak intensities at 1245, 1540, and 1740 cmMn and Mw for both the cast and porous samples suggesting severe degradation of samples. These findings were reinforced by the ATR-FTIR results, which found reductions in peaks at 1730, 1540, 1240, and 1165 cm−1 indicating chain scission in both the hard and soft segments.There is no evidence to suggest any cross-linking is taking place in the POSS-PCL samples. GPC data showed significant reductions in The effect of polymer degradation was seen in the mechanical testing of the samples. The UTS and Young’s modulus decreased significantly for the casted samples of both POSS-PCU and POSS-PCL. The porous samples did not display any significant changes in their mechanical properties. This is probably due to the highly porous nature of the samples meaning that the true cross sectional area on which force was applied was considerably lower than the measured area resulting in lower than expected values. The force range on which the samples were examined may not be large enough to determine any detrimental effects on the polymer chains.It should be noted, however, that the polymer samples were exposed to gamma irradiation for a continuous 10 h at a dose of 28.4 kGy. Alternative protocols for gamma irradiation may influence the materials differently, for example, higher doses at shorter exposure time periods.in vivo use of biodegradable scaffolds.Sterilizing POSS-modified polyurethanes via EtOH resulted in no observable damage but the sterilization process was inefficient. On the other hand, both autoclaving and gamma irradiation resulted in effective sterilization of samples; although the high temperatures involved in autoclaving resulted in the structural collapse of the biodegradable POSS-PCL samples, the nondegradable POSS-PCU withstood autoclaving well. Gamma irradiation however led to mild degradation of both POSS-PCU and POSS-PCL samples and had a cytotoxic effect on EPC culture. The results indicate that autoclaving is the optimal sterilization procedure for POSS-PCU; however, more suitable methods to sterilize biodegradable materials are needed. Tissue engineering scaffolds are purposefully designed to degrade over time; however, their fragile nature also makes them unsuitable for the standard forms of sterilization. Therefore, new methods capable of sterilizing without affecting them chemically, mechanically, or morphologically are needed for


MET exon 14 deletion (METex14del) has been postulated to be one potential mechanism for MET protein overexpression. We screened for the presence of METex14del transcript by multiplexed fusion transcript analysis using nCounter assay followed by confirmation with quantitative reverse transcription PCR with correlation to MET protein expression by immunohistochemistry (IHC) and MET amplification by fluorescence in situ hybridization (FISH). We extracted RNAs from 230 patients enrolled onto the prospective molecular profiling clinical trial (NEXT-1) (NCT02141152) between November 2013 and August 2014. Thirteen METex14del cases were identified including 3 gastric cancer, 4 colon cancer, 5 non-small cell lung cancer, and one adenocarcinoma of unknown primary. Of these 13 METex14del cases, 11 were MET IHC 3+ and 2 were 2+. Only one out of the 13 METex14del cases was MET amplified (MET/CEP ratio > 2.0). Growths of two  METex14del+ patient tumor derived cell lines were profoundly inhibited by both MET tyrosine kinase inhibitors and a monoclonal antibody targeting MET. In conclusion, METex14del is a unique molecular aberration present in gastrointestinal (GI) malignancies corresponding with overexpression of MET protein but rarely with MET amplification. Substantial growth inhibition of METex14del+ patient tumor derived cell lines by several MET targeting drugs strongly suggests METex14del is a potential actionable driver mutation in GI malignancies.Recently,  MET amplification is rare in gastroesophageal/gastric cancer ., 6.MET aMETex14del was initially described in 2006 in non-small cell lung cancer (NSCLC) and was caused by mutation in the splice donor site in intron 14 and intronic sequence deletions around MET exon 14 [METex14del in NSCLC has subsequently been confirmed by RNA sequencing and whole genome sequencing [METex14del has been reported in gastric cancer (GC) cell line Hs746T [ exon 14 . The prequencing , 8. Addie Hs746T , 10 and e Hs746T  indicatie Hs746T .METex14del using multiplexed fusion transcript detection assay and then confirmed with reverse transcription PCR (RT-PCR) correlated the MET protein expression and MET amplification in METex14del+ cases. We further generated patient derived tumor cell lines and screened them for the presence of METex14del and investigated the consequence of MET inhibition in these METex14del+ cells lines.We investigated patients with metastatic solid malignancies primarily gastrointestinal (GI) and lung malignancies for the presence of METex14del transcript , which is an actively enrolling clinical trial for genomic profiling in cancer patients, was used : 3 gastric carcinoma (GC), 4 colon, 5 non-small cell lung cancer (NSCLC) and one adenocarcinoma of unknown primary (ACUP). Among these 13 METex14del cases, 11 cases were MET IHC 3+ and 2 cases were MET IHC 2+. Only one of the 13 METex14del+ cases had concomitant MET amplifications , 219 were finally included in the analysis as 11 samples failed to pass QC . With initial screening of multiplexed nCounter fusion transcript analysis, 26 were detected as potential positive cases for pression  and 13  values of RT-PCR showed relatively high around 32, there was definite amplification of target sequences. Deep sequencing targeting whole MET gene including intron using DNAs from GI cancers, there were many mutations in the introns  but no MET amplification by FISH , Table METex14del case was a 67-year old male patient who also presented with poorly differentiated adenocarcinoma with concomitant MET amplification (MET/CEP7 ~12.8) and strong MET overexpression. For colon cancer, 4 patients were METex14del positive (Tables METex14del+ (or positive) colon cancer patients were not MET amplified and all but one were MET IHC 3+. KRAS was wild-type in all 4 colon cancer patients but BRAF V600E was detected in two of the 4 cases. Interestingly, all 4 colon cancer METex14+cases were left-sided colon cancer. For NSCLC, 5 of 51 (9.8%) patients were METex14del+ and none of the patients had concomitant MET amplifications. The median age for the five patients was 49 years and four patients (80%) were never-smokers  and SAIT301  [METex14+ GC and CRC PDCs whereas lapatinib, an EGFR and HER2 inhibitor, and cetuximab (Erbitux), an EGFR targeting monoclonal antibody exhibited no such effects (METex14del+ patient derived cell lines.We identified 5 with high CR Table . Of the ntibody) . In Figuntibody) –14, led ntibody) . PHA-665 effects . Other PMETex14del+ GC and CRC PDCs to further confirm its Cbl-independent Met degradation mechanism. As shown in Figure METex14del+ PDCs following SAIT301 treatment  according to the manufacturer's instruction in duplicate. Total RNA was extracted from fresh tumor tissue (tumor content > 70%) or from one to four FFPE tissue sections (4 μm thick) using the High Pure RNA Paraffin kit . After RNA extraction according to the manufacturer's protocol, we added additional DNase treatment. RNA concentration was measured with the Nanodrop 8000  and stored −80°C until use. Briefly, 300 ng of total RNA was hybridized to nCounter probe sets for 16 hours at 65°C. The target sequence for G CAGTAT . For conMET, GFPT1 F (5′-TGGGTTTTTCCTGTGGCTGAA-3′), reverse primer for exon 15 of MET (5′- GCATGAACCGTTCTGAG ATGAATT-3′) and probes overlapping an exon 13-exon 15 junction (5′- AAGCAAATTAAAGATCAGTTTCC-3′). GAPDH gene  was used as an endogenous control. TaqMan probes were labelled with the reporter dye molecule FAM (6-carboxyfluorescein) at the 5′ end and with TaqMan minor groove binder non-fluorescent quencher (MGB-NFQ) probe at the 3 the 3in) at the 5′ end and with GATCAGTT TaqMan Universal PCR master mix with AmpErase UNG (Applied Biosystems), 900 nm primers (forward and reverse), 250 nm TaqMan probe, and 5 μl of cDNA sample in a total reaction volume of 20 μl. PCR conditions were 95°C for 10 min followed by 40 cycles of amplification at 95°C for 15 s and 60°C for 1 min on the ABI PRISM 7500HT Fast Real-time PCR. Ct values < 33 were considered as METex14del+ and ≥ 34 were negative for METex14del.RNAs were reverse transcribed using a superscript III first-strand synthesis system . For validation of nanostring results, we designed forward primer for exon 13 of METex14del. The kit is intended to detect the presence of alternatively spliced (METex14del) MET transcript in RNA from FFPE tissue sections in accordance with the provided protocol . For the MET WT control, Ct from MET WT P/P mix should be in range between 22 and 28, and Ct from METex14del P/P mix should be “undetermined”. For the METex14del Control, Ct from MET WT P/P mix should be between 16 and 22; the Ct from METex14del P/P mix should be between 26 and 32. If the Ct values for controls fall outside the expected range then that run should not be used for evaluation of test samples.To validate our results additionally, we used a qRT-PCR based kit for detection of alternatively spliced variant of MET to detect For MET immunohistochemistry, we used CONFIRM anti-Total MET SP44) rabbit monoclonal primary antibody  with a Ventana BenchMark XT automated slide processing system according to the manufacturer's protocol as previously described 4 rabbit . Both meMET/CEP7 probes  as described previously [MET and chromosome 7 centromere probe (CEP7) signals  in 20 inter-phase tumor cell nuclei, and the mean number of MET and CEP7 copies per nucleus were determined, along with the ratio. Normal MET/CEP7 signals (one to two copies per cell) in the various non-neoplastic cells served as the internal positive control. We defined MET gene amplification as a MET/CEP7 ratio > 2.0 in 20 tumor nuclei and polysomy-7 were regarded as negative for gene amplification.FISH was performed using dual-color DNA-specific eviously . Two patTotal proteins from PDCs were isolated using RIPA buffer  containing a protease inhibitor cocktail  and phosphatase inhibitor cocktail (Roche), and protein concentrations were determined according to Bradford procedure using a Quick Start Bradford Protein Assay . Thirty μg of proteins were subjected to 10% SDS-polyacrylamide gel electrophoresis, and electro-transferred onto nitrocellulose membranes. The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.1% v/v Tween 20, and probed overnight at 4°C with a Specific antibodies: pMET (Tyr 1234/1235), pAkt (Ser473), Akt(C67E7), pERK1/2 (Thr202/Tyr204), ERK1/2 (Thr202/Tyr204), GAPDH from Cell Signaling Technology , and MET from Abcam  and MET (C-28) from Santa Cruz biotechnology , and beta actin from Sigma Aldrich. Horseradish peroxidase-conjugated anti-rabbit or mouse IgG  were used as a secondary antibody, and signals were detected by chemiluminescence using ECL Western Blotting Substrate , and visualized by using LAS-4000 .SAIT301 was produced using a recombinant CHO stable cell line . Crizoticlinicaltrials.gov). The protocol was approved by the Institutional Review Board at Samsung Medical Center. The cells were cultured in RPMI media supplemented with 10% fetal bovine serum, 0.5 μg/ ml of hydrocortisone (Sigma Aldrich), 5 μg/ ml of insulin, 5 ng of EGF and FGF (PeproTech). Cell proliferation in response to antibody treatment in vitro was assessed by a CTG  assay according to manufacturer instructions. Cells were plated at a density of 5 × 105 cells in FBS 10% (v/v) RPMI 1640 medium onto a 96-well plate . After 24 h incubation, treated antibodies or small molecules diluted in 10% FBS (v/v) RPMI medium were added. After 5 days incubation, 100 μl of the CTG reagent was added to each well followed by incubation at RT for 30 min. The luminescence signal was recorded using Envision 2104 Multi-label Reader .Patient derived tumor cells (PDCs) were isolated from malignant effusions, surgical tissues or biopsies after obtaining informed consent form , including 126 distinct sequence variants in lung adenocarcinoma (3.0%), other lung neoplasms (2.3%), brain glioma (0.4%), and tumors of unknown primary origin (0.4%) [METex14del has not been reported in either gastric or colon cancer patients. This is the first report that identified METex14del at a frequency of approximately 5% in both gastric and colon cancer in addition to NSCLC. All METex14del+ cases also over-expressed MET protein with only one case showed MET amplification consistent with the hypothesis that METex14del leads to MET over-expression without the need for concurrent MET amplification. In addition, METex14del occurs exclusively to ALK, ROS1, NTRK1, NTRK3 or RET fusions indicating METex14del is likely a driver mutation and defines a unique molecular subset of gastric and colon cancers.The Met proto-oncogene is encoded by 21 exons spanned by 20 introns . The tragenicity . The incin NSCLC . RecentlMET amplification with MET protein overexpression [METex14del and strong MET protein overexpression by IHC. We performed MET exon 14 Sanger sequencing with gDNAs and cDNAs, but failed to detect mutations in our GI METex14del+cancer samples (data not shown). Given the low sensitivity of Sanger sequencing (<12%) and low Ct values in our RT-PCR results, we postulate that METex14del+ tumor population is present in small subpopulation of tumors. Furthermore, we also report c.3082+811A TTTTAACA > GGTTTGAT mutations on intron 14 region of MET with variant allele frequencies around 10%. This intronic mutation is a novel mutation, which has not been reported in COSMIC and TCGA lung adenocarcinomas  cases.4 In IHC-positive cases, we perform FISH to exclude MET amplification as a cause of MET overexpression. In cases without MET amplification, we perform custom-designed and RT-PCR using mRNAs from tumor to detect METex14del transcripts. For RT-PCR positive cases, we sequence them to find underlying cause of METex14 alterations at the DNA level.In two PDC cell lines with In summary this report supports that the aberration in Cbl-mediated negative regulation of MET can indeed result in MET protein overexpression and subsequent addiction of tumor cells to MET signaling and may serve as an actionable driver mutation in a subset of GI malignancies.


Clonostachys rosea. Addition of Met to culture media led to the production of MTL and downregulation of STR3, while incubation of Met with surrogate substrate α-ketoglutaric acid enhanced the synthesis of MTL and triggered the upregulation of STR3. Subsequent biochemical analysis with recombinant STR3 showed that STR3 directly converted both Met and its transamination product KMBA to MTL. These results indicated that STR3 as rate-limiting enzyme degrades Met and KMBA into MTL. Our findings suggest STR3 is a potential target for therapeutic agents against Met-dependent tumors and aging.Eukaryote-derived methioninase, catalyzing the one-step degradation of methionine (Met) to methanethiol (MTL), has received much attention for its low immunogenic potential and use as a therapeutic agent against Met-dependent tumors. Although biological and chemical degradation pathways for Met-MTL conversion are proposed, the concrete molecular mechanism for Met-MTL conversion in eukaryotes is still unclear. Previous studies demonstrated that α-keto-methylthiobutyric acid (KMBA), the intermediate for Met-MTL conversion, was located extracellularly and the demethiolase STR3 possessed no activities towards Met, which rule out the possibility of intracellular Met-MTL conversion pathway inside eukaryotes. We report here that degradation of Met resulted in intracellular accumulation of KMBA in  S-adenosylmethionine. Thus, it can be assumed that Met synthesis, accumulation, and transfer play fundamental roles in cell growth and development1235Methionine (Met) is employed at multiple levels in cellular metabolism: as a protein constituent, in the initiation of mRNA translation, and as a regulatory molecule in the form of In eukaryotic cells, Met was catabolized to α-keto-methylthiobutyric acid (KMBA) by aminotransferase, KMBA was hypothesized to be converted to methional and methionol via Ehrlich pathway and methanethiol (MTL) via demethiolation pathway, respectively67AAT1, AAT2, ARO8, ARO9, BAT1, BAT2, and α-ketoacid decarboxylase gene PDC are annotated in fungi based on the homology to Saccharomyces cerevisiae genes6891112CYS3 only exited in the genome of S. cerevisiae, its homologue in C. rosea were not obtained after searching C. rosea genomeS. cerevisiae CYS3 and STR3 expressed and purified from E. coli displayed a broad specificity toward cysteine-S-conjugates except Met13Met degradation pathway genes are highly conserved, Ehrlich pathway genes including aminotransferase genes Methioninases, catalyzing the one-step degradation of Met to MTL, α-ketobutyrate (α-KB) and ammonium, have received much attention as therapeutic agents against various Met-dependent tumors and aging1516171819161719172223Clonostachys rosea. Our findings support an effective approach against various Met-dependent tumors and aging.In this study, we located the biodegradation pathway of Met, and then used genetic, biochemical and metabolite–based analysis to prove that STR3 has the ability for one-step degradation of Met and its transamination product KMBA in C. rosea Tang19, liquid chromatography and LTQ-Orbitrap MS analyses demonstrated that the metabolite showed similar molecular weight as KMBA  to retrieve the sets of aminotransferase and demethiolase, and conducted multiple alignments of amino acid sequences by ClustalX. Based on the analysis of the highly conserved regions, degenerate PCR primers were designed for cloning Met-MTL pathway gene homologs in C. rosea  by 24.46%, and the mRNA levels of aminotransferase genes BAT and ARO8-2 by 5.80 and 8.85 folds .Recombinant STR3 was expressed in −1) and 16.68 mM for KMBA (Vmax = 1.56 μM min−1), showing that STR3 can degrade Met and KMBA, but preferred the substrate KMBA to its precursor Met. This suggests that one-step degradation of Met into MTL exists, but the conversion of KMBA to MTL is the primary pathway in C. rosea.To further support our conclusion that STR3 is a rate-limiting enzyme controlling Met-MTL conversion, Met and its transamination product KMBA were used as substrates for STR3 in biochemical reactions. Except the controls, MTL was detected in reaction systems either with Met or KMBA, indicating that STR3 has the ability to convert Met and KMBA to MTL. STR3 had a Km of 196.37 mM for Met  and To validate that PLP is a cofactor in STR3 activity, different molar ratios between PLP and STR3 in enzymatic reactions were investigated to determine the demethiolation activity of STR3. Demethiolation activity was detected even without exogenous PLP addition, but the addition of exogenous PLP significantly enhanced STR3 activity , which iβ, γ-S bonds were further assayed as potential substrates for the STR3 enzyme  with a higher catalytic efficiency for L-cystathionine than other physiological substrates. In this catalytic reaction, cystathionine is cleaved by STR3 to produce homo-Cys, the direct precursor of Met in eukaryotes2STR3 expression repressed by Met addition may be related with feedback effects. Thus, STR3 is a potential key target for the biosynthesis of Met and MTL in eukaryotes by metabolic engineering40Biochemical analyses indicated that STR3 exhibited the ability for one-step degradation of Met and its transamination product KMBA, revealing a novel enzymatic role distinct from its previously identified features as a eukaryotic family member. Met restriction suppressed cancer cell metabolism and extended lifespan of normal cell151920C. rosea. Our findings provide a rationale for Met depletion in vivo or dietary supplementation of methioninase in cancer therapy and anti-aging, and the regulation of essential amino acid Met content and volatile flavor constituent MTL in plants and yeast, respectively.In conclusion, STR3 displayed demethiolase activities to Met and its transamination product KMBA. Together with genetic and metabolite data, we demonstrated that the direct conversion of Met and its transamination product KMBA into MTL controlled by STR3 occurs in Chemical standards of MTL and its derivatives DMS and DMTS with over 98% purity were purchased from J&K Scientific Ltd . All analytical grade reagents for STR3 enzymatic assay were purchased from Biosharp , Tokyo Chemical Industry  and Sigma-Aldrich .E. coli DH5α cells  were used for amplification of pYES2 plasmid (Invitrogen). Growth and selection were carried out in Luria-Bertani medium supplemented with 100 mg/L ampicillin. Commercial S. cerevisiae strain INVSc1 (Invitrogen) was used as a host strain for the expression of STR3. Yeasts were cultivated at 30 °C in either a rich medium YPD  yeast extract, 2% peptone, and 2% glucose), or a synthetic minimal defined medium SC ). Uracil (SC-U) was omitted to obtain selective plates for growing pYES2 transformants.Chemically competent 2PO4 ·H2O, 1 g/L; MgSO4·7H2O 0.5 g/L; Vitamin B1, 0.05 g/L), and then their ability to degrade Met was tested by inoculating them in fermentation medium added with 5 g/L Met. Met transamination product KMBA was only detected in the culture inoculated with Clonostachys rosea Tang 19, which was identified by ITS sequence (Genbank accession number: KT007105). This strain was selected for further study.Thirty isolated fungal strains were grown on potato-agar-dextrose slants, precultured in fermentation medium  from Sepax Technologies . Targeted samples were identified by comparison with the standard compounds, followed by tandem mass spectrometry (MS/MS) on a Finnigan TSQ Quantum Ultra AM instrument . The metabolites were confirmed by standard MS using positive electrospray ionization and scanned in the normal mass range from 50 m/z to 180 m/z. Characteristic fragment ions were detected by second-order MS.AAT1, AAT2, BAT and ARO8, and demethiolase gene STR3 of C. rosea Tang 19 were amplified by PCR using degenerated forward/reverse primers  L-Met or 0.098% (w/v) α-KG (Sigma-Aldrich) with respective controls included. After culturing for 3 to 5 days in the fermentation medium, C. rosea mycelia were collected and the transcriptional expression of Met-MTL pathway genes was analyzed by quantitative real-time PCR. RNA was isolated using the MasterPure Fungal RNA Purification Kit (Biozym Scientific GmbH). RNA concentration and purity (A260/A280 ratio) were measured by using the Take3 micro-volume plate of BioTek Epoch Microplate Spectrophotometer. Complementary DNA (cDNA) was synthesized using a PrimeScriptTM 1st Strand cDNA Synthesis Kit  Co., Ltd) with 0.5 μg of total RNA as the template. The 18S rRNA served as the reference gene for normalization of gene expression. Quantitative real-time PCR was performed on a StepOnePlus Real-Time PCR system in duplicate for at least three independent experiments. SYBR dye  Co., Ltd) was used to visualize gene amplification. The internal primers for RACE cloning were used to amplify the target sequence from different Met-MTL pathway genes  device was inserted into the headspace of vial to extracte MTL and its derivatives for 20 min at 40 °C. MTL and its derivatives were analyzed by gas chromatography (GC-2010 with a flame ionization detector (FID) from Shimadzu Technologies Inc., Tokyo, Japan) with an oven temperature program. First, the temperature was maintained at 35 °C for 3 min. Subsequently, the temperature reached 100 °C, with an increment of 8 °C/min. Then, the temperature was raised to 220 °C, with an increment of 10 °C for 1 min. Three independent cultures were analyzed for each condition.C. rosea were performed using E.Z.N.A Fungal DNA kit (Omega) according to the manufacturer’s protocol. Primer pairs YSTR3-5H and YSTR3-3BH were designed to amplify the complete ORF of STR3 from chromosomal DNA of C. rosea (HindIII (in the forward primer) and BamHI (in the reverse primer) restriction sites were introduced to ensure that STR3 was correctly inserted into yeast expression plasmid pYES2. For purification of STR3, a His-tag encoding fragment was included before the stop codon (TTA). After the PCR product was cloned into pYES2, the resulting plasmid, pYES2-STR3, was transformed into S. cerevisiae strain INVSc1 as describedThe isolation and manipulation of DNA from C. rosea . HindIIIAfter 20 h of galactose induction, yeast cultures were pelleted by centrifugation at 2,000 g for 5 min at 4 °C. Intracellular proteins were extracted by vigorously grinding the pelleted yeast cells cooled in liquid nitrogen. The lysates were centrifuged at 13,000 g for 1 h at 4 °C and filtered through a 0.22 μm (Millipore) filter. The resulting clear lysate was then exposed to nickel affinity chromatography using a 1-mL Ni-Sepharose 6 Fast Flow matrix according to the manufacturer’s instructions (GE Lifesciences). STR3 was purified by washing with 3 column volumes of NiA buffer containing 50 mM Tris, pH 8.5, 250 mM imidazole.TM Pure 25 . The flow rate was 0.4 mL/min. Proteins were separated by electrophoresis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).The collected sample was loaded on a Mono Q 10/100 GL (GE) equilibrated with buffer A . Only one peak was detected by using a linear gradient method, and was eluted with 516 mM NaCl. Subsequently, Size exclusion chromatography was carried out with a Superdex 200HR 10/30 analytical column (GE Lifesciences) using an AKTASTR3 resolved by SDS-PAGE was subjected to a trypsin digestion and analyzed by the 4700 Proteomics Analyzer MALDI-TOF/TOF . Based on the combined MS and MS/MS spectra, STR3 was identified based on a 95% or higher confidence interval of their scores in the MASCOT V2.0 search engine . Functional roles of STR3 as demethiolase involved in the conversion of Met to MTL were measured using Met and its transamination product KMBA as substrates. Enzymatic reaction was conducted in a total reaction volume of 5 mL containing a final concentration of 1 mg/mL of STR3, 50 mM Tris-HCl (pH8.0), 5 μM PLP and 20 mM of Met or KMBA. Reaction mixtures were incubated for 1 h at pH 8.0. MTL production via demethiolation of Met and KMBA was determined by GC according to the method mentioned above. Control experiments referred to the complete assay with either omission of STR3 or with denatured STR3 (boiled for 10 min) and MTL and its derivatives were not detected after enzymatic reaction. Meanwhile, different concentrations of cofactor PLP  were added to the standard enzyme assay mixture to investigate the effect of cofactor on STR3 activity toward KMBA. Demethiolating activities of STR3 were measured using the other sulfur-containing amino acids including S-methyl-L-cysteine, L-cystine, L-cystathionine and L-djenkolate as the substrates, as previously describedHow to cite this article: Jia, K.-Z. et al.Clonostachys rosea demethiolase STR3 controls the conversion of methionine into methanethiol. Sci. Rep.6, 21920; doi: 10.1038/srep21920 (2016).


CONSTANS (CO) and FLOWERING LOCUS T (FT) are two genes playing critical roles in flowering time control in Arabidopsis. Through homology-based cloning and rapid-amplifications of cDNA ends (RACE), we obtained full-lengths cDNA sequences of Prunus persica CO (PpCO) and Prunus persica FT (PpFT) from peach (Prunus persica (L.) Batsch) and investigated their functions in flowering time regulation. PpCO and PpFT showed high homologies to Arabidopsis CO and FT at DNA, mRNA and protein levels. We showed that PpCO and PpFT were nucleus-localized and both showed transcriptional activation activities in yeast cells, consistent with their potential roles as transcription activators. Moreover, we established that the over-expression of PpCO could restore the late flowering phenotype of the Arabidopsis co-2 mutant, and the late flowering defect of the Arabidopsis ft-1 mutant can be rescued by the over-expression of PpFT, suggesting functional conservations of CO and FT genes in peach and Arabidopsis. Our results suggest that PpCO and PpFT are homologous genes of CO and FT in peach and they may function in regulating plant flowering time.Flowering is an essential stage of plant growth and development. The successful transition to flowering not only ensures the completion of plant life cycles, it also serves as the basis for the production of economically important seeds and fruits.  Arabidopsis thaliana [CONSTANS (CO) plays a key role in the photoperiod-dependent induction of flowering in Arabidopsis and loss-of-function mutations of CO cause a distinctive late flowering phenotype [CO under the constitutive cauliflower mosaic virus (CaMV) 35S promoter exhibit an early flowering phenotype [CO encodes a putative zinc finger transcription factor and CO protein features two conserved domains [CONSTANS, CONSTANS-like and TOC1 (CCT) domain in the C terminus, which may function as a nuclear-localization signal [FLOWERING LOCUST (FT) and SUPPRESSOR OFOVEREXPRESSION OFCO1 (SOC1) [CO, loss-of-function mutations of FT result in delayed flowering, and the over-expression of FT can accelerate flowering, indicating that FT is also a flowering activator [LEAFY (LFY) and APETALA1 (AP1) [The developmental transition from vegetative to reproductive growth is one of the most fundamental events during the life cycles of flowering plants. Given the central role of flowering, it came as no surprise that a large number of genes act in a coordinated manner to ensure the successful induction of the flowering process in the model system thaliana –6. Amonghenotype –10. Conshenotype . CO enco domains , 12. The domains , 14. Then signal , 12. CO 1 (SOC1) –16. Simictivator –18. FT ictivator –19. FT cA1 (AP1) , 20–23. A1 (AP1) .CO and FT work during flowering induction and has laid the foundation for our current understanding of the mechanisms of flowering control. However, the regulation of flowering in non-model systems such as woody perennial trees remains largely unexplored [CO and FT may work as inhibitory signals in non-inductive conditions [Solanum species [The information derived from model plants such as Arabidopsis has been extremely useful to define the molecular mechanisms of how key flowering regulators such as explored , 25. Feaexplored . For exanditions , and the species .Prunus persica (L.) Batsch) is one of the most economically important fruit producing species and is also emerging as a potential model woody species for genetic studies within the Rosaceae family [CO and FT homologs, PpCO and PpFT. Gene structure, mRNA and protein sequence analyses showed that PpCO and PpFT are closely related to Arabidopsis CO and FT, respectively. We determined that both PpCO and PpFT can be targeted to the nucleus and that PpCO and PpFT have transcription activation activities. Moreover, the ectopic expressions of PpCO and PpFT can functionally complement the late flowering phenotypes of Arabidopsis co-2 and ft-1 mutants, respectively. Our results provide new insight into the functions of PpCO and PpFT in peach and may enable the future utilization of these genes in the artificial manipulation of peach developmental programs, particularly flowering time regulation.Peach , MdCO (NCBI accession number AF052584) and MdFT (NCBI accession number AB161112), as the query sequences and searched the NCBI expressed sequence tag (EST) database. We identified one peach EST sequence (NCBI accession number BU044758) homologous to MdCO and one peach EST sequence (NCBI accession number BU042239) homologous to MdFT. However, these ESTs covered only partial coding sequences of PpCO and PpFT, respectively.To identify potential expressed peach sequences related to PpCO and PpFT, we carried out RACE experiments based on the partial PpCO and PpFT sequences. Using cDNAs synthesized from RNAs extracted from peach mature leaves as templates, we successfully amplified a 353 bp potential PpCO fragment and a 188 bp potential PpFT fragment that were identical in sequences to BU044758 and BU042239, respectively. Next, based on these two fragments, we obtained the 5′ and 3′ sequences of PpCO and PpFT using the RACE strategy and assembled open reading frames (ORFs) of the two genes. We next experimentally confirmed the deduced sequences through the direct amplification of full-length cDNAs of PpCO and PpFT and determined that the ORFs of PpCO and PpFT were 1032 bp and 525 bp, respectively. Genomic DNA sequences of PpCO and PpFT were also amplified and PpCO and PpFT were 1440 bp and 2015 bp in length at the DNA level, respectively  revealed the presence of conserved domains . These structural features are similar to those of FT protein from Arabidopsis (AtFT) [Prunus mume and MdFT (95.04%) from apple  in exon 2, Gln-139 (PpFT amino acid numbering), as well as the segment B in exon 4 . These rom apple . These dCO and FT members vary among different plant species [PpCO and PpFT genes in peach genome, southern blotting analyses were conducted using full-length PpCO and PpFT cDNA as probes. Peach genomic DNAs were digested with EcoRI and BglII restriction enzymes, which do not cut either PpCO or PpFT DNA sequences. Southern blotting results indicated that two EcoRI fragments and one BglII fragment hybridized to the PpCO probe  were obtained for each transformation, and 5 lines were selected for further analyses. At least 15 plants per line were grown to score the flowering time phenotype. The time of flowering was determined by counting the days to flowering after sowing and the number of rosette leaves at the time of blooming.To further validate the roles of erecta (Ler) plants took 25.9±0.2 days to bloom after sowing, while co-2 mutants (in Ler background) needed 34.3±0.5 days  background, it took 39.6±1.4 days to flower, whereas the wild type Col-0 plants flowered at 23.0±0.0 days  Batsch. cv. Bayuecui was used in this study. The peach trees were grown in the Institute of Forestry and Pomology, Beijing Academy of Agricultural and Forestry Science, P. R. China . Because it is one of the research partners of China Agricultural University, there is no specific permission required for research sampling in this institute. In addition, the peach cultivar we used is not an endangered or protected species. Peach leaves were harvested on May 30, 2012, frozen in liquid nitrogen immediately and stored at -80°C until use. Leaves at the tip of the shoot were designated as young leaves while those at the base of the shoot were designated as mature leaves.Peach variety Arabidopsis thaliana ecotypes Columbia-0 (Col-0) and Landsberg erecta (Ler) were used in this study. Arabidopsis flowering mutants co-2 (Ler background) and ft-1 (Col-0 background) have been described [-2 s-1) in a controlled growth room maintained at ~22°C.Wild type escribed . ArabidoTotal RNAs were extracted from the mature leaves using the Trizol RNA reagent . CTAB method was used for total genomic DNA purification. Southern blot analysis using digoxin (DIG)-labeled probes was carried out according to manufacturer’s manual .PpCO and PpFT gene specific primers PpCOF1, PpCOR1, PpFTF1 and PpFTR1 were designed according to the peach CO and FT EST sequences  deposited in the National Center for Biotechnology Information (NCBI) and used to amplify partial cDNAs of PpCO and PpFT. Full-length cDNAs of PpCO and PpFT were then obtained via 5′ and 3′ RACE methods using a Generacer kit  following the manufacturer’s manual. Primers PpCOF2, PpCOF3, PpCOR2, PpCOR3 were used to obtain PpCO full-length cDNAs and primers PpFTF2, PpFTF3, PpFTR2, PpFTR3 were used to obtain PpFT full-length cDNAs. Amplified fragments were cloned to pMD18-T vector  and sequenced in both directions. Detailed information of primers used in this study was listed in First strand cDNAs were synthesized using the PrimeScript reverse transcription kit  according to the manufacturer’s instructions. -Δct. Primers used  in real-time PCR experiments were listed in PpACTIN2 and AtACTIN2 genes were used as controls.For Real-time quantitative RT-PCR analysis, total RNAs were extracted using Trizol RNA reagent  and 1 μg DNase I treated RNA was used for cDNA synthesis. Real-time PCRs were performed with a Bio-Rad CFX96 Real-Time PCR System. Relative expression levels of the target genes were calculated with 2PpCO and PpFT were predicted with the NCBI ORF Finder program. The isoelectric point and molecular weight of PpCO and PpFT protein were calculated using the PeptideMass program (http://web.expasy.org/peptide_mass/). Multiple protein sequence alignments were carried out using Clustal x 1.83 and GeneDoc software (http://iubio.bio.indiana.edu/soft/molbio/ibmpc/genedoc-readme.html). Phylogenetic trees were constructed by the MEGA5 program using the Neighboring-Joining method (http://www.megasoftware.net/).Open reading frames (ORFs) and protein sequences of PpCO and PpFT were obtained by PCR with gene specific primers  and cloned into the EcoRI and PstI sites of BD vector pGBKT7 to generate pGBKT7-PpCO and pGBKT7-PpFT. Sequences of primers used were showed in MEL-1 reporter genes were assayed following the procedures described in Yeast Protocols Handbook .The ORFs of PpCO and PpFT were obtained by PCR with gene specific primers  and cloned into the SalI site of the pTF486 vector [P35S:PpCO-GFP and P35S:PpFT-GFP for protoplast transient expression. Detailed primer information was described in The ORFs of 6 vector  to generPpCO and PpFT cDNAs were amplified using gene specific primers  and cloned into NcoI and BglII sites of pCAMBIA1301 binary vector  to generate P35S:PpCO and P35S:PpFT. Primers sequences was showed in P35S:PpCO and P35S:PpFT were introduced into the Arabidopsis co-2 and ft-1 mutant background, respectively. The floral dip method was used for Arabidopsis transformation [–1 hygromycin. Phenotypes of the transgenic lines were examined in T2 generation.Full-lengths ormation . TransgeS1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


SCID mice and significantly inhibited by cabozantinib, as was tumor phospho-Met content. These studies indicate the prevalence and functionality of the HGF/Met signaling pathway in UC cells, suggest that paracrine HGF may contribute to UC tumor growth and progression, and that support further preclinical investigation of Met inhibitors for the treatment of UC is warranted.There is mounting evidence of oncogenic hepatocyte growth factor (HGF)/Met signaling in urothelial carcinoma (UC) of the bladder. The effects of three kinase inhibitors, cabozantinib, crizotinib and EMD1214063, on HGF-driven signaling and cell growth, invasion and tumorigenicity were analyzed in cultured UC cell lines. SW780 xenograft growth in SCID and human HGF knock-in SCID (hHGF/SCID) mice treated with cabozantinib or vehicle, as well as tumor levels of Met and pMet, were also determined. Met content was robust in most UC-derived cell lines. Basal pMet content and effector activation state in quiescent cells were low, but significantly enhanced by added HGF, as were cell invasion, proliferation and anchorage independent growth. These HGF-driven effects were reversed by Met inhibitor treatment. Tumor xenograft growth was significantly higher in hHGF/SCID mice  In 2013, 72,570 new cases of bladder cancer (BCa) and 15,210 bladder cancer-related deaths were estimated in the U.S. alone . These cin vivo [FGFR3 (13%), ERBB2 (6%), FGFR1 (6%) and MET (2%) [et al. [MET or HGF genes in 3% and 2%, respectively, of cases in the Nature Genetics 2013 dataset (n = 99) [n = 131) [n = 201). Together these studies identified genetic aberrations in HGF and MET at an average combined frequency of 10% in 528 BCa cases.The cell surface receptor tyrosine kinase (RTK) for hepatocyte growth factor (HGF), known as Met, is widely present in cells of epithelial origin. HGF/Met signaling is frequently implicated in cancer, driving tumor invasiveness and metastasis . Evidencin vivo ,10,11. Ain vivo . The RTKMET (2%) . The RASMET (2%) . A prelio [FGFR3 %, ERBB2 MET (2%)  further  [et al. , as well(n = 99) , in 10% n = 131) , and in While several Met kinase inhibitors are in preclinical and clinical development, there use for the treatment of BCa has been limited. Here, we focused on three small molecule Met inhibitors, crizotinib, cabozantinib and EMD1214063. Crizotinib is an orally bioavailable small molecule inhibitor of Met and anaplastic lymphoma kinase (ALK), which has been US FDA approved for the treatment of ALK-positive non-small cell lung cancer . CrizotiAccurately selecting therapies that will be effective against a specific tumor is the ultimate goal of clinicians treating BCa. In a prior study we found that levels of soluble Met ectodomain (sMet), which are readily measured in the urine of bladder cancer patients and those with no evidence of cancer, could distinguish patients with BCa from those without, and patients with or without muscle-invasive BCa, suggesting the potential utility of urinary sMet as a BCa biomarker for surveillance following initial treatment . In paralow), intermediate (Metint) or high (Methigh) Met content  for multiple human tumor derived cell lines  and 12 UC derived cell lines as measured by electrochemiluminescent two-site immunoassay are listed in  content A. This o50 and IC90 values, respectively; 50 and IC90 values, respectively; None of the UC derived cell lines produced detectable levels of HGF, as determined by two-site electrochemiluminescent immunoassay (200 femtomole limit of detection), suggesting that autocrine HGF/Met signaling is rare in UC of the bladder. Treatment with exogenous HGF (1 nM) induced significant Met kinase activation (pMet/Met SI ratio) in all UC derived cell lines within 20 min, which was suppressed by the small molecule Met kinase inhibitors crizotinib at 5 and 10 nM,  and MAPK pathways downstream of Met mediates HGF-driven cell survival, migration, invasion and proliferation. HGF stimulated both Akt and Erk phosphorylation in all UC derived cell lines . Treatme TCC-SUP A, left, nd SW780 A, right d HT1376 C. Treatm TCC-SUP B, top, t5, SW780 B, bottom5, SW780 C, left, ines J82 B, bottomines J82 C. These low lines RT4, T24M2, T24M3 and TCC-SUP, the Metint lines UMUC5 and SW780, and the Methigh line J82 Aveo). Growth of SW780 cell xenografts in SCID mice was significantly slower than in hHGF/SCID mice (3 (~day 18), significantly blocked, and later reversed, SW780 tumor growth in both SCID  overexpression of ET genes ,14, and ET genes . EpithelET genes . ReinforET genes . HGF proET genes . A priorET genes . FinallyET genes . These sMET can confer resistance to crizotinib and other Met kinase inhibitors [MET gene mutation in the four TCGA bladder cancer datasets (11/569 cases or 1.9% combined), the small molecule Met inhibitors crizotininb, cabozantinib and EMD1214063 uniformly and potently suppressed Met and Met-effector activation, and HGF-driven invasion, growth and soft agar colony formation in all UC cell lines tested. Our cell-based assay results do not reveal a relationship between Met abundance and biological response to HGF, or between Met abundance and drug suppression of HGF-induced responses, in the 12 UC-derived cell lines studied. In this regard BCa is not similar to certain other cancers, such as gastric and lung adenocarcinomas, where frequent MET gene amplification leads to ligand-independent activation and enhanced sensitivity to Met inhibition [Mutations in hibitors . Consisthibition  are suggSubcutaneously implanted SW780 cells grew significantly faster in hHGF/SCID mice relative to SCID mice, revealing the ability of paracrine delivered HGF to drive UC tumor growth. Cabozantinib fully suppressed SW780 xenograft growth in mice, and prevented the elevated tumor pMet observed in vehicle-treated hHGF knock-in mice. Additional studies will be needed to discern meaningful differences in efficacy among these agents against UC tumor growth, particularly whether the broader target spectrum of cabozantinib and, to a lesser degree, crizotinib, is potentially advantageous, and whether drug effects on the host, such as on tumor-stroma interactions and the tumor vasculature, also contribute to anti-tumor efficacy.Tissue culture media and supplements were obtained from Invitrogen . Antibodies against pMet (1234/1235), pErk, tErk, pAkt, and tAkt were obtained from Cell Signaling Technology . Anti-Met (C-28) was obtained from Santa Cruz Biotechnology . Purified recombinant human HGF was obtained from ProSpec . Crizotinib, cabozantinib and EMD1214063 were obtained from the Open Chemical Repository Collection, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.SW480, SW620, CRL 7636, CRL 7637, HTB 125, MCF7, 786-0, ACHN, PC3, UMUC3, UMUC5, H596, A549, MKN45, RT-4, TCC-SUP, T24, HT1197, SW780, J82, 5637 and HT1376 cells were obtained from ATCC . PC3M was obtained from MD Anderson Cancer Center, University of Texas . T24M2 and T24M3 cell lines were derived from metastases of T24 xenografts in mice. Cells were cultured in DMEM medium containing 10% FBS and antibiotic-antimycotic. Cells were grown in 5% CO2 at 37 °C.Cells were washed with cold PBS, extracted in Laemmli buffer, sonicated, heated for 5 min at 95 °C prior to SDS-PAGE and electrophoretic transfer to nitrocellulose. Membranes were blocked with 5% milk in TBST  for 1 h at 25 °C then incubated 16 h at 4 °C with primary antibody in TBST/0.5% milk. Membranes were washed three times with TBST, incubated with horseradish peroxidase-labeled secondary antibody for 1 h at 25 °C, washed for 3 h with TBS prior to ECL detection . Phospho- and total Met content in Triton X-100 cell extracts were analyzed by electrochemiluminescent two site immunoassay using a SectorImager 2400 plate reader  as described . The asshttp://imagej.nih.gov/ij/, 1997–2012). For proliferation assays, cells were plated in triplicate at a density of 5 × 104 cells/35-mm dish in defined medium. HGF ± crizotinib or cabozantinib were added on days 1, 2, and 4. Cells were trypsinized and counted using a hemocytometer on days 3 or 6.Invasion assays were performed using 8 um pore size BD Bioscience BioCoat Matrigel Invasion chambers  according to the manufacturer’s instruction. Images were captured by light photomicrography and quantitation was performed using Image J software V1.47  in phenol-red free DMEM was added to 96 well plates. Cells in 0.3% agarose in phenol-red free DMEM were added on top of the base layer. Cells were fed with DMEM with or without HGF and crizotinib or cabozantinib every other day. After one week, MTT was added to quantify the viable colonies by absorbance using a Victor plate reader .http://www.informatics.jax.org) were acquired from The Jackson Laboratory. SW780 cells (3 × 106 per site) suspended in matrigel were injected subcutaneously into both flanks of 5 mice per treatment group, and tumor volumes were calculated from caliper measurements. When tumor volume reached 150 mm3, mice were treated with either vehicle or cabozantinib once daily by oral gavage. Tumors excised from mice were disrupted in non-ionic detergent containing buffer, lysates were cleared by centrifugation, and analyzed using two-site immunoassays for Met and pMet [Experiments with SCID and hHGF/SCID mice were performed in accordance with National Institutes of Health Guidelines for Care and Use of Laboratory Animals using protocols approved by the Institutional Animal Care and Use Committee of the Center for Cancer Research, National Cancer Institute. hHGF/SCID mice (HGF/SCID: Rideout WM 3rd Mouse Genome Database (MGD) at the Mouse Genome Informatics website, The Jackson Laboratory (Bar Harbor, MN, USA; and pMet .p = 0.05 was the threshold for statistical significance.Significant differences between two groups were determined by One Way ANOVA with post hoc comparison of all groups using GraphPad Prism 5 software, where Met protein content in 12 UC-derived cell lines ranged from 12 ng/mg to 133 ng/mg total protein, comparable to that of several other well-studied tumor-derived cell lines from other prevalent cancers. HGF production was conspicuously absent, but exogenous HGF stimulated the activation of Met and known downstream effectors, and significantly enhanced UC cell invasion, proliferation and anchorage-independent growth. Concomitant treatment with cabozantinib, crizotinib or EMD1214063 effectively suppressed these HGF-driven effects. Cabozantinib also significantly inhibited HGF-driven tumor xenograft growth and Met activation in SCID and, more dramatically, in hHGF/SCID mice. Our results suggest that further studies are warranted to fully define the prevalence and oncogenic impact of HGF/Met signaling in bladder cancer, and to identify reliable and practical biomarkers of pathway activation for patient selection and predicting response to treatment with HGF/Met targeted therapeutics.


PLZF is a transcription repressor, which plays a critical role in development, spermatogenesis and oncogenesis. Down-regulation of PLZF has been found in various tumor cell lines. There has been virtually no tissue study on the expression of PLZF in prostate cancer (PCa). PCa is a heterogeneous disease, most of which are indolent and non-lethal. Currently there are no biomarkers that distinguish indolent from aggressive PCa; therefore there is an urgent need for such markers to provide clinical decision support. This study aimed to investigate the expression of PLZF by immunohistochemistry in different grade as well as metastatic PCa and to correlate the alteration of PLZF expression with PCa aggressiveness. We studied a total of 83 primary PCa from biopsies, 43 metastatic PCa and 8 paired primary and metastatic PCa from radical prostatectomies with lymph node dissection. Our results demonstrated that PLZF was strongly expressed in almost all (~100%) benign luminal cells (n=77) and low grade (Gleason pattern 3) PCa (n=70) and weak or absent (100%) in basal cells (n=70). Decreased or lost expression of PLZF was evidenced in 26% of high-grade (Gleason 4 and 5) primary PCa (n=70) and 84% metastatic PCa (n=43). The primary high grade PCa in the prostatectomies shared similar PLZF loss/decrease and histomorphology to that of paired parallel lymph node metastases. These data demonstrated that down-regulation of PLZF is an important molecular process for tumor progression and loss of PLZF expression detected by routine immunohistochemistry is a promising and valuable biomarker for PCa aggressiveness and metastasis in the personalized care of PCa. Prostate cancer (PCa) is the most common cancer among men in the United States . With thPromyelocytic leukemia zinc finger protein (PLZF), also known as Zbtb16 or Zfp145, first identified in a patient with acute promyelocytic leukemia, is a zinc finger transcription factor belonging to the POZ-Krüppel (POK) family that binds to specific DNA sequences with its carboxy-terminal zinc fingers and suppresses transcription by recruiting co-repressors with its aminoterminal POZ domain . FunctioThis study was performed after approval by the institutional review board and in accordance with an assurance filed with and approved by the ethics committee/institutional review board of the University of Rochester and Mount Sinai School of Medicine. This is an exempted immunohistochemical study of archived samples and contains no any identifiable patient information and the need for written informed consent was waived. The prostate gland is a solid fibromuscular organ with ingrowth of glandular epithelia, which it is not readily permeable to formalin fixative. To avoid the possible effect of uneven and suboptimal tissue fixation of the antigen preservation and retrieval as well as on the subsequent immunohistochemical results, biopsy material of primary as well as metastatic prostate cancer was chosen for this study, except for eight cases of radical prostatectomies with pelvic lymph node dissection. All the prostate biopsy specimens and prostatectomies and the majority of metastatic prostate Ca tissue were collected and studied at University of Rochester Medical Center, NY. Twenty percent of the metastatic PCa cases were from Mount Sinai Medical Center, New York. All the cases were from different patients. The specimens were routinely fixed in 10% neutral buffered formalin and paraffin embedded.There were a total of 83 prostate biopsies, each with a tumor volume ranging from 10 to 100%, among which 13 cases had a total Gleason’s score 6, 57 cases had a score 7, 7 cases has a score 8, and 6 cases had a score 9/10. Benign glandular epithelia were present in 77 of the 83 prostate biopsies and were absent in the remaining 6 biopsies. Forty three cases were metastatic prostate cancer with a tumor volume ranging from 5% to 95%, among which 22 were bone metastases, 11 lymph nodes metastases, 7 liver metastases and 3 lung metastases. Eight paired PCa from prostatectomy specimens and their parallel lymph nodes were also included in the study. The eight primary prostate carcinomas in radical prostatectomies had a total of Gleason’s score ranging from 7 to 9 with a tumor stage pT3a or pT3b. The lymph node metastases ranged from 1 mm to 3mm in greatest dimension. The case distribution are summarized in 2O2) to eliminate endogenous peroxidase activity, then processed for antigen retrieval with 10-mM citrate buffer pH 6.0 using a pressure cooker  for 1 minute at 125°C, followed by slow cooling. The rest of the procedure was done in a DAKO automated instrument. All sections were rinsed with phosphate-buffered saline  and reacted with mouse anti-PLZF antibody  for 1.5 hours at 1:500 dilution in phosphate-buffered saline containing 1% bovine serum albumin (BSA) and 5% normal goat serum at room temperature. The sections were then incubated for 20 minutes with EnVision+ System horseradish peroxidase-labeled polymer conjugated with biotinylated anti-mouse secondary antibody and 3,3'-diaminobenzidine substrate. All slides were counterstained with hematoxylin, dehydrated, and cover slipped.Following deparaffinization and rehydration, charged slides with 5-μm thick sections of tissue were treated with 3% hydrogen peroxide , +1 (weak), +2 (moderate), or +3 (strong/intense). Each individual Gleason’s pattern was separately evaluated for their nuclear labeling with PLZF for tumor in the prostate, and for simplicity, Gleason’s pattern 4 (G4) and Gleason’s pattern 5 (G5) were combined at analysis. Gleason’s score was not used for metastatic PCa. The strong lunimal cell staining and weak or non-reactive basal cells (see results section for detail) were used as positive and negative control, respectively.The Jockheere-Terpstra Test  was usedAmong the 83 prostate biopsies, seventy seven contained benign prostate glandular epithelia. PLZF was moderately or strongly expressed in the nuclei of the luminal cells of benign prostatic glands, however, weak or no expression was detected in either the nuclei or cytoplasm of the basal cells or stroma cells in all the 77 cases .Among the primary PCa in the prostate biopsies, there were a total of 70 individual Gleason pattern 3 and 70 individual Gleason’s pattern 4/5. Gleason patterns 4 and 5 were analyzed together mainly due to their similar biologic behavior as well as the absence of obvious immunostaining difference among these two patterns. PLZF was expressed differentially in intensity in the primary prostatic adenocarcinoma of Gleason’s patterns 3 and 4/5. Similar to the luminal cells of benign prostatic glands, PLZF was moderately or strongly expressed in all the tumor glands of Gleason’s pattern 3 : 45 showVast majority of the metastatic PCa did not or had only weakly PLZF expression . Of the The Jockheere-Terpstra Test revealed a negative trend , indicating that more aggressive metastatic cancer had the lowest protein expression scores, while Gleason scores of 3 or 4/5 had higher protein expression scores. Similarly, highly significant negative associations were found when the collapsed protein score was compared between G3 and G4/5, G3 and metastatic cancer, and G4/5 and metastatic cancer  PCa and decreased or lost expression in focus/foci of high grade (G4/5) PCa  The focuAlthough tremendous efforts have been devoted in search of effective prognostic biomarkers that are able to predict the aggressiveness of prostate cancer, little progress has been made toward practical use. By far the most studied biomarker for this purpose is PTEN, a well-known tumor suppressor –23. GenoPLZF, a gene modulator similar to PTEN, functions as a transcription repressor. Previous studies of PLZF have been mainly focused on its role in spermatogenesis, stem cell maintenance and promyelocytic leukemia ,7,11,12.To further elucidate whether or not the decrease or loss of PLZF expression in primary PCa correlated to that of the metastatic PCa, we studied the immunohistochemical expression pattern of PLZF in both primary PCa in the prostate and the paired metastatic PCa in dissected pelvic lymph nodes of the same patient. Our results showed the changes of PLZF expression as well as the morphology in the focus/foci of primary high grade PCa in the prostate was correlated to that of the pelvic lymph node metastatic PCa. This finding strengthened the association of PLZF loss with PCa progression and implicated a prognostic value of PLZF loss in primary PCa in predicating its metastatic potential. In the paired prostatectomy and lymph node dissection, the variable PLZF expression (-/+1/+2/+3) in the primary G4/5 PCa glands may reflect the variability of antigen presentation due to suboptimal fixation of the prostatectomy specimens, nevertheless, proportionally, the PLZF in the foci of G4/5 PCa are less expressed than the foci of G3 PCa.As demonstrated in this study, PLZF was almost exclusively expressed in the nuclei of luminal cells and weak or absent in the basal cells and stromal cells of the prostate. This clean background not only made it easy to interpret the immunostaining results, but also served as excellent positive and negative controls. These were the obvious advantages of PLZF over PTEN. In addition, compared to the loss of PTEN in 10–70% of PCa –23, the We have examined the expression of PLZF using Oncomine (Life Technologies) as well as the underlying GEO data in five prostate cancer datasets stratified by Gleason score. No apparent association is evident, neither when examining the stratified expression box plots ., nor thin vitro study of PCa cell lines, PLZF is found to be a prostatic androgen-responsive gene [Although the exact role of PLZF in prostate epithelium is unknown, The constitutive expression of PLZF in the luminal cells and absent in basal cells of benign prostatic glands is reminiscent of androgen receptor, and strongly infers an association of PLZF with androgen receptor. In fact, in an ive gene . PLZF isive gene . Overexpive gene . Endogenive gene , 42. TheIn summary, our study demonstrates that the immunohistochemical loss of PLZF is a sensitive and specific biomarker in detecting aggressive and metastatic PCa. Nevertheless we would also like to emphasize that the scope of this study is limited and a larger scale study to validate the results is needed. If further validated, due to the simplicity and cost-effectiveness, immunohistochemical testing of PLZF expression in PCa will have a significant impact and be of clinical utility in the management and personalized care of PCa patients in daily practice. PLZF immunohistochemistry may also be combined with PTEN to provide increased predictive power for progressive PCa. Prospectively restoring PLZF in these aggressive PCa may also have potential therapeutic values by reversing their courses.S1 FigPLZF (ZBTB16) expression was examined in five prostate cancer datasets stratified by Gleason score, available in Oncomine. Only datasets with 10 or more cases of Gleason score 8 and 9 were analyzed. Box plots depict mean expression, stratified by Gleason score. Stratification in columns by Gleason score indicated in legend below . Note that the appropriate comparison is between Gleason score 7 and lower versus Gleason score 8 and or 9, and not the first column for sets 1–4, which depicts samples for which a score was not available.(TIFF)Click here for additional data file.S2 FigPLZF (ZBTB16) expression was examined in five prostate cancer datasets stratified by Gleason score, available in Oncomine. Only datasets with 10 or more cases of Gleason score 8 and 9 were analyzed. Waterfall plots portray the underlying individual data analyzed in (TIFF)Click here for additional data file.S3 FigDisruptions of the gene encoding PLZF (ZBTB16) were examined in the indicated five datasets available at cBioportal. Alterations are indicated in plots below study name: homozyogous deletion (blue bars); amplification (red bars); mutation (green bars). Sum of all detected alterations was 0%, 4%, 1%, 2%, and 4% (top to bottom) in the studies depicted.(TIFF)Click here for additional data file.


Numerous opportunities are available in primary care for alleviating the crisis of increasing antibiotic resistance. Preventing patients from developing an acute respiratory infection (ARI) will obviate any need for antibiotic use downstream. Hygiene measures such as physical barriers and hand hygiene, and possibly vaccination and exercise, may be effective. Also, a large range of complementary and alternative medicines  are proposed for preventing and treating ARIs, but evidence for efficacy is scarce.General practitioners’ (GPs) attitudes towards antibiotic prescribing are a major factor in the prescribing for ARIs. Professional interventions with educational components are effective, although they have modest effects, and are expensive. GPs’ perceptions – that mistakenly assume as a default that patients want antibiotics for their ARIs – are often wrong. Shared decision making might be a solution, as it enables clinician and patient to participate jointly in making a health decision, having discussed the options together with the evidence for their harms as well as benefits.a priori clearly defined actions to take if the course of the illness deviates from the expected.Furthermore, GPs’ diagnostic uncertainty – often leading to an antibiotic prescription “just in case” – might be addressed by exploiting strategies such as safety-netting, e.g., establishing with the patient None of these strategies or interventions on their own will greatly improve the use of antibiotics for ARIs. However, used in concert, combinations are likely to enable clinicians and health care systems to implement the strategies that will reduce antimicrobial resistance in the future. In the past 70 years, antibiotics have been essential in the fight against infectious diseases and have been a contributing factor in the rise in life expectancy . HoweverThe majority of antibiotics are prescribed in primary care  and mainThe solution seems at first to be straightforward: targeting the use of antibiotics in the community – which is where the greatest tonnage is prescribed and the efficacy of their use is most limited –15. WithUnderstanding the events of decision points surrounding an antibiotic prescription for ARIs in primary care can inform strategies to address the problem at each of these points. This paper considers the path from a healthy person, to a person with symptoms of ARIs, to a person consulting a GP, who may or may not be prescribed an antibiotic Figure . The autOf course, prevention of ARIs in the first place will reduce the whole downstream use of antibiotics.Haemophilus influenzae type b (Hib) is an important cause of pneumonia and meningitis in children, and the Hib vaccine is safe and effective in reducing Hib disease  1.11; 95% CI 1.04–1.18] in a Cochrane review . AnotherA large range of complementary and alternative medicine is available for symptomatic treatment of ARIs. Vitamin C has been proposed for treating respiratory tract infections and weak evidence suggests regular supplementation of vitamin C might be effective in reducing the duration of common cold by 8% (4–12%) in adults and 14% (7–21%) in children . EchinacZinc inhibits replication of virus and has been tested in trials for the treatment of the common cold. A Cochrane review demonstrated that when zinc is administered within 24 h of onset of symptoms, it reduces the duration (days) of common cold symptoms in healthy people  . HoweverCaffeine as an analgesic adjuvant has been discussed for many years. In a Cochrane review based on 19 studies with a total of 7238 patients with different pain conditions, caffeine enhanced the efficacy of paracetamol, ibuprofen, or aspirin with a NNT of about 15 . HoweverAlthough the body of existing evidence is limited, it suggests that there is minimal benefit for the use of these non-antibiotic alternatives and larger, high quality, primary care-based studies are needed to further explore these alternatives and to develop and test new non-antibiotic treatments for ARIs.P < 0.01)] ] , even at< 0.01)] . A signi< 0.01)] . However< 0.01)]  and a re< 0.01)] . Althoug< 0.01)] .General practitioners often feel patients with ARIs are consulting with an expectation of antibiotics , 53. BacThe relationship between diagnostic uncertainty and antibiotic misuse has been demonstrated in several studies . CliniciIn nearly 30 years, the rapid antigen detection test has been used for detection of Lancefield group A β-hemolytic streptococci (GABHS) in patients with a sore throat. Not all patients with a positive test and throat symptoms have an infection caused by GABHS , 67, andProcalcitonin is a promising biomarker for identification of bacterial infections. So far, meta-analyses have mainly investigated the use of procalcitonin as a diagnostic marker for sepsis , 71, butGeneral practitioners may prescribe antibiotics to prevent complications like quinsy, mastoiditis, and pneumonia, although the rates of serious complications are low in modern developed countries , 74. HisBusy GPs are more liberal with antibiotics for ARIs than less busy GPs, and use a greater proportion of broad-spectrum antibiotics . UnfortuHow can GPs manage the therapeutic vacuums left by not prescribing antibiotics? Over-the-counter drugs, complementary and alternative medicines, or steroids might be recommended for patients with some ARIs. Others might benefit from advice on preventing future ARIs by routine handwashing. Most patients are satisfied with a good explanation of their symptoms , and infOften patients end up with leftover antibiotics because there is a mismatch between guidelines and the packet size of antibiotics. This is a problem because leftovers are saved and used for self-medication . AntibioThere are numerous opportunities available in primary care for alleviating the crisis of increasing antibiotic resistance. None of these strategies or interventions on their own will greatly improve the use of antibiotics for ARIs. However, used in concert, combinations are likely to enable clinicians and health care systems to implement the strategies that will reduce antimicrobial resistance in the future.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The authors report a case that is started with a simple upper third molar's surgical extraction and a broken 3.0 suture needle tip incident occurred. Broken fragment's localization has been detected with 3D cone beam computed tomography (CBCT). Needle tip has been reached with the help of CBCT vision. CBCT's benefits have been discussed on these types of cases and further migration of the needle tip during surgical procedure is reported. Accidental breakage of suture or injection needles during oral surgical procedures is a rare but serious complication. It commonly occurs due to excessive force applied on the tip or sudden movement of the patient, especially if their mechanical properties are weak . FindingHerein, we report a case that is started with a simple upper third molar's surgical extraction and required further interventions to find out an accidentally broken 3-0 suture tip. A lost broken suture needle was located using cone beam computed tomography scan and retrieved via surgery.27-year-old male patient referred to our clinic complaining about his right upper third molar. According to clinical and radiographical examination, moderate infection was inspected, caused by wisdom molar. In order to cure infection and its symptoms the tooth was decided to be extracted. Suture needle was found to be broken while passing through the palatinal mucosa. Although it was tried to localise the broken needle fragment, it could not be managed to reach it in deep mucosal area. The broken fragment could not be prevented from migrating to deeper tissues while attempting over reaching. The flap was closed and the patient was gone under further diagnostic examination.3D cone beam computed tomography (CBCT)  and conventional OPTG were performed to localize the needle. According to tomographic images, location of needle was about under the palatinal mucosa, the region of right maxillary tuberosity, nearly just below the medial pterygoid plate. The broken fragment was detected distant from the localization it had been broken at the first place Figures , and 3.After detecting the exact location of the needle, the patient has gone under a second surgery immediately and the needle was reached with a more extensive incision of 35 mm from former horizontal incision of third molar surgery through buccal mucosae inferiorly. Blunt dissection was made through oropharyngeal region and the broken fragment was removed successfully .A broken suture needle may be clinically visible or fully embedded after breakage. Visible fragments may be retrieved immediately using a hemostat . SeveralAs the CT scanning is considered an accurate method of imaging that indicates the position of a needle in relation to recognizable anatomical landmarks  and giveCBCT can be able to detect the fragment accurately, leading to a more specific surgical procedure and decreasing operating time but handling the surgical procedure gently is of great importance. Surgical access may be limited by the presence of vital structures like nerves and arteries. Otherwise, the procedure might get more complicated by the migration of the broken fragment to deeper portions of oropharyngeal tissues.


Objective: To evaluate by survey the comfort upon instillation of timolol hemihydrate compared to timolol maleate with potassium sorbate.Design: A prospective, multicenter, observational, non-interventional study.Participants: One hundred and three patients of open-angle glaucoma or ocular hypertension who were ≥21 years old and were currently prescribed timolol hemihydrate (once or twice daily) or timolol maleate with potassium sorbate once daily as monotherapy or as a part of two-drug therapy.Materials and methods: Study was performed at seven clinical sites in the United States. Patients were surveyed on comfort upon instillation of timolol hemihydrate compared to timolol maleate with potassium sorbate.Results: A difference between timolol hemihydrate and timolol maleate with potassium sorbate for questions 1  and 4  was noted. There were no differences between treatment groups for any other question (p > 0.05).Conclusion: This survey suggests that timolol hemihydrate is associated with less stinging/burning and tearing than timolol maleate with potassium sorbate.How to cite this article: Stewart WC, Oehler JC, Choplin NT, Markoff JI, Moster MR, Ichhpujani P, Nelson LA. A Comfort Survey of Timolol Hemihydrate 0.5% Solution Once or Twice Daily vs Timolol Maleate in Sorbate. J Current Glau Prac 2013;7(1):11-16. At that time it was shown that it could be used, like timolol maleate, once daily with equal 24-hour efficacy as monotherapy or added to latanoprost.3Timolol hemihydrate  as an alternative once daily prescribed beta-blocker. This preparation contains potassium sorbate which enhances ocular bioavailability, allowing for once daily dosing.4 However, timolol maleate with potassium sorbate has been shown to sting upon instillation in approximately 42% of patients.5Several years ago a new timolol maleate preparation became available  the comfort upon instillation of timolol hemihydrate compared to timolol maleate with potassium sorbate.This was a prospective, multicenter, observational  study that was performed at seven clinical sites in the United States. The trial was registered with Clinicaltrials.gov, NCT00823043. This study was approved by the Institutional Review Board—Schulman Associates Institutional Review Board, Inc. (SAIRB), 4290 Glendale-Milford Road, Cincinnati, Ohio 45242—and adhered to the tenets of the Declaration of Helsinki. We included patients of open-angle glaucoma or ocular hypertension who were ≥21 years old and were currently prescribed timolol hemihydrate (once or twice daily) or timolol maleate with potassium sorbate once daily as monotherapy or as a part of two-drug therapy. With timolol hemihydrate if one eye was treated QD and the other BID, the QD eye was chosen as the study eye. Similarly, if dosed with two medicines in one eye and one medicine in the other, the lower dosed eye was chosen for the study.Appropriate consecutive patients who agreed to complete the survey were entered into the trial. After signing informed consent, subjects provided demographic information, medication and ophthalmic history, and then completed a 10-question survey about the comfort and influence on activities of daily living of their current glaucoma medication.Subjects who met any of the following criteria were not qualified to be enrolled in the trial: Inability to understand the trial procedures and thus inability to give informed consent. Inability to understand, read or write English. Best corrected visual acuity of 20/200 or worse in each eye. Current moderate to severe infectious or inflammatory condition of the eye or eyelids including: Blepharitis, conjunctivitis, keratitis or uveitis. Current moderate to severe dry eye syndrome. Current chronic use of ocular corticosteroids. Ocular surgery or intraocular laser surgery to either eye in the prior 3 months. Treated with three or more glaucoma medicines in both eyes.All data collected in the study were from the survey and no patient identifying information was kept. No randomization of treatment assignments was conducted but in general, investigational sites balanced the surveys collected between both groups of treated patients. Investigators were instructed regarding the protocol and related materials during a Site Initiation Teleconference conducted by the Clinical Research Associate (CRA) who had been trained by the CRO. Investigational sites were instructed to review each subject's survey to ensure all questions had been answered before collecting from the subject. The same CRA conducted a study close-out teleconference.No safety and efficacy variables were measured in the study. The endpoints evaluated were each subject's response to survey questions about their current timolol treatment.vs timolol maleate with potassium sorbate with a Student's t-test except question 9 which was evaluated with a Chi-square test.6 All analyses were two-tailed, with a p-value of 0.05 used to declare statistical significance.Patient survey shows the survey questionnaire. A 5 point scoring (0 to 4) was done for each of the questions. All questions were analyzed between timolol hemihydrate The study included 103 patients from seven centers. The demographic and baseline data is elucidated in The results for all patients for the survey questions are shown in vs timolol maleate with potassium sorbate, or mono- or adjunctive treatment with timolol hemihydrate vs timolol maleate with potassium sorbate. In contrast, no differences were observed for any question between timolol hemihydrate once daily vs twice daily treatment (p > 0.05)   to 7.7 Timolol hemihydrate eliminates the maleate buffer and simplifies the purification step for the R-enantiomer.Timolol maleate formulation combined with sorbate has the advantage of better penetration due to enhanced lipophilicity, but maleate is a salt of an organic acid that has several types of cellular effects. It reduces glutathione, a free radical scavenger, in both epithelial and endothelial cells and in corneal endothelial cells specifically.® is an isotonic, microbiologically preserved aqueous solution which has monosodium and disodium phosphate dihydrate to adjust its pH (6.5-7.5) and an osmolality of 260 to 320 mOsmol/kg. Timolol maleate too has a pH of 6.5 to 7.5 and an osmolality of 275 to 330 mOsmol/kg. Despite similar pH and osmolaity, stinging is more marked with the latter.BetimolThe reason for the differences between these medications in comfort were not clear by the results of this survey. Past research has also not been able to elucidate if the irritation from timolol maleate results from the active ingredient or the maleate. The lack of effect on activities of daily living may have been related to the resolution of any post-instillation stinging/burning or tearing before these daily activities needed to be performed.8Sonty S et al compared the symptoms and tolerability of once daily timolol hemihydrate 0.5%, timolol in sorbate 0.5%, and timolol gel-forming solution 0.5% in patients with glaucoma and/or ocular hypertension and found that the short-term (3-day) administration of timolol in sorbate was associated with more stinging or burning compared with timolol hemihydrate and timolol gel-forming solution.Our survey did not evaluate symptoms or signs after instillation or adherence in a randomized prospective clinical trial. Further research is needed to analyze anterior segment safety between these two beta-blockers as well as to describe the mechanism of stinging associated with timolol maleate with potassium sorbate.This survey demonstrates that topical timolol hemihydrate is associated with less stinging/burning and tearing than timolol maleate with potassium sorbate.