D,L-lactic-co-glycolic acid)-block-poly(ethylene glycol)-block-poly (PLGA-PEG-PLGA) was used as a sustained drug delivery carrier for metformin (MET) and levofloxacin hydrochloride (LFH). Both drugs and PLGA-PEG-PLGA copolymers could be easily dissolved in water at low or room temperature and the mixed solution could form a drug-loaded thermosensitive hydrogel in terms of body temperature response. The in vitro release investigation displayed a sustained release of MET and LFH from the formulation for one month. The in vivo efficacy of subconjunctival injection of the MET + LFH loaded thermosensitive hydrogel in inhibiting CNV was evaluated on a mouse model of corneal alkali burn. Compared with the single administration of MET or LFH loaded thermosensitive hydrogel, the MET + LFH loaded thermosensitive hydrogel remarkably inhibited the formation of CNV. The sustained release of MET and an antibiotic (LFH) provides synergistic therapeutic outcome. As a result, the co-delivery of MET and LFH using PLGA-PEG-PLGA thermosensitive hydrogel by subconjunctival injection has great potential for ocular anti-angiogenic therapy.Corneal neovascularization (CNV) is one of the major causes of severe disorders in ocular surface. Subconjunctival administration provides a localized and effective delivery of anti-angiogenic agents to inhibit neovascularization. In the present study, the ABA triblock copolymer of poly( Joe et al. , a typical drug used for the treatment of type 2 diabetes, works by stimulating the adenosine 5’-monophosphate (AMP)-activated protein kinase and influences the glucose uptake from the blood  under inflammatory condition , stannous octoate (Sn(Oct)2) of purity 95% and LFH were purchased from Sigma-Aldrich . D,L-lactide (LA) and glycolide (GA) were provided by Jinan Daigang Biomaterial Co., Ltd. , China) and recrystallized three times with reflux in toluene and dried under a vacuum for 24 h before use. MET was obtained from Selleck Chemicals . Proparacaine eye drops (0.5%) and 3% sodium pentobarbital were purchased from Santen Pharmaceutical Co., Ltd., . Human corneal epithelial cells (HCECs) lines were provided by the laboratory of Professor W. Chen . Cell Counting Kit was purchased from Peptide Institute Inc., . Chemicals and reagents for immunofluorescence and hematoxylin-eosin (H&E) staining were all provided by Service Biotechnology Co., Ltd. . Distilled water was prepared by the Milli-Q Plus system . All the chemicals used in this research were of analytical grade and without further purification.Poly were obtained from Guangdong Medical Experimental Animal Center . The mice were housed under specific pathogen free (SPF) standard condition  in the Animal Experimental Center of Zhongshan Ophthalmic Center, Sun Yat-Sen University, China. The animals were fed with a standard animal food and free access to water. The procedures for animal care and experiments complied with the Association for Research in Vision and Ophthalmology (ARVO) Statement Regarding the Use of Animals in Ophthalmic and Vision Research, and the guidelines of the Animal Experimental Ethics Committee of Zhongshan Ophthalmic Center, Sun Yat-Sen University, China.2.2.D,L-LA and GA initiated by PEG-OH as macroinitiator under dry nitrogen, with Sn(Oct)2 used as catalyst. Briefly, PEG was added into a branched-neck round bottom flask and dried under vacuum with stirring at 130 °C for 4 h. Subsequently, a certain amount of LA, GA, and Sn(Oct)2 were added under dry nitrogen. The reaction system was kept at 140 °C under stirring for 12 h. Then, the reaction system was cooled to room temperature under nitrogen atmosphere. The products were washed by 80 °C water to remove soluble low molecular weight polymers. The final product was dried by lyophilization and stored at −20 °C until use.The PLGA-PEG-PLGA triblock copolymers were synthesized through ring-opening polymerization of 1 H NMR, using a 400 MHz nuclear magnetic resonance (NMR) spectrometer . Deuterated chloroform (CDCl3) was used as the solvent and tetramethylsilane (TMS) was used as the internal reference to determine chemical shifts (δ). Molecular weight (MW) and molecular weight distribution (MW/Mn) of the copolymers were determined using a gel permeation chromatography (GPC) system  with a differential refractometer. Tetrahydrofuran was used as a mobile phase at a flow rate of 1 ml min−1. Narrow molecular weight distribution polystyrene standards were used for MW calculation.The chemical structure of the copolymers was determined by 2.3.μm filter at 4 °C.A certain amount of PLGA-PEG-PLGA copolymers (0.25 g) were added into stroke-physiological saline solution  to obtain 20 wt% polymer/water system at low temperature. Then, a given amount of MET  and 3 mg LFH were completely dissolved in the polymer/water system under ultrasound to form a homogeneous mixed system. The resultant solution was sterilized through a 0.22 G’) and viscous modulus (G’’) were recorded as a function of temperature.The sol-gel transition of the MET + LFH@Thermogel was detected using a HAAKE Rheometric  equipped with a 60 mm parallel plate. About 1 ml of polymer/water systems (20 wt % in SPSS) with/without drugs were injected in the parallel plate. The measurements were carried out at a fixed oscillatory frequency of 10 rad/s with a heating rate of 0.5 °C/min from 10 to 40 °C. Storage- was added into the centrifuge tube as the release medium. The shaking rate was 80 rpm. At predetermined time intervals, the release medium was withdrawn and replaced by the same amount of fresh PBS. The amount of released MET and LFH was determined by high performance liquid chromatography (HPLC) system (Waters1525) with a C18 column . For MET, the mobile phase was composed of methanol and 0.02 M phosphoric acid  at a flow rate of 1 ml/min and the temperature of 30 °C. And for LFH, the mobile phase was composed of acetonitrile and 0.02 M phosphoric acid  at a flow rate of 1 ml/min and the temperature of 40 °C. The accumulated release amount was calculated by the calibration curve.The drug loaded polymer/water system (1 g) was injected into a dialysis bag  and a centrifuge tube containing the dialysis bag was incubated in an air bath at 37 °C. After the formation of in vitro cytotoxicity of the PLGA-PEG-PLGA thermosensitive hydrogel and MET + LFH@Thermogel with different concentrations of MET  and the same concentration of LFH (3 mg/ml) were evaluated via Cell Counting Kit-8 (CCK-8) assay. HCECs(2.5 × 104) were cultured on 24-well plates at 37 °C with 5% CO2. The polymer/water systems loaded with/without drugs (0.1 ml) were added into transwell chambers and transformed into in situ gels at 37 °C. After 24 h incubation, the cells were washed with PBS, then the transwell chambers containing different concentrations of MET + LFH@Thermogel and PLGA-PEG-PLGA thermosensitive hydrogel were added to the 12-well plates. After another 24 h incubation, the medium was removed and the cells were washed with PBS and 10 μl of CCK-8 solution was added to the 24-well plates and reincubated at 37 °C for 1 h. The assay was performed by a microplate reader  at 450 nm to measure cell viability, and the cells without any treatment were served as the blank control.The 2.5.All procedures and tests were performed under general anesthesia by intraperitoneal injections of 3% pentobarbital (1 ml/kg) and topical administrations of 0.5% proparacaine. Alkali-burn injury to the cornea was induced by placing a sterilized filter paper (2 mm in diameter) soaked with 1 N sodium hydroxide solution for 30 s. Then, the ocular surface and conjunctival fornixes were extensively flushed with 10 ml of sterilized PBS solution. One day after alkali-burn injury, CNV were imaged under a slit lamp microscope . Subsequently, the CNV mice were randomly divided into 4 groups, the first group received single subconjunctival injections of PLGA-PEG-PLGA thermosensitive hydrogel without drugs, the second group received single subconjunctival injections of LFH loaded thermosensitive hydrogel (LFH@Thermogel); the third group received single subconjunctival injections of MET loaded thermosensitive hydrogel (MET@Thermogel); and the fourth group received single subconjunctival injections of MET + LFH@Thermogel .2.6.Seven days after alkali-burn injury, corneas were imaged by the slit lamp microscope  and the CNV areas were analyzed using ImageJ 1.40 image analysis software . According to the previous study, the vessel area (VA) was calculated by the using 2.7.μm-thickness. The sections were stained by H&E and examined under a light microscope . To analyze the expression of F4/80 and VEGF in corneas, the sections from paraffin-embedded corneas were deparaffinized, then they were exposed to ethylenediaminetetraacetic acid (EDTA) antigen repair buffer (pH 8) for antigen retrieval and blocked in albumin from bovine serum (BSA). The tissues were incubated with a rabbit polyclonal anti-F4/80 antibody and a goat polyclonal VEGF antibody separately overnight at 4 °C, then the sections were incubated with fluorescently labeled secondary antibody (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) or Donkey Anti-Goat IgG H&L (Alexa Fluor® 647)). Subsequently, the tissues were incubated with a 4’,6-diamidino-2-phenylindole (DAPI) solution and analyzed under fluorescence microscope .Mice were euthanized after seven days and then the excised corneas were imbedded in paraffin and sectioned to 4 2.8.β (IL-1β), IL-6, IL-17, Ang-2, VEGF-A, VEGF-C, and tumor necrosis factor (TNF-α) were quantified by Milliplex MAP Rat Cytokine/Chemokine Magnetic Bead Panel  and Luminex® xMAPTM technology , the data were analyzed by MILLIPLEX® Analyst 5.1 software  .On the seventh day after alkali-burn injury, mice were euthanized and the corneas were carefully enucleated, then the proteins were extracted using cell lysis buffer with the addition of protease inhibitors. The expression levels of mice interleukin-12.9.p < .05 (*), while p < .01 (**) and p < .001 (***) were considered as highly significant.All experiments were performed in triplicate. The data were statistically analyzed using SPSS Statistic 17.0 software . The data were expressed as the mean ± standard error. A statistically significant difference was shown when 3.3.1.1 H NMR spectrum and GPC curve of PLGA-PEG-PLGA copolymer are shown in 2–in PEG block. The chemical shifts at 5.2 and 1.6 ppm were assigned to –CH– and –CH3 in the LA unit, the 4.8 ppm was –CH2– in the GA unit, and the 4.2 ppm was –CH2– in ethylene glycol neighboring to the GA unit. Based on the calculation results of 1 H NMR spectrum, the block weight ratio of PLGA:PEG:PLGA was about 6:5:6. The GPC results showed the weight-average molecular weight (Mw) and Mw/Mn of the triblock copolymer were 6700 and 1.28, respectively. The 3.2.G’ and G’’ of the thermosensitive hydrogels loaded with/without drugs. As shown in G’ represents the elastic behavior or the elastic component of the complex shear modulus, whereas G’’ represents the viscous behavior. The G’ values were lower than G’’ at low or room temperature, demonstrating the mixed solution was free-flowing and suitable for injection. With the increase of temperature, both G’ and G’’ increased, and G’ was equal to G’’ at approximately 31 °C. The sol-gel transition was observed by naked-eye as well. After the formation of in situ thermosensitive hydrogel, conversely, the G’’ was lower than G’, and both G’ and G’’ reached maximum near physiological temperature of 37 °C. Moreover, there was no obvious difference in rheological behavior between the four groups, indicating that the addition of MET and LFH did not affect the sol-gel transition of the thermosensitive hydrogel. It is beneficial for ocular drug delivery of such formulations.Dynamic rheological measurements were performed to assess the changes in in vitro drug release profiles of MET + LFH@Thermogel were displayed in in vitro release profile demonstrated the MET + LFH@Thermogel can provide an extended and sustained drug release for the treatment of ocular disorders.The in vitro cytotoxicity of PEG-PLGA-PEG thermosensitive hydrogel and MET + LFH@Thermogel with different drug concentrations was evaluated via CCK-8 assay. After incubation of the PEG-PLGA-PEG hydrogel for 24 h, the cell viability was approximately 100%, indicating that the thermosensitive hydrogel exhibits very low cytotoxicity and good biocompatibility. With the MET concentration increasing from 1–5 mg/ml, the cell viability of MET + LFH@Thermogel decreased from 96 to 80%. This decreased trend was attributed to the high concentration of MET and the cell viability was dependent on drug concentration. These results implied that the MET + LFH@Thermogel with long-term release and good biocompatibility is appropriate for ocular drug delivery.The 3.4.in vivo evaluation, which was based on the considerations of drug dosage required to inhibit CNV and anti-infection, the in vitro drug release profile and the in vitro cytotoxicity of MET + LFH@Thermogel. The drug concentrations of LFH@Thermogel and MET@Thermogel were 3 mg/ml as well. The anti-angiogenic effect of conjunctival injection of LFH@Thermogel, MET@Thermogel, and MET + LFH@Thermogel were first evaluated on mice with CNV. As shown in 2, respectively. The MET + LFH@Thermogel group markedly reduced the area of CNV more than the other groups. Mice treated with MET + LFH@Thermogel led to ∼88% reduction in CNV area compared with blank control group. The efficacy of local ocular drug delivery using thermosensitive hydrogel is much better than topical administration and conventional intraocular injection . Besides, VEGF staining was also analyzed to demonstrate the anti-VEGF effect of MET + LFH@Thermogel. Although MET alone suppressed VEGF to a lesser extent (p < .05). Previous studies revealed that macrophages play an important role in neovascularization by secreting matrix metallopreoteinase-2 (MMP-2), MMP-7, MMP-9, MMP-12, and cycloocygenase-2 , expressed by macrophage, can regulate the expression of VEGF by binding IL-1R. Factors related to the biological behavior of macrophages during inflammation may affect the extent of CNV . LFH expressed strong inhibitory effect on inflammatory cytokines levels , although MET has a certain inhibitory effect on the expression of IL-6. The inflammatory cytokines levels in MET + LFH@Thermogel treated group were significantly lower than those in LFH@Thermogel treated group (p < .05). This result indicated that the treatment of MET + LFH@Thermogel has the most significant effect on reducing the inflammatory cytokines, which is consistent with our results of macrophage infiltration shown in We further detected the concentrations of inflammatory and angiogenic cytokines in corneas after treatment, including Ang-2, IL-14.in vitro for one month and the drug release profile was able to be modulated by the drug loading amount. In vitro evaluation of cytotoxicity confirmed the low toxic and good biocompatibility. Moreover, in vivo results further demonstrated the sustained co-delivery of MET + LFH reduced the neovascularization area and suppressed the expression levels of inflammatory and angiogenic cytokines, indicating that such a system is more effective in the treatment of neovascularization. In summary, this novel MET + LFH@Thermogel system, which provides a localized, efficient, and cost-effective manner, has great potential for the treatment of ocular neovascularization.In this study, we developed a sustained co-delivery system of MET and LFH using thermosensitive PLGA-PEG-PLGA hydrogel by subconjunctival injection to inhibit CNV. The free-flowing drug loaded polymer/water system could be prepared by simply mixing at low or room temperature and it exhibited a sol-gel transition to form a drug delivery gel depot at body temperature. The prepared MET + LFH@Thermogel exhibited a sustained drug release


This paper proposes a simple model for predicting the development of deleterious expansion from alkali-silica reaction (ASR) in long-service concrete structures. This model is based on some composition and reactivity parameters related to ASR, including the long-term alkali contribution by aggregates to concrete structures. This alkali contribution was estimated by means of a laboratory extraction test, appositely developed in this study in order to maximize the alkali extraction within relatively short testing times and with low leaching solution/aggregate ratios. The proposed test is a modification of the Italian Standard test method UNI 11417-2  and it consists of subjecting an aggregate sample to leaching with saturated calcium hydroxide solution in a laboratory autoclave at 105 °C. Nine natural ASR-susceptible aggregates (seven sands and two coarse aggregates) were tested and the following optimized test conditions were found: leaching solution/aggregate weight ratio = 0.6; solid calcium hydroxide/aggregate weight ratio = 0.05; test duration = 120 h. The results of the optimized alkali extraction tests were used in the proposed model for predicting the potential development of long-term ASR expansion in concrete dams. ASR predictions congruent with both the field experience and the ASR prevention criteria recommended by European Committee for Standardization Technical Report CEN/TR 16349:2012 were found, thus indicating the suitability of the proposed model. Deleterious expansions associated to Alkali-Silica Reaction (ASR) have been repeatedly reported in the last decades among the main causes of deterioration of concrete structures ,2.ASR is a slowly expansive reaction between certain forms of alkali-reactive silica  and/or certain silicate minerals present in concrete aggregates and the hydroxyl ions in concrete pore solution, mainly associated with the alkaline metal ions (sodium and potassium) ,4,5. ThiMost of the alkalis are available in the concrete since the construction phase of the structures and come primarily from the cement, with eventual minimal contributions from the mix water and the chemical admixtures. However, enhancement of the alkali concentration in concrete may arise during the service life of concrete structures when aggregates with alkali-bearing minerals  release their alkalis into the concrete pore solution, over a long time ,10,11,12The concrete alkali enrichment is a consequence of the alkaline cations release from aggregates. In order to maintain the electroneutrality of the pore solution, it is assumed that the hydroxide ion concentration increases as well , thus trThis chemical mechanism is generally accepted to explain the observed cases of discrepancy between measured and expected alkali contents in field concrete structures showing late or progressive ASR-induced distress, such as, for example, the case of concrete dams, in consideration of their very high content of aggregates and their required long service life. In concrete dams containing alkali-reactive aggregates, deleterious ASR expansion may occur long time after construction (initiation phase of more than 20 years) even if low-alkali Portland cement is used as an ASR preventive measure . Slowly However, based on their own experimental results, some authors  recentlyEnhancement of the alkali concentration in concrete may also arise during the service life of concrete structures when they are exposed to external sources of potential alkalis  ,24,25.Differently from the alkali release from concrete aggregates, the alkali contribution from external sources concerns only some concrete structures and it is mainly localized at structure surface layers of some components . Moreover, it is very difficult to quantify this contribution in terms of increased concrete alkali content, also due to surface leaching phenomena . TherefoFor such structures, the knowledge of the amount of alkalis releasable from aggregates represents an issue of particular concern in developing models aimed at predicting eventual long-term deleterious ASR expansion of mass concrete.Considering that the determination of the alkali release from aggregates in the field conditions is very difficult, laboratory test methods have been proposed in the literature for estimating the long-term alkali contribution by aggregates to concrete structures.+ and K+ ions, and solid calcium hydroxide dissolve, thus supplying OH- ions to maintain the solubility equilibrium and electroneutrality with the alkali ions. This ion exchange with Ca+2 can potentially increase the alkali leaching [These tests are directly carried out on concrete aggregates, by using caustic solutions such as saturated calcium hydroxide solution or 0.7 M NaOH and 0.7 M KOH solutions, as extraction media. The use of saturated calcium hydroxide solution at boiling temperature is specified by the Italian standard test method UNI 11417-2 (test duration 6 h)  and the leaching .The above-mentioned leaching tests provide different results, depending on the aggregate fineness, the solution adopted, the operating conditions . If carried out at the same temperature, saturated calcium hydroxide solution is much less aggressive compared to sodium and potassium hydroxide solutions, due to a lower pH of the solution and the formation of reaction products able to incorporate some of the released alkali ions ,30. HowePresently, alkali releases obtained through all laboratory tests proposed in the literature still need to be verified with those in field conditions, and procedures for this verification need to be developed. By the way, this is within the present plan of activity of RILEM TC 258 AAA.In the present study, in order to maximize the alkali release from concrete aggregates within relatively short testing times and with low leaching solution/aggregate ratios, an appositely modified version of the current Italian UNI standard test method was developed using nine natural ASR-susceptible aggregates (seven sands and two coarse aggregates) with different petrographic and alkali-reactivity characteristics.Moreover, a simple model was developed to predict the potential effect of alkali release from the aggregates investigated on the development of deleterious ASR expansion in long-service structures, such as concrete dams. In this model, the results of the proposed alkali extraction test were used together with some parameters that are characteristic of alkali-silica reaction and related to the composition of concrete.The proposed model was based on the knowledge of four key parameters: (1) the initial alkali content of the concrete mix used for the structure construction, 2Oeq/m3) is defined as the minimum alkali content of a standard concrete mix made with Portland cement and the aggregate under examination, above which deleterious expansion of concrete will occur. The higher is the aggregate reactivity, the lower the As reported in our previous papers ,34, the contents .2Oeq/m3) is defined as the maximum alkali consumption by a blended cement in correspondence of the maximum concrete alkali content, The efficacy parameter xpansion ,36. This cements ,38 and cIn concrete structures containing ASR-susceptible aggregates, deleterious expansion will occur when the amount of alkali-silicate gel formed by alkali-silica reaction and the concomitant water absorption by this gel are sufficient to develop deleterious swelling pressures.2Oeq/m3) not producing deleterious ASR expansion in concrete can be expressed as:Based on the 2Oeq/m3), related to the alkali contributions by the various concrete mix components  can be expressed as:2Oeq/m3) is the alkali content of the concrete mix at the time of structure construction  and 2Oeq/m3) is the long-term alkali contribution from the aggregates.The concrete alkali content,The value of As anticipated, the value of According to Equations (1) and (2), deleterious ASR expansion in concrete will develop only if the following condition is satisfied:Combining Equations (1) and (3) yields:Equation (4) holds for blended cements containing active mineral additions  and the long-term alkali contribution by aggregates , the other three sands, known for their high alkali contents, came from Norway, Portugal and Spain . Two coarse aggregates (size gradation 12–32 mm), coming from the same quarries of sands S1 and S2 and exhibiting similar lithological composition as the respective sands, were also tested and designated as aggregates C1 and C2, respectively.According to the RILEM AAR-1 petrographic method , all the3 solution; (4) appropriate dilution of the resulting solution with deionized water and determination of the sodium and potassium ion concentrations by flame Atomic Absorption Spectrophotometer (AAS) according to the procedure reported in [Each aggregate was analyzed for the contents of sodium and potassium by the following test procedure: (1) grinding of the aggregate sample to fineness below 45 μm; (2) alkaline fusion of a powdered aggregate-lithium metaborate mixture (weight ratio 1:6) in platinum crucible at a temperature of 1000–1200 °C; (3) quenching of the melt with water and dissolution of the solid with 10% HNOorted in .2O, K2O, Na2Oeq)/kg dry aggregate.Prior to leaching tests , all the aggregates were characterized for their grain size distribution by dry sieving. The two coarse aggregates were used as received, while the different sands were used with the same grain size gradation that was obtained by appropriate recombination of the various grain size fractions.As anticipated, the release of alkalis from aggregates was evaluated by using the extraction test method reported in the Annex A of the Italian Standard UNI 11417-2 , both inct, of 6 h, at the boiling temperature. The weight ratio between the saturated Ca(OH)2 solution (leachant) and the dry aggregate sample (L/S ratio) is equal to 0.6 g leachant/g dry aggregate and the weight ratio between solid calcium hydroxide (CH) and dry aggregate (CH/S ratio) is equal to 0.05 (5 g CH/100 g dry aggregate). The solubilizing attack is due to the OH- ions action on the siloxane and silanol groups of the reactive silica contained in the aggregate and to the subsequent ion exchange between alkaline metals (Na and K) and calcium ions arising from calcium hydroxide with formation of NaOH and KOH (base exchange) [The current UNI 11417-2 test method consists of subjecting the aggregate sample under examination to a solubilizing attack by reflux with a saturated calcium hydroxide solution for a contact time, xchange) . TherefoIn the modified version of the UNI test method, the glass boiler equipped with the reflux condenser was replaced by a laboratory autoclave having a volumetric capacity of 1.3 L  placed iUsing the two different equipment (glass boiler or autoclave) and the different amounts of aggregate, leaching tests on sand S1 were first performed under the test conditions, specified in the UNI standard test method.The main reasons for the choice of testing sand S1 were the great availability of this material in both fine and coarse grain size gradations and its known relatively high alkali-reactivity in field conditions.ct, over the range from 6 to 240 h.Successively, leaching tests with the above test modifications were performed on sand S1 by changing the CH/S ratio from 5 to 16 g CH/100 g dry aggregate, the L/S ratio from 0.6 to 3.3 g leachant/g dry aggregate and the contact time, At the end of each leaching test, the autoclaved sample was rapidly cooled, filtered on 0.45 μm paper filter and the solid residue was washed with deionized water in order to quantitatively remove the leachate. The resulting solution (filtrate + washing) was acidified and then analyzed for the concentrations of sodium and potassium ions by AAS. In some cases, pH measurements were also performed on filtrate solutions and blank samples .ct values were also performed on the other aggregates investigated in order to better characterize their alkaline metal releases.Finally, under the optimized CH/S and L/S ratio conditions, leaching tests at different In the case of coarse aggregates C1 and C2, due to their much lower specific surface area as compared to the respective sands, the release of sodium and potassium was evaluated only at the ultimate testing time investigated (240 h). Moreover, in order to guarantee the complete immersion of coarser grains of the aggregates in the leachant solution, the L/S ratio was equal to 3.3 g leachant/g dry aggregate, corresponding to the upper value of the L/S ratio range investigated for sands. The CH/S ratio was kept equal to 5 g CH/100 g dry aggregate.For each aggregate and test condition considered, three replicate tests were performed and the results obtained were averaged. For each test specimen, the average value was obtained from measurements on at least three solution sampling, by discarding results giving a coefficient of variation higher than 10%.ct) were adopted. The releases of sodium or potassium, expressed in terms of mg metal released/kg dry aggregate, The results of preliminary leaching tests on sand S1 showed that the release of sodium and potassium determined according the UNI standard test method  or the modified version of the UNI standard test method  were similar where the same operating conditions .rM lower than about 12% for both sodium and potassium), at least within the range of CH/S values considered (5–16 g CH/100 dry aggregate). This was because the amount of solid CH used in the leaching test was largely in excess of the quantity needed to maintain the lime saturation condition in the leaching solution throughout the test.These results indicated that the CH/S ratio did not significantly affect the alkaline metal release from the aggregate  and CH/S ratio (5 g CH/ 100 g dry aggregate) of the UNI Standard test method. Maximum percentage variations of about 14% for sodium and 8% for potassium were observed.As shown in ct, irrespective of the L/S ratio used. This effect is highlighted by ct curves for K and Na are reported for two different L/S ratios (0.6 and 3.3 g leachant/g dry aggregate) and the same CH/S ratio (5 g CH/100 g dry aggregate). Exception made for the series , for which the points were interpolated with a polynomial equation by the method of least squares (R2 = 0.9782), all the curves in this figure were drawn joining experimental points. For both sodium and potassium, the ct curves exhibited an asymptotic trend. Values of In contrast, the release of the two alkaline metals was found to greatly increase with increasing contact time, ct = 6 h.As expected, at long contact times (120–240 h), the Based on the results of ct, in order to evaluate the alkaline metal releases from these sands and, at the same time, to confirm the asymptotic trend of the ct curves.Using the values of CH/S = 5 g CH/100 g dry aggregate, and L/S = 0.6 g leachant/kg dry aggregate of the UNI standard test method, leaching tests were performed on the other six sands by varying the contact time, ct curves obtained for sodium and potassium relevant to sands S2–S7. For comparison, the results relevant to sand S1  and the reduction of the amount of aggregate tested (from 400 g to 200 g), the only substantial modification of the operating parameters of UNI standard test method consisted of prolonging significantly the duration of the extraction test from 6 h to 120 h.ct = 120 h). In this table, the percentage release (wt %) of sodium and potassium and the alkali release, 2O, K2O, or Na2Oeq/kg dry aggregate are also reported.The results in ng media ,30,31.− ions consumed by ASR, through lime dissolution, balancing alkaline metals released by aggregates.In order to confirm the assumed mechanism of alkali release reported in the Introduction section, pH measurements were performed on filtrate solutions obtained for sands S1 and S5, whose relatively high reactivity in the field was known. The measurements performed at different contact times between solid specimens and saturated calcium hydroxide solution revealed negligible changes in the pH values (pH = 12.16–12.36) in comparison to blank samples (pH = 12.22–12.32). Due to the alkali reactivity of these sands, a reduction of pH would have been expected with increasing testing time. Therefore, the results obtained appeared to be consistent with the hypothesis of a progressive replacement of OHct = 240 h). In this table, the percentage releases of sodium and potassium and the alkali releases  were found to be much lower than those obtained for the respective sands .2Oeq/kg dry aggregate) from coarse aggregates and sands coming from the same source ranged from 0.13 to 0.14.Comparing the data in These results show how important is the role of the specific surface area of aggregate in the releasing process by attack of the saturated calcium hydroxide solution, as already pointed out in the literature .3 depending on the type of structure) and the high percentage of the coarse aggregate in the combined aggregates (55–65% by weight).Although the release of alkalis from coarse aggregate is very low, this release cannot be neglected in calculating the alkali contribution by combined aggregate (sand + coarse aggregate) to concrete mix. This is because of the high combined aggregate content in the concrete mixes –(7) yields:The percentage increase of sand content Using the As can be noted from Using the average The values of The model described in 3; water content = 120 kg/m3; aggregate content = 2100 kg/m3, of which 35% as sand and 65% as coarse aggregate. Use of two types of cement was considered: a Portland cement (CEM I) with Na2Oeq = 1.0% and a pozzolanic cement made with natural pozzolan (CEM IV/B-P) with Na2Oeq = 2.20% and 2Oeq/m3 [A typical concrete mix composition for concrete dams is as follows: cement content = 200 kg/ma2Oeq/m3 .2Oeq/m3 for Portland cement concrete and 4.4 kg Na2Oeq/m3 for pozzolanic cement concrete.Based on the assumed concrete composition, the An alkali release from coarse aggregate equivalent to that resulting from an increase of the sand content equal to 23% by weight  was cons2Oeq/kg dry aggregate (2Oeq/m3) values were calculated for each combined aggregate.Using the values of alkali releases from each sand determined by laboratory extraction tests and expressed in terms of g Naggregate  and the In The lable in , those olable in  while thorted in . No info2Oeq/m3) were much lower than those obtained for the three aggregates coming from other countries (0.62–1.12 kg Na2Oeq/m3). This was related to the lower initial alkali content of sands S1–S4 (1.07–2.62% as Na2Oeq), compared to sands S5–S7 (4.44–6.41% as Na2Oeq)  .2Oeq/m3), the significance of such contributions was very different: marginal significance for concretes made with aggregates 1–4  obtained from petrographical examination .Comparing the 2Oeq/m3 was obtained, corresponding to an alkali content of cement equal to 0.84% Na2Oeq, for concrete mix with 200 kg/m3 of cement.In the case of the Portland cement concrete mix made with aggregate 5, for which deleterious ASR expansion was predicteted in the long term, Equations (2) and (4) were used to calculate the maximum 2Oeq/m3) in concrete made with aggregate 5 (2Oeq/m3) would yield a 2Oeq/m3 that was much lower than the 2Oeq/m3 The modified alkali extraction test with saturated calcium hydroxide solution is suitable for maximizing the alkali extraction from concrete aggregates. With respect to the Italian Standard test method UNI 11417-2, the modifications consist of replacing the glass boiler with reflux by a laboratory autoclave operated at 105 °C, reducing the amount of aggregate tested from 400 to 200 g, and prolonging the test duration from 6 to 120 h. No change of the liquid/aggregate ratio (L/S = 0.6 g leachant/g dry aggregate) and the solid lime/aggregate ratio (CH/S = 5 g CH/100 g dry aggregate) was found to be necessary.(2)With the use of the modified extraction test, the amounts of alkaline metals released from the sands investigated were found to vary from 46 to 690 mg/kg dry aggregate for sodium and from 71 to 576 mg/kg dry aggregate for potassium, corresponding to percentage releases of 0.93–2.84% for Na and 0.41–3.35% for K. These results are congruent with those available in the literature for concrete aggregates subjected to leaching tests using sodium hydroxide and potassium hydroxide as leaching media.(3)For coarse aggregates, much lower releases of alkaline metals were found (6–11 mg/kg dry aggregate for both sodium and potassium), due to their lower specific surface area, compared to the respective sands. However, these very low releases may not be neglected in calculating the alkali release from combined aggregates (sand + coarse aggregate), in consideration of the very high content of coarse aggregates in concrete mixes. As demonstrated in this study, if the alkali release from sand is only known, it is possible to estimate the alkali release from coarse aggregate in terms of an equivalent sand content of 23 wt %.(4)A simple model is proposed to predict the potential effect of alkali release from aggregates on deleterious ASR expansion development in long-service concrete structures. This model is based on the knowledge of four key parameters relevant to the components of the concrete mix, such as the initial alkali content of the mix used for the structure construction ((5)Application of the above model to a typical dam concrete mix leads to ASR expansion predictions that are congruent with both the field experience and the ASR prevention criteria recommended by European Technical Report CEN/TR 16349:2012 and by RILEM Specifications, thus indicating the suitability of the proposed model.Based on the results of this study, the following conclusions can be drawn:


The emergence, spread, and expansion of antibiotic resistance and increasing restrictions on the use of antibiotics in animal agriculture have created a need for efficacious alternatives that remains unmet. Prioritizing research needs in the development of alternatives is key to ensuring that scarce research resources are dedicated to the most promising approaches. However, frameworks to enable a consistent, systematic, and transparent evaluation of antibiotic alternative candidates are lacking. Here, we present such an evaluation framework. Traditional antimicrobial drugs, or antibioticsFor the purpose of this study, and consistent with other established definitions , altPublic and private sector funding for research on antibiotic alternatives in animal agriculture is scarce . PrioritA pre-workshop survey of over 40 experts in animal agriculture identified antibiotic alternatives as the top research priority related to antibiotic stewardship, prompting its selection as the workshop topic (details available upon request). For the purposes of the workshop, antibiotic alternatives were defined broadly as animal feed additives  as well as animal drugs  and veterinary biologics  that prevent, control, or treat infectious diseases. In contrast, management practices such as nutritional or backgrounding strategies, improvements in housing, or more stringent biosecurity were excluded from the discussion.For the workshop, a panel of 23 subject matter experts from academia, industry, governmental, and non-governmental organizations convened for a 1 day in-person meeting in December 2018. The goal was to identify strategic priorities for funding research and development on antibiotic alternatives in animal agriculture. The workshop explored factors critical to the success or failure of new antibiotic alternatives and identified associated data gaps and research needs that, if addressed, could help grow the pipeline of safe and effective product candidates. The workshop consisted of two facilitated discussions to reach consensus on key factors important in the evaluation of research approaches and funding decision-making for antibiotic alternatives. This manuscript highlights key themes that emerged during the workshop and, in certain instances, develops them further. All workshop participants were given an opportunity to review this manuscript before publication.Successful antibiotic alternatives solve a substantial real-world infectious disease problem and provide an economic and animal health benefit. An entity or entities must be willing to invest in scientific research to bring the product to market, and someone must be willing to purchase and use it. However, that alone is not sufficient for successful adoption. The ability of farmers and veterinarians to use the product relies on additional factors such as the logistics of delivery and storage and whether the product aligns with their own and their customers' values and expectations. Ultimately, many factors influence whether an alternative is successful. Profitability is foundational to the success of an antibiotic alternative; farmers and veterinarians cannot adopt economically unsustainable products. Similarly, without a viable business model, investors and pharmaceutical companies are unlikely to provide sufficient funding to bring the concept to market. Economic viability is therefore the first framework criterion, although it can be difficult to predict. For instance, in 2018, <2 years after gaining FDA approval, the animal pharmaceutical company that developed Imrestor®, an antibiotic alternative addressing mastitis in dairy cattle, decided to suspend its commercialization , 8.To determine economic viability, animal health companies and investors evaluate the potential product's expected revenue and probability of success, compared to anticipated costs and risks . These eThe initial discovery and development of a new animal health product typically incurs substantial costs, as outlined in To predict product revenue, investors analyze both the market and the product's expected performance in it. The predicted market size for an antibiotic alternative ultimately depends on the number of farms and animals affected by the disease and on how likely the producer or veterinarian is to proactively take steps to address it through prevention, control, or treatment. Economic factors play a role here as well, including short term disease impacts as well as long-term consequences on animal health and productivity , 11. MasOther market characteristics, such as segmentation of the existing market and predicted market growth, factor into the revenue calculation as well. In addition, market access may be greater in more highly integrated industries and for products with more internationally harmonized regulatory requirements.Product specific considerations include the expected return on investment (ROI) for the livestock producer, and the competitiveness of the product compared to alternatives. As outlined in Even if these initial economic considerations are favorable, an antibiotic alternative candidate may fail for many reasons.In vitro and in silico models have been developed to help assess the pharmacokinetics and predict the safety of veterinary drugs  or else the rates of natural disease occurrence are observed, and whether animals are managed under real world conditions . More tightly-controlled studies—such as those experimentally infecting a small number of healthy, genetically homogenous animals with one pathogen strain at one point in time, can use smaller experimental group sizes for statistical significance than less closely controlled studies, but they often do not adequately capture population-level variations that can impact efficacy. For instance, the experimental animals may be more uniform with regard to factors such as age, breed, health status, management, and disease history than animals in commercial settings (Predicting product efficacy early in R&D can be challenging.  studies –30. Howesettings . Study cin vivo studies are scarce—an issue that is compounded by lack of information regarding the products' mechanism of action (For many antibiotic alternatives, conclusive data from large, well-controlled f action , 32–34. f action . In the f action , 36. WheThe acceptability of new alternatives by farmers and veterinarians, who often have vast experience using antibiotics, is also key to success. Studies have shown that many farmers and veterinarians are skeptical about the efficacy of antibiotic alternatives –39. BehaThe success of an alternative also requires that the animal products derived using the alternative are acceptable to consumers. Generally speaking, the biological function of an alternative should be easy to explain to a layperson and must align with consumers' beliefs and expectations concerning food production and their conceptualizations of risk and adulteration. Alternatives may be preferable over antibiotics to some consumers, to the extent that they alleviate concerns regarding their use . In factThe widespread adoption of an antibiotic alternative requires that they be practical to use for farmers and veterinarians. This means any such product must be integrated into current production practices without causing major disruptions. Products that require disruptive shifts in the infrastructure or systems under which livestock commodities are raised are unlikely to succeed, at least in the shorter- to medium-term. In addition, products that are not readily compatible with current animal production practices—for instance, because of their application frequency, mode of administration, stability or timing of use—may face obstacles to adoption. Side-effects of the product or the stress associated with handling an animal to apply the product may also reduce adoption. As with antibiotics, farmers may need to observe specific withdrawal times that may limit their ability to market animal-derived products.Antibiotic alternatives represent a major unmet need for the livestock sector. However, the factors predicting their success or failure are complex. Here, we outline a framework for the evaluation of alternative candidates that may empower federal agencies, philanthropic organizations, and other key stakeholders to consistently and transparently prioritize investments in antibiotic alternatives. Our framework first considers the overall costs and benefits related to the new alternative, because economic viability is foundational to ultimate commercial success and this information may be readily available prior to or early during R&D.Ultimately, bringing an alternative to market is an extremely complex process, involving evaluation of product safety, efficacy, acceptability and practicality. Therefore, the potential success of a new alternative may be best evaluated from multiple perspectives, an approach that we replicated in our original survey and workshop design and encourage in the evaluation of alternatives. Research funders may, for instance, start to involve farmers, veterinarians and farm advisors more closely in early funding decisions. Developing new antibiotic alternatives is a challenging issue but holds considerable promise for animal health and the fight to combat antibiotic resistance. This framework will empower research funders to evaluate alternatives early during R&D, and to dedicate scarce funding to the most promising alternatives.The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


It has been suggested that manipulation of gut microbiota using antibiotics can inhibit colitis-associated colorectal cancer (CAC) in a mouse model. We investigated whether timing of gut microbial manipulation using antibiotics affects colon tumorigenesis in the azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced CAC model. CAC was induced in C57BL/6 mice by injection of 12.5 mg/kg AOM followed by three rounds of 1.7% DSS exposure. There were six groups based on timing of antibiotic administration. Colonic inflammation, proliferation, and tumorigenesis were evaluated after animal sacrifice. High-throughput sequencing of the mice feces was performed to characterize changes in gut microbiota. Full-time antibiotic treatment significantly decreased the number and size of tumors, histological scores, and expression of pro-inflammatory cytokines compared to the AOM/DSS group without antibiotic treatment. The early and late antibiotic groups, antibiotic administration from the first and second rounds of DSS to the end of the study, showed significantly lower histological scores and tumor burden. In contrast, the pretreatment antibiotic group, antibiotic administration from 3 weeks prior to AOM to the first round of DSS, did not exhibit decreased tumorigenesis. Principal coordinate analysis showed similar gut microbial community structures among the full-time, early, and late antibiotic groups, whereas other groups showed distinct gut microbial profiles. There was a positive correlation between number of tumors and number of operational taxonomic units. Colonic tumorigenesis was attenuated by antibiotic administration, except for that only prior to DSS administration, suggesting that gut microbial changes should be maintained throughout the entire period of inflammation to suppress tumorigenesis. Inflammatory bowel disease (IBD), characterized by chronic relapsing inflammation of the gastrointestinal tract, has emerged as a substantial public health challenge worldwide . IBD cauEscherichia coli, Bacteroides fragilis, and Fusobacterium nucleatum, have been reported to be associated with colonic tumorigenesis; however, a single causative organism has not yet been identified ..10].Total RNA was extracted from colon tissues using Hybrid-R Total RNA Isolation Kit  and quantified using a Biospec-nano spectrophotometer . RT-PCR was performed 30 times at 95°C for 2 min, 95°C for 30 sec, 55–65°C for 30 sec by each primer, 72°C for 1 min, and 72°C for 5 min. The PCR products were resolved by electrophoresis on 1.5% agarose gels that contained Safe-Pinky DNA gel staining solution (GenDEPOT), and bands were visualized using a ChemiDoc XRS+ System . The primer sequences used for PCR are shown in Mouse fecal samples were collected before antibiotic administration and before the mice were sacrificed and were immediately stored at -80°C. To extract bacterial DNA, bacterial walls were pulverized using the phenol/chloroform extraction and bead beating method, and the purified DNA was extracted using PowerFecal DNA Isolation Kit .TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG; reverse, GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC. Gene amplification conditions were initial denaturation at 95°C for 5 min, denaturation 35 times at 95°C for 40 sec, primer annealing at 57°C for 40 sec, extension at 72°C for 60 sec, and final elongation at 72°C for 60 sec. Amplified 16S rRNA PCR products were quantified, purified, and sequenced using the Miseq platform (Illumina). The short or extra-long reads in the sequences were trimmed, and the filtered sequence was classified using CD-HIT-DUP. Chimeric reads were identified, and small noise sequences were removed. Sequences with 97% accordance among the remaining representative readings were classified as operational taxonomic units (OTUs). We then assigned a taxonomy for each OTU representative sequence using the QIIME pipeline. Alpha diversity of each group was compared with Chao 1 richness index. In addition, to determine the total bacterial load in the gut, quantitative 16S rRNA PCR on stool samples was performed. The real-time PCR was performed by the 7500 Real-Time PCR System  in a 2-step procedure using TOPreal qPCR 2X PreMIX . The following primers were used: forward, GTGSTGCAYGGYTGTCGTCA; reverse, ACGTCRTCCMCACCTTCCTC. All reactions were performed in a 96-well plate using the following cycling conditions: 40 cycles of 95°C for 30 sec, 63°C for 30 sec, and 68°C 1 min. Using the Ct (ΔΔCt) method, the value of each control sample was set at 1 and used to calculate the fold-change of target genes. Principal coordinate analysis was performed to evaluate the structural changes of the gut microbial community. The complete genome sequence data have been deposited in the NCBI Sequence Read Archive under BioProject accession number PRJNA560633.To amplify the extracted DNA, primers for the V3-V4 region of the 16S rRNA gene were used as follows: forward, p value <0.05 was considered statistically significant. All statistical procedures were conducted using IBM SPSS Statistics 20.0  and R .Continuous and categorical variables were compared among groups using the Mann-Whitney test and the chi-square or Fisher’s exact tests, respectively. A The overall weight of the mice in the study decreased at the onset of antibiotic treatment and during DSS administration. Weight loss due to the antibiotic treatment gradually recovered over time. At the end of the study, there was no significant difference in body weight between groups .p = 0.016). All groups that received antibiotic treatments had longer colonic lengths compared with the AOM/DSS group .p<0.001). The decrease in number of tumors was more prominent in the full-time antibiotic group than in the late antibiotic group . In contrast, there was no significant difference in number of tumors between the pretreatment antibiotic group and the AOM/DSS group   .These results indicate that the degree of suppression of tumorigenesis varies according to the timing of microbial manipulation with antibiotics.p<0.001). However, there was no significant difference in histologic score between the pretreatment antibiotic group and the AOM/DSS group . Contrary to the results of the most marked reduction of tumor burden in the full-time antibiotic group, the alleviation of colonic inflammation was most pronounced in the early antibiotic group. Overall, however, the histologic scores and number of tumors were positively correlated (Pearson correlation coefficient R2 = 0.585).These results demonstrate that the alleviation of colonic inflammation also varies with the timing of antibiotic administration, and there was a positive correlation between colonic inflammation and tumor burden.The mRNA expression profiles of TNF-α, IFN-γ, IL-17A, and IL-6 were increased and higher in the AOM/DSS group than in the control group. Antibiotic treatment decreased mRNA expression of proinflammatory cytokines in the colonic tissue explants. The full-time, early, and late antibiotic groups showed significant reductions in mRNA expression of TNF-α and IL-17A compared with the AOM/DSS group. The expression of IFN-γ was significantly decreased in the full-time and early antibiotic groups, but not in the late antibiotic group. On the other hand, expression of IL-1β, IL-22, and IL-6 was significantly decreased in the early antibiotic group but not in the full-time antibiotic group. In addition, there was no significant difference in mRNA expression of any cytokines between the pretreatment antibiotic group and the AOM/DSS group .These results demonstrate that the antibiotic administration reduce the expression of pro-inflammatory cytokines such as TNF-α, IFN-γ, and Th17 cytokines. The expression of cytokines differed according to the timing of antibiotic administration.At the phylum level, the relative abundance of Proteobacteria was significantly higher in the full-time, early, and late antibiotic groups than the other groups. Principal coordinate analysis showed similar gut microbial community structures among the full-time, early, and late antibiotic groups. In contrast, distinct intestinal microbial profiles were present between the control, AOM/DSS, and pretreatment antibiotic groups. In addition, there was a tendency toward a positive correlation between number of tumors and number of OTUs. The relative abundance of the Bacteroidales order and Lachnospiraceae family tended to be positively related to tumor burden.These findings suggest that the gut microbial diversity and community structure altered by the antibiotic treatment may be involved in colon tumorigenesis.In this study, we demonstrated that manipulation of the gut microbiota using antibiotics attenuates colon tumorigenesis in the AOM/DSS-induced murine CAC model. In addition, there were significant differences in development of CAC according to timing of gut microbial change through antibiotic administration. Colon tumorigenesis was most markedly suppressed when antibiotics were administered before onset of inflammation induced by DSS and throughout the whole period of inflammation. In contrast, colon tumorigenesis was not suppressed when antibiotics were administered only before DSS administration.Previous studies have shown that inflammatory cells and related mediators, including IL-6, TNF-α, and IL-23, enhance DNA damage in intestinal epithelial cells to form a carcinogenic microenvironment during development of CAC –13. PersCAC develops through a sequence of no dysplasia-indefinite dysplasia-low grade dysplasia-high grade dysplasia-carcinoma . In otheStreptococcus gallolyticus, Enterococcus faecalis, colibactin-producing Escherichia coli, enterotoxigenic Bacteroides fragilis, and Fusobacterium nucleatum, are involved in development of CRC through modulation of the tumor immune environment and promotion of DNA damage [Several specific microbes, including A damage . HoweverA damage , 9, 22. A damage , 23, 24.In our results, the number of OTUs was significantly reduced in the groups in which tumorigenesis was suppressed, resulting in an overall positive correlation between number of OTUs and tumor burden. Additionally, Chao 1 index, one of the alpha diversity indices, was reduced in the antibiotic treated groups than in the control and AOM/DSS groups. These results suggest that gut microbial diversity may play an important role in tumorigenesis. However, caution is needed in interpreting results, because the total amount of microorganisms may also be reduced by the antibiotic treatment. Increased bacterial load in colon tissue is known to contribute to intestinal carcinogenesis . HoweverIn conclusion, we found that manipulation of gut microbiota using antibiotics can attenuate colon tumorigenesis in the AOM/DSS model. In particular, gut microbial changes should be maintained throughout the entire period of inflammation to effectively suppress colon tumorigenesis. Manipulation of gut microbiota using antibiotics may be considered as a potential therapeutic option for CAC.S1 TableThe humane endpoint was determined to have a total score of the sum of each item ≥8, or the highest points in two or more of the individual items.(PDF)Click here for additional data file.


Plants being sessile in nature are constantly exposed to environmental challenges resulting in substantial yield loss. To cope with the harsh environment, plants have developed a wide range of adaptation strategies involving morpho-anatomical, physiological, and biochemical traits . In receQuercus ilex as a model species for other forest tree species. Regarding the progress of techniques in proteomics with other plant species, the research in Q. ilex moved from a gel-based strategy to a gel-free shotgun workflow. New directions in Q. ilex research leads to the identification of allergens in pollen grains/acorns and the characterization of wood materials, which are objectives clearly approached by proteomics [In this special issue, out of the 27 original proteomic publications, 21 articles use the gel-free technique, in which nine are label-free and 12 are label-based. Progress has been fueled by the advancement in mass spectrometry techniques, complemented with genome-sequence data and modern bioinformatic analysis; however, until now the two-dimensional electrophoresis based proteomic technique was used  summarizoteomics Arabidopsis plasma-membrane associated candidate proteins in response to fungal treatment as well as those possibly interacting with the microbe-associated molecular pattern as ligands. They identified defense-related proteins and elucidated unknown signaling responses to this microbe-associated molecular pattern, including endocytosis. Furthermore, proteomic techniques were used to identify the mechanism in crops such as tomato [The impact of diseases on crop production negatively reflects on sustainable food production and the overall economic health of the world. Five publications focus on biotic stress using various proteomic techniques. Khoza et al.  used a ps tomato , sugarcas tomato , potato s tomato , and whes tomato  under biClematis terniflora DC, respectively.Wang et al.  and Gao Morus alba [Brunfelsia acuminate [Phalaenopsis [To facilitate the biotechnological improvement of crop productivity, genes, and proteins that control crop adaptation to a wide range of environments will need to be identified. This special issue includes many functional mechanisms of plants with nitrogen utilization , ammoniurus alba , pea culrus alba , maize [rus alba , tea [25cuminate , potato cuminate , and Phaaenopsis . Due to aenopsis .RGG4/DEP1/DN1/qPE9-1/OsGGC3 mutants exhibited dwarfism, the tissues that accumulated Gγ4 corresponded to the abnormal tissues observed in RGG4/DEP1/DN1/qPE9-1/OsGGC3 mutants [RGG3/GS3/Mi/OsGGC1 mutants show the characteristic phenotype in flowers and consequently in seeds, the tissues that accumulated Gγ3 corresponded to the abnormal tissues observed in RGG3/GS3/Mi/OsGGC1 mutants [Despite recent advancements, more emphasis needs to be given to the protein-extraction protocols, especially for proteins that are not abundant. Matsuta et al.  and Nish mutants . On the  mutants . An amalFurthermore, organelle function, post-translational modifications, and protein-protein interactions, which are progress of proteomic research, provide deeper insight into protein molecular function. The major subcellular organelles and compartments in plant cells are nucleus, mitochondria, chloroplasts, endoplasmic reticulum, Golgi apparatus, vacuoles, and plasma membrane. The intracellular organelles and their interactions during stressful conditions represent the primary defense response. Subcellular proteomics has the potential to elucidate localized cellular responses and investigate communications among subcellular compartments during plant development and in response to biotic and abiotic stresses. This special issue includes the proteomic results in plasma membrane ,30,31, cFurthermore, proteomic data will be improved with convention regarding metabolomics and transcriptomics . AlthougThe guest editor hopes that this special issue will provide readers with a framework for understanding plant proteomics and insights into new research directions within this field. The guest editor thanks all of the authors for their contributions and thanks the reviewers for their critical assessments of these articles. Moreover, the guest editor renders heartiest thanks to the Assistant Editor, Ms. Chaya Zeng for giving me the opportunity to serve “Plant Proteomic Research 2.0” as guest editor.


The tobacco control community has raised some concerns about whether studies on electronic cigarettes (e-cigarettes) published in scientific journals hide conflicts of interest (COI) and whether such reports are biased. This study assessed potential COI in the e-cigarette scientific literature.Cross-sectional study was conducted on e-cigarette publications indexed in PubMed up to August 2014. We extracted information about the authors , publication characteristics , results and conclusions, presence of a COI statement, and funding by and/or financial ties to pharmaceutical, tobacco, and/or e-cigarette companies. An algorithm to determine the COI disclosure status was created based on the information in the publication. Prevalence ratios (PRs) and confidence intervals (CIs) were calculated to identify associations with COI disclosure, controlling for several independent variables.Of the 404 publications included in the analysis, 37.1% (n=150) had no COI disclosure statement, 38.6% declared no COI, 13.4% declared potential COI with pharmaceutical companies, 3.0% with tobacco companies, and 10.6% with e-cigarette companies. The conclusions in publications with COI, which were mainly tied to pharmaceutical companies, were more likely to be favourable to e-cigarette use . Publications that supported the use of e-cigarettes for both harm reduction  and smoking cessation  were more likely to have conclusions that were favourable to e-cigarettes.One-third of the publications reporting studies on e-cigarettes did not have a COI disclosure statement, and this proportion was even higher in news articles, editorials and other types of publications. Papers with conclusions that were favourable to e-cigarette use were more likely to have COI. Journal editors and reviewers should consider evaluating publications, including funding sources, to determine whether the results and conclusions may be biased. COI occurs when authors, reviewers or editors have interests that are not fully apparent and that could influence their judgment on what they publish or review3.A conflicts of interest (COI) is ‘a set of conditions in which professional judgment concerning a primary interest  tends to be unduly influenced by a secondary interest ’5. In recent years, COI disclosure policies have become a regular part of biomedical research. The International Committee of Medical Journal Editors’ guidelines recommend that authors should disclose the study’s funding sources and any financial ties to core companies, such as pharmaceutical or tobacco companies6. In addition, since 2013 the Committee on Publication Ethics (COPE) established two levels of obligation for its members. The first is a Code of Conduct which Journal Editors are expected to practise (and will consider complaints against members who do not follow it), and the second is a set of Best Practice recommendations, which are voluntary, but are suggested to include into the journal’s policies and practices7. Among these practices, ‘Editors should have systems for managing COI by authors and reviewers, as well as their own COI, including their staff and editorial board members’7.Efforts to provide transparency regarding COIs date back to 19844 or tobacco companies may be biased11 due to vested economic interests. In the 1970s, tobacco companies downplayed the harm of second-hand smoke, for example, by hiding damaging data and distorting research findings12. Nowadays, the four major tobacco companies are involved in the manufacturing of electronic cigarettes . Considering that this electronic device resembles cigarette use, it is plausible that the same strategies could be used for manipulating the scientific evidence about the use of this relatively new product12. In fact, some in the scientific community are concerned that publications on e-cigarettes may hide COI and thus their results could be biased16. A systematic review of the health effects of e-cigarettes revealed that 34% of the 76 reviewed articles had declared COI, mostly funding from the manufacturers of e-cigarettes or authors who had acted as consultants for the manufacturers of smoking cessation treatments17. Another systematic review that addressed passive exposure to e-cigarettes showed that 30% of the 40 reviewed papers had potential COI18. One report on the ‘evidence update’ of e-cigarettes19 claimed that ‘e-cigarettes are 95% less harmful to health than normal cigarettes’; this claim was contested because its conclusions appeared to be biased due to COI15. That report also raised serious questions because of its lack of a peer-review process, and thus several organizations put this issue in the spotlight19.Nevertheless, evidence shows that tobacco research sponsored by pharmaceutical20. This decision has been criticised by some authors, who argue that these journals have passed a strong rule without enough evidence to justify it and that the journals themselves have some countervailing reasons to do so20. Although full disclosure of financial ties is common in scientific journals when publishing an original report, it seems to be less a priority for other types of articles, such as commentaries, news items, etc7. There are hundreds of publications about e-cigarettes, but, considering its novelty, less than half are original articles or reviews, with the rest being mainly notes, editorials or opinions21.Some journals do not accept manuscripts with links to tobacco companiesThus, our objective was to conduct an assessment of potential COI in the emergent scientific literature on e-cigarettes. Specifically, we aimed to analyse the declaration of potential COI in any type of publication , according to the criteria proposed by COPE.www.pubmed. gov) to identify all publications on e-cigarettes listed in Medline and indexed until August 2014. We included all types of publications whose primary or secondary aim or topic was related to e-cigarettes.We performed a search on PubMed (The full search strategy used was as follows: (‘electronic cigarettes’[All Fields] OR e cig[All Fields] OR e cigar[All Fields] OR e cigarettes[All Fields] OR e cigarette[All Fields] OR e cigarette’s[All Fields] OR e cigs[All Fields] OR ‘electronic nicotine delivery system’[All Fields] OR ‘electronic nicotine delivery devices’[All Fields]) AND (‘0001/01/01’[PDAT]: ‘2014/08/31’[PDAT]). No other restrictions were established.The search returned 445 publications. Five pairs of reviewers assessed the title and abstracts (if available) of about 90 publications each. Of the retrieved publications, 30 were not related to e-cigarettes, 2 were duplicate publications, and 2 anecdotally mentioned e-cigarettes. Thus, 411 publications were eligible for full assessment. After reading the full text, 7 were excluded: 1 was not related to electronic cigarettes, 3 mentioned them as a secondary topic with no specific comments, and 3 were unavailable to retrieve (including unsuccessful contact with the corresponding authors); thus, 404 publications were included in the final analysis .ad hoc Excel spreadsheet to register the information for each publication. They were pilot-tested using 5 full-text publications that were assessed by all of the researchers. When there were any questions, the reviewers discussed the papers with the assigned partner until agreement was reached. If they could not come to an agreement, two other researchers were assigned and resolved the issue.Some tools were created to extract the information, including a protocol to guide the pull out of data, an algorithm  to assess the publication’s disclosure status, and an Based on information in the publication several variables were assessed, including information about the authors, the publications, COI, results and conclusions.Authors: The surname and initials for all authors were collected.Institutional affiliation: All affiliations from all the authors were considered. This information was classified as follows: no affiliations were provided for any of the authors; some of the authors had their affiliations specified; all authors had their affiliations specified. Affiliations were classified into the following categories: 1) academy/university, 2) governmental/public agency, 3) health organization, 4) private company, 5) pharmaceutical company, 6) tobacco company, 7) e-cigarette company, and 8) other. If the affiliation was related to more than one core company , then it was classified based on its main scope .Country of origin: The country with which the first author was affiliated was recorded and was reclassified according to continent for the analysis.Year of publication: The search included all publications indexed in PubMed up to August 2014 .Type of publication: Publications were classified as original article, review, editorial, letter, news, commentary, or other, based on the information in the full publication; when this information was not provided, the classification was based on the structure of the publication, following the descriptions of different types of publications on the journal’s website.Aim: The presence of a specific research aim was captured .Empirical data: The reporting of empirical data was determined . For publications reporting empirical data, they were classified as follows: 1) the main topic/s of the study-allowing for multiple responses such as prevalence of use, toxicology , health effects, smoking cessation, topography, attitudes and knowledge, regulation and marketing, clinical advice, other, and miscellaneous (different or mixed topics), and 2) the main subject of the study-allowing for multiple responses such as no main subject, humans, e-cigarettes themselves, including the device, liquids and emissions, regulation and marketing, and other.COI: The presence of a COI section was determined , using this or similar wording (e.g. competing interests). In addition, we recorded whether the authors reported their COI status in any section of the publication  and the literal wording. Also, we recorded whether the potential COI reported was related to pharmaceutical (P), tobacco (T) and/or electronic cigarette (EC) companies (PTEC_CO) .Acknowledgments: The presence of an acknowledgments section was assessed . If yes, we recorded whether the authors acknowledged any of the PTEC_CO.Funding: The disclosure of any funding or sponsorship for the study  in any section of the publication was assessed . If yes, we classified the source(s) of funding as being from a university, governmental/public organisation, private organisation, PTEC_CO, and/or other sources.Financial ties: We determined whether the authors had other financial ties to PTEC_CO  and the type of these financial ties, meaning personal payments for: 1) services, travel, training, etc.; 2) money for patents, conferences, etc.; and 3) other financial ties. This information was drawn from any part of the publication, main affiliation, COI statement, funding and acknowledgements.Overall view of disclosure status: Based on the information about the funding sources and the authors’ financial ties, we classified the disclosures of potential COI into 5 categories as follows: the authors did not disclose any COI within the manuscript (code 0); the authors disclosed no COI for the study, and no funding from any PTEC_CO are mentioned (code 1); the authors disclosed no COI for the study, but they acknowledged that some of the authors or previous studies conducted by the authors received funding from PTEC_CO in the past (code 2); the authors disclosed no COI for the study, but acknowledged receiving funding for the study from PTEC_CO (code 3); and the authors disclosed COI with PTEC_CO (code 4). For multivariate Poisson regression analysis, we excluded publications with no disclosures (code 0) and collapsed the publications into two categories: those that disclosed no COI at all (code 1) and those that disclosed any COI (codes 2–4).Results: For empirical publications, the reported results were categorised as being ‘favourable’ to the use of e-cigarettes, ‘not being favourable to their use’, ‘neutral’ (not in favour nor against), or ‘unclear’ (undefined tendency). The classification was done according to the findings provided in the study, avoiding any interpretation of them. For the analysis, we combined publications in the ‘neutral’ and ‘unclear’ categories into one category.Conclusions: For all publications, with and without results, the specific conclusions in this section, or in the abstract if unclear, were also categorised as being ‘favourable’ to the use of e-cigarettes, ‘not being favourable’ to their use, ‘neutral’ or ‘unclear’. For assessing this variable we searched for authors’ conclusions and recommendations in the corresponding section, in the discussion section or in the abstract if it was not clear, in the case of empirical publications, and within the publication for non-empirical publications. We also combined the ‘neutral’ and ‘unclear’ conclusions into one category.Overall publication tendency: Based on the results and conclusions, we determined whether the publication showed a tendency to support some of the following (multiple responses were possible): the use of e-cigarettes for harm reduction (reduction of tobacco products by using e-cigarettes), stronger regulation of e-cigarettes, and the use of e-cigarettes for tobacco smoking cessation.We report the frequency and percentage of COI disclosure (codes 0–4), the potential COI with PTEC_CO and the tendency of the main conclusions of publications with empirical data according to the publication characteristics, the main study subjects and the topics studied. We also describe the COI disclosure (codes 0–4) according to the conclusion and the publication tendency, for all publications and for empirical studies. In addition, we calculated prevalence ratios (PRs) and their 95% confidence intervals (CIs) using multivariate Poisson regression analysis with robust standard error estimations (White-Huber robust standard errors) to identify associations with COI disclosure, controlling for the continent of the first author, the year of publication, empirical data, main subject and main topic.A total of 404 publications were analysed. The more recent the publication year, the higher the number of publications on e-cigarettes, with a tendency towards an exponential increase . Of all publications, 36.9% were original articles . Almost We examined the factors associated with reporting potential COI in the scientific literature on e-cigarettes. We found that over a third of the publications did not have any COI disclosures; these were mostly news, editorials and other types of publications. Authors who had COI (disclosed or not) and publications in which the main topics included toxicology/health effects or smoking cessation/clinical advice were more likely to report favourable conclusions about e-cigarettes. In contrast, studies in which the main subject was e-cigarettes themselves were less likely to have favourable conclusions about e-cigarettes. There are a few possible explanations for these findings. First, the authors who published studies with favourable conclusions about e-cigarettes could have some ties to PTEC_CO. These authors may have framed the research question, conducted the study, or reported the results and conclusions, either consciously or unconsciously with the interests of their sponsors in mind. Second, it is possible that the authors failed to disclose their sponsorship or research constraints, and the editors were not as strict as with studies that reported non-favourable conclusions.We found that the conclusions of reports on e-cigarettes are associated with their authors’ COI. In addition, we observed that publications that supported the use of e-cigarettes for smoking reduction and smoking cessation were more likely to be from authors with COI with PTEC_CO. Our study shows that publications that report favourable conclusions about e-cigarettes come from authors who had received financial support from PTEC_CO. This pattern is seen in publications with both empirical and non-empirical data.1. Studies measuring undisclosed COI suggest that between 43% and 69% of study reports and other publications fail to include disclosures of COI3. In our study, we found that 37.1% of publications did not include any disclosure about COI. Disclosure only reveals the possibility of bias, without providing any guidance for resolving it3. Biases introduced by COI are difficult to deal with and should be treated like any other confounder that could affect the results of a study3; however, treating COI as a confounder is challenging. The failure of the scientific community to effectively address COI has serious repercussions for public health policy7. The scientific community must provide transparency, supervision and accountability to help ensure that research is not biased by undisclosed COI7. Some suggested methods for improving the disclosure of potential COI include the introduction of clinical trial registries, public accessible registers for declarations of interest, and the creation of an Ethics Centre that monitors and reports the enforcement of ethical standards7. A compulsory detailed statement of COI, including some of the variables studied in this analysis, may disclose them in a more effective manner. This would not completely mitigate the risk of bias, but it would facilitate a better understanding of how COI are related to biases and how they can influence research consensus3.A systematic review of the prevalence of COI found that a third of biomedical researchers in academic institutions had COI22. Articles published in peer-reviewed journals are generally of higher quality than those published in non-peer-reviewed journals2. However, we did not include this independent variable in our assessment. Additional research is needed to clarify possible associations between the journal’s quality and the disclosure of COI in e-cigarette publications.We also found that the type of publication can influence its tendency to e-cigarettes. Practically all reviews were neutral in terms of the tone of their conclusions, but letters and other non-empirical publications were more likely to be either against or favourable to the regulation of e-cigarettes. The journal characteristics may also influence the results and conclusions of the studies, as the quality of their reporting may vary according to the journal’s standards24. In our study, we observed that only publications in which the authors reported ties with pharmaceutical companies were associated with favourable conclusions on e-cigarettes. However, we did not find that authors openly reported COI in all cases; sometimes, this information was extracted based on the author affiliation, funding received in previous studies or from the acknowledgments section. We observed that some authors reported no COI but declared financial support from or even were employees of PTEC_CO (code 3 in our classification); thus, they did not acknowledge this potential COI. Whether non-reporting of COI was consciously done or not is beyond the aims of this study. Lack of transparency at the time of submission on these issues is likely to lead to a perception by readers and editors that the research is tainted25, thus is must be avoided.There is evidence that financial COI lead to systematic bias26. This type of study is difficult to design and conduct, since peer review is usually blinded and any potential COI are thus unclear. This raises the question of whether current journal policies are sufficient to ensure objectivity in the publication process and whether the current COI definitions and restrictions on publication should be expanded.Our analysis was limited to the information reported in the papers; thus, any financial ties or other interests that were not reported in the paper could not be assessed, so the COI may be underestimated. Also, not all journals specify whether all their publications are peer-reviewed; not conducting peer review may contribute to a lack of declaration of potential COI. In addition, we only searched publications in the PubMed database. However, PubMed is the core database for the biomedical sciences. Another limitation is the cross-sectional nature of this study, so finding causal relationships was beyond the scope of the study. In the search strategy, we included publications up to August 2014; this time-frame represents seven years after e-cigarettes launched into the market (2007 to 2014). Nevertheless, given the growing number of papers on e-cigarettes, it is possible that more recent publications would show different tendencies. However, we were interested in the situation when e-cigarettes first began to gain popularity, so the study period covered those years. Since some of the authors of this report have conducted or are conducting research on e-cigarettes, their judgments could also have some bias. However, all publications were assessed by pairs of reviewers who achieved a consensus; also, a protocol and algorithm were created to facilitate the objective assessment of each publication, and the procedures were pilot-tested. In addition, we only identified COI when authors reported them in any of the sections of the papers . This way, we were able to establish their ties with pharmaceutical, e-cigarette and tobacco companies. Future bibliographic and social science studies should be done to explore the several sources of COI in e-cigarette publications, besides what authors have reported in papers. Moreover, we only studied potential COI related to the authors, but COI related to the editors and the reviewers might also be relevant7. In addition, it classified COI disclosures into 5 categories and created an algorithm to facilitate the evaluation, thus increasing internal validity. The algorithm allowed us to detect COI that could be misleading or that were not well-described. This tool might be useful for identifying situations in which authors do not correctly report their COI. In addition, the results of this study represent an exhaustive review of potential COI for a highly relevant topic, taking into account several publication-related variables.The study also has some strengths. It pioneered new methodology to describe COI according to the criteria proposed by the COPEA third of the assessed publications about e-cigarettes did not have any COI disclosure, and this proportion was even higher in certain kinds of publications, such as news, editorials and other types of articles. Publications with favourable conclusions about e-cigarettes were more likely to include a COI disclosure. Furthermore, favourable conclusions about e-cigarettes were more frequent in publications that had COI declarations related to pharmaceutical companies. Journal editors and reviewers should consider evaluating publications, including the funding sources, to determine whether the results and conclusions may be biased and to determine whether there are any relationships with PTEC_CO.Click here for additional data file.


MET) and anaplastic lymphoma kinase (ALK), which has been considered as a multi-targeted tyrosine kinase inhibitor (TKI). The objective of this study was to explore the efficacy of crizotinib in advanced non-small-cell lung cancer (NSCLC) with concomitant ALK rearrangement and c-Met overexpression.Crizotinib can target against mesenchymal-epithelial transition  were screened for ALK-rearranged cases, with the incidence of 10.0% (16/160). A total of 116 ALK-rearranged patients received the treatment of crizotinib. Objective response rate (ORR) was 86.7% (13/15) in ALK-rearranged patients with c-Met overexpression and 59.4% (60/101)in those without c-Met overexpression, P = 0.041. Median PFS showed a trend of superiority in c-Met overexpression group . Median overall survival (OS) showed a significant difference for ALK-rearranged patients with c-Met overexpression group of 33.5 months with the hazard ratio (HR) of 3.2.Sixteen patients were identified with c-Met overexpression in 160 ALK rearrangement in a small population of advanced NSCLC. There may be a trend of favorable efficacy of crizotinib in such co-altered patients.C-Met overexpression co-exists with  EML4) and anaplastic lymphoma kinase (ALK) resulted in the activation of ALK kinase, which was identified as a driver oncogene in non-small-cell lung cancer (NSCLC) in 2007 [ALK fusion accounted for only 3 to 13% patients with advanced NSCLC, it has made significant effect on the treatment of advanced NSCLC as precise targeted treatment [ALK rearrangement can be identified with fluorescent in situ hybridization (FISH), rapid amplification of cDNA ends coupled polymerase chain reaction (RACE-coupled PCR) and Ventana immunohistochemistry (IHC). In China, all the three methods were recommended by Chinese expert consensus and have been approved as companion diagnostic tests by the Chinese Food and Drug Administration [Targeted therapy has led to a therapeutic paradigm shift in lung cancer, which brought the treatment of lung cancer into the era of precision medicine . At leasstration .c-Met gene encodes a high-affinity receptor for hepatocyte growth factor (HGF) [MET mutations, MET amplification and c-Met overexpression in many solid and hematological malignancies [MET amplification occurred in 7.3–10.4% of patients with untreated NSCLC [MET exon 14-alteration accounted for 0.9–3.0% of lung adenocarcinoma, while c-Met protein was reportedly overexpressed in about 22.2–67.2% of NSCLC and associated with poor prognosis [MET amplification is rare and difficulty in the detecting method of FISH, MET IHC acts as the most robust predictor of overall survival and progression-free survival to all examined exploratory markers [ALK-rearranged patients.Moreover, cellular-mesenchymal-epithelial transition (c-Met) has recently been discovered as a novel promising target in NSCLC . The c-Mor (HGF) –10. HGF or (HGF) . The actgnancies , 12. METed NSCLC –15. MET rognosis –23. Sinc markers . C-Met o markers –27, whil markers . The act markers . HoweverALK rearrangements have previously been considered mutually exclusive with epidermal growth factor receptor (EGFR) and kirsten rat sarcoma viral oncogene homolog (KRAS) mutations [EML4-ALK rearrangements and EGFR mutations have been identified [ALK rearrangement and c-Met overexpression can also occur [ALK, has been considered as the first-line treatment for ALK-rearranged advanced NSCLC patients with remarkable response in a series of clinical trials [ALK-rearranged patients, compared with 45% and 7.0 months of chemotherapy respectively [utations –32. Howeentified –35. Simiso occur , 36. Cril trials –40 .The ectively .ALK rearrangement and c-Met overexpression (ALK/c-Met) co-existence [ALK-rearrangement co-existing with c-Met overexpression in advanced NSCLC, as well as the efficacy of crizotinib for such co-altered patients.Of 19 patients with c-Met overexpression treated with crizotinib, 11 achieved partial response (PR) . Howeverxistence , 42. ThuALK rearrangement with any of these 3 methods: FISH, RACE-coupled PCR and Ventana IHC; (3) sufficient tissue for the analysis of c-Met IHC. Patients’ clinical and treatment information were extracted from electronic medical records at the Guangdong Lung Cancer Institute and the Peking University Cancer Hospital. Tumor histology was assessed by pathologists and staging was classified based on American Joint Committee on Cancer 7th edition of tumor, node, metastasis staging criteria [This retrospective study was conducted on 4622 advanced patients from two institutes, including a cohort of 3762 patients with NSCLC at the Guangdong Lung Cancer Institute, the Guangdong General Hospital from January 2011 to December 2016 and a total of 860 at Peking University Cancer Hospital from January 2015 to December 2016. The inclusion criteria were: (1) pathologically confirmed advanced NSCLC with at least one measurable lesion; (2) identified with criteria . This stALK-rearranged patients were enrolled for the analysis of c-Met expression  [In the present study, 160 (RECIST) . PFS wasALK (D5F3) rabbit monoclonal primary antibody  according to the manufacturers’ instructions. ALK FISH analysis was performed on formalin-fixed paraffin-embedded tissues using a commercially available ALK probe  according to the manufacturer’s instructions. Patients were diagnosed with ALK FISH-positive when 15% or more of scored tumor cells had split ALK 5′ and 3′ probe signals or had isolated 3′ signals. Total RNA was extracted from tissue samples with the RNeasy Kit (Qiagen). Reverse-transcriptase PCR and 5′ rapid ampli6fication c-DNA ends coupled PCR plus sequencing was performed out as ever reported [ALK reference sequence (NM_004304.3) to determine if a novel fusion was present.All tissue samples were routinely assessed with sectioning, hematoxylin and eosin staining, and visualization with a microscope to ensure tumor content was at least 50%. IHC was carried out on 4-mm thick slides using Ventana anti-reported . PCR proMET amplification was detected with the method of FISH. Dual-color FISH was performed in deparaffinized sections 4 μm thick using a c-Met/CEN7q Dual Color FISH Probe . After the immersion of tissue sections and TRIS-EDTA (pH = 8.0), sections were washed in PBS. Then they were digested in a protease solution at 37 °C for 7–8 min and washed in PBS once more. The sections were co-denatured with probe at 80 °C for 5 min and then hybridized at 37 °C for 14–18 h and subsequently counterstained with DAPI. The results of positivity were assessed according to the 2 criteria: A c-Met: centromere 7 ratio ≥ 2.0 and the criterion of Cappuzzi  [C-Met protein expression was evaluated by IHC. Sections of 4 μm thick were cut from paraffin tissue blocks of NSCLC tumors. Staining was performed on a Ventana Benchmark XT automated immunostainer (Ventana) with a CONFIRM anti-total c-Met rabbit monoclonal primary antibody  and an ultra View Universal DAB. A standard protocol for immunostaining of samples was used. A four-tier (0–3) intensity scoring system has been used in our present study, as shown in Fig.  nuclei) .Fig. 2ReEGFR exons 18 to 21 and KRAS exons 2 and 3. These exons were amplified by PCR as previously described [Genomic DNA from each sample was used for sequence analysis of escribed , and the2 test, or Fisher exact test when necessary. Survival curves were constructed with the Kaplan–Meier method and differences analyzed by the log-rank test. Multivariable predictors were assessed by a forward step-wise likelihood ratio Cox proportional hazards model. Statistical analysis was performed using SPSS version 22.0 software . All statistical tests were two-sided, with P value < 0.05 considered statistically significant.Categorical variables were compared using XALK gene status, of whom 282 were identified with ALK rearrangement. The study recruited 160 ALK-positive advanced NSCLC patients with sufficient tissue for the detection of c-Met expression. Sixteen patients were identified with c-Met overexpression, accounted for 10.0% (16/160) of ALK-rearranged patients. A total of 116 patients treated with crizotinib were investigated for the response to crizotinib. Baseline characteristics are presented in Table ALK-rearranged patients with c-Met overexpression were relatively young with less than 45 years old, and most of them were female and non-smokers with adenocarcinoma. The baseline clinicopathologic characteristics were well balanced between ALK-rearranged patients with c-Met overexpression and those without c-Met overexpression. MET amplification was identified as negative by FISH in 8 ALK-rearranged NSCLC patients with c-Met overexpression, while 7 patients were unknown due to insufficient tissue  with complete electronic records were screened for ALK-rearranged patients were enrolled in our study to assess the efficacy of crizotinib, including 101 patients without c-Met overexpression and 15 cases with c-Met overexpression. Objective response rate (ORR) was 86.7% (13/15) and 59.4% (60/101) in patients with and without c-Met overexpression groups, respectively. (P = 0.041)    were independent prognostic factors of OS.Median PFS showed a trend of superiority in c-Met overexpression group  in our research [ALK-rearranged Western NSCLC patients with early or advanced stages, whereas ours was in 160 ALK-positive Eastern metastatic cases. The variations in the frequency of c-Met overexpression in ALK-positive patients were likely related to different definitions of c-Met overexpression and population investigated.The frequency of patients with c-Met overexpression were 29.2 and 58.8% of research , 36. Theresearch , 36. Howresearch , 41. ThuALK and c-Met pathway. ALK-rearranged patients obtained ORR of 74% and median PFS of 10.9 months with first-line crizotinib, while those with de novo c-Met overexpression experienced ORR of 58% [ALK-rearranged tumors with c-Met overexpression may be shrunk largely with the treatment of crizotinib. However, only few limited cases were reported the response to crizotinib for ALK-rearranged patients with c-Met overexpression. Feng et al. study demonstrated 2 CRs and 4 PRs with crizotinib in 6 ALK-rearranged patients with c-Met overexpression [ALK-rearranged patient with c-Met overexpression and Her-2 amplification [ALK- rearranged NSCLC patients were undiscovered. Our results showed that ALK-rearranged patients with c-Met overexpression obtained significant objective response rate and median OS and a trend of superiority in terms of PFS with treatment of crizotinib, compared with those who without c-Met overexpression. Regardless, it would be interesting to further explore the response to the potential combination therapy of c-Met inhibitor plus ALK inhibitor, especially the second-generation ones, such as ceritinib or alectinib, which does not possess c-Met inhibitory activity.Crizotinib simultaneously inhibits the activation of R of 58% , 41 . Thpression . Howeverpression . Diseasefication .The clinALK-rearranged patients with c-Met overexpression. In addition, this was a retrospective study. Furthermore, the status of c-Met exon 14 skipping mutations in patients of our research was not detected for the lack of tissue samples. Finally, it is difficult to compare each ALK variants or other gene profiles for insufficient tissues in this study.However, there were several limitations in the present study. First, we focused on only a small sample-size of ALK-rearranged NSCLC. There may be a trend of favorable efficacy of crizotinib for advanced ALK-rearranged patients co-existing with c-Met overexpression. Further investigations are warranted to validate our findings and to elucidate molecular mechanisms.Our study demonstrated that c-Met overexpression of ≥50% of tumor cells of strong staining exists in a small population of advanced