A review of the research activities and achievements at Shenzhen University is conducted in this paper concerning the creation and further development of novel microcapsule based self-resilience systems for their application in concrete structures. After a brief description of pioneering works in the field starting about 10 years ago, the principles raised in the relevant research are examined, where fundamental terms related to the concept of resilience are discussed. Several breakthrough points are highlighted concerning the three adopted comprehensive self-resilience systems, namely physical, chemical and microbial systems. The major challenges regarding evaluation are emphasized and further development concerning self-resilience in concrete structures will be addressed. Being a typical composite material, modern concrete (mostly reinforced by steel) has already been used as a construction material in civil engineering for more than a century, and it is believed to do so for another century because of the widespread availability of its constituents, and its cost-effectiveness, versatility, durability and adaptability. However, there are two major issues that raise serious concern among civil engineers worldwide ,2, i.e.,The first one is related to the performance degradation of a concrete structure under environmental actions. Over the years, there has been a growing understanding that all concrete structures deteriorate over time. Deterioration will change the performance of a concrete structure under various actions. Numerous forms of performance degradation have been observed in concrete structures due to the attack of corrosive ions, gases and other types of aggressive actions in the marine environment. When deterioration occurs, expensive repairs and maintenance have to be undertaken in order to maintain an adequate level of performance and the service life of concrete structures. It has been estimated that the annual cost of repair of concrete structures in Europe is in excess of $20 billion . In ChinDue to the shortage of natural resources, the increase in population and deterioration of environmental conditions, the second issue concerning sustainability emerges. It is often the case that the large-scale demolition of obsolete structures and their replacement with new ones are out of the question either due to economic reasons or a shortage of resources .In recent years, so-called self-healing materials have been exploited in civil engineering, which have the built-in capability to repair structural damage autogenously or with the minimal help of an external stimulus ,6. By apA microcapsule based novel intelligent resilience system for concrete structures has been recently established and further developed at Shenzhen University. Its short history and development will be briefly reviewed in this paper. Starting from the principles of self-resilience, the logical path of development of such a system is examined. Fundamental terms related to the resilience concept and strategy are discussed. Remarkable breakthroughs regarding material systems, design procedures and numerical models are highlighted. The major challenges and further development concerning self-resilience in concrete will be addressed.Since 2008, attempts have been made to introduce a novel microcapsule based self-healing system for concrete at Shenzhen University ,10,11. TThe urea formaldehyde resin was used for the wall of the microcapsule, and bisphenol—an epoxy resin E-51 diluted by n-butyl glycidy ether (BGE)—was adopted as the healing-agent inside the microcapsule. A combination of latent curing agent MC120D and tetraethylene penamine (TEPA)—a type of liquid curing agent functioning at normal temperature was used for curing the healing product. There were basically four stages in forming microcapsules as shown in Results showed that the microcapsule obtained with the adopted production process can be used for the self-healing system in concrete. In the broken specimen of concrete after hardening, it could be clearly seen that the microcapsules remain undamaged during the producing process see c. The trIt was found that the diameter, shell thickness, and surface texture of the microcapsules play major roles regarding the performance of self-healing system ,16,17,18The shell thickness and the surface texture of microcapsules can be controlled by varying core/shell ratio and synthesis temperature, respectively. This is illustrated in On the basis of the fundamental understanding of characteristics of microcapsules and their influences on the performance of a self-healing system, further optimizations and improvements have been made since 2008 at Shenzhen University. One way is to narrow the size distribution in order to improve the efficiency of the physical trigger . BesidesThe healing efficiency with respect to the mechanical and permeability related performance of the adopted self-healing system in concrete was evaluated by experiments ,10,11,12As shown in The two different healing mechanisms were evaluated, namely mechanical performance recovery  and permeability related performance recovery. Details of evaluation can be found in references ,10,11,12From Dafterpreloading/Dhealed) − 1) × 100%, where Dafterpreloading and Dhealed are the diffusion coefficient obtained after the preloading and after healing, respectively. The tendency of recovery shows a linear increase for the adopted parameters, such as the dosage, size of microcapsules and the pre-loading level, whereas the healing temperature demonstrates as strong an effect on the recovery  test. The recovery is calculated as  are classified and it is clear that the degradation of performance of a concrete structure starts at the material level. Anything related to durability should be implemented at this point. Therefore, degradation-related resilience is paramount and forms the foundation for the strategy development of the targeted self-resilience system ,4,21.The principle of resilience consists of two important aspects see a: namelyIt has to be emphasized that the self-resilience system is based on the microcapsule technology. In other words, the container of healing materials is restricted to the microcapsule, however, the type of materials of microcapsules could be essentially different , depending on the targeted recovery performance index.As far as the healing-agent is concerned, there are a large range of options, from organic, bacteria to in-organic materials.With respect to the trigger, not only is the physical trigger  further explored, but the so-called chemical trigger has drawn major attention in our current research. This is in turn related to the resilience principle, namely, healing and recovery at a multi-scale.Physical system Chemical system Microbial system In summary, three relatively independent but strategically integrated approaches have been adopted aiming at the formation of a microcapsule based comprehensive self-resilience system. The targets are to heal the mechanical damage or to recover lost functionality corresponding to impermeability or corrosion protection b:PhysicRelying on several cohesive research projects funded by the Nature Science Foundation of China, several breakthroughs have been made recently at Shenzhen University. They will be briefly highlighted in the following section.A microcapsule based self-recovery system with a chemical trigger (in this case pH sensitive) has firstly been proposed and later on further developed at Shenzhen University ,25,26,27To prepare the microcapsules, sodium monofluorophosphate and microcrystalline cellulose were mixed into Polysorbate 80. Then, the small spherical particles were molded by the extrusion–spheronization method, which is able to control the diameter of a microcapsule. Finally, the spray drying method was adopted to fabricate microcapsules, spraying the liquid PS onto the surface of spherical particles. Three types of microcapsules with different amounts of shell materials were fabricated; the mass percentages of shell materials were 10%, 20% and 43%. For example, 10% refers the percentage of increased weight of microcapsules, according to that of spherical particles molded by the extrusion–spheronization method.The results of studies on the surface damage of microcapsules at different pH values by means of SEM are illustrated in 2+ released from the capsule cores.The release mechanism is further studied after the trigger is excited . To examAccording to the calibration curve, the amount of healing material released from the microcapsules at every designated pH level was calculated, as the pH value was measured. 2PO3F) as the healing material was adopted. The system is triggered by pH value variation. The study focused on examining the healing mechanism of the system for chloride-induced corrosion of rebar in a simulated concrete environment [To evaluate the recovery efficiency, a self-recovery system with sodium monofluorophosphate /calcium hydroxide was used to recover the protection ability against corrosion based on increasing hydroxyl content in concrete . The capA novel idea related to the performance self-recovery was first proposed and patented by Shenzhen University. By means of a chemical trigger (chloride sensitive), a self-resilience system based on the microcapsule technology has been proposed and developed, where the capacity to resist the penetration of harmful agent (chloride ion) into concrete will be restored. As the concentration of this harmful agent reaches a certain level, the microcapsule will be broken and the healing material inside the microcapsule will take action to remove the harmful agent. A remarkable breakthrough was made in 2015 related to this . Various+, Pb2+). When these microcapsules come into contact with chloride ions, the metal ions will be extracted to disintegrate microcapsules (see 2+) to form hydrogel. In our first try, Ag+ is chosen to be coordinated with alginate to form the wall of microcapsules. Silver alginate  can also form a characteristic “egg-box” structure. Each alginate molecular chain can be linked with other chains, forming a three-dimensional gel network  was employed to detect concrete specimen embedded with Ag-alg microcapsules. 3) precipitation, which is a common phenomenon in the natural environment. Certain mineralization bacteria can make use of carbon source to produce CO2 or CO32−, which subsequently reacts with calcium ions to form calcium carbonate precipitate on the surface of concrete cracks, thus sealing the concrete cracks. At the same time, the metabolism of mineralization bacteria creates an alkaline environment, which is further in favor of the process of calcium precipitation. Moreover, bacteria also provide nucleation sites for calcium precipitation. In addition to the ability to induce calcium precipitation, self-healing bacteria should be alkaliphilic and spore-forming due to the harsh environment inside concrete.Self-healing of concrete cracks by bacteria is based on microbial-induced calcium carbonate  of the strains were evaluated , and strThe features of the precipitated products were further studied . As show3, indicating that the crystal is CaCO3. Furthermore, the XRD analysis confirmed that the crystal induced by H4 is calcite [Element composition analysis via energy dispersive spectroscopy (EDS) revealed that the crystal is primarily composed of calcium, carbon and oxygen with a weight ratio closely matching that of CaCO calcite .2+ concentration were also studied [2. Therefore, H4 is a promising bacterium for self-healing of concrete cracks, considering that its CPA can remain at greater than 80% even if the pH reaches 11.0. In addition, it was found that the presence of excessive Ca2+ not only inhibits the bacterial-induced calcium precipitation process but also results in waste of the Ca2+ resource, and maintenance of a Ca2+ concentration lower than 30 mM is a good strategy. Fortunately, due to the poor solubility of calcium hydroxide, the free Ca2+ concentration of the pore solution inside the concrete is normally less than 30 mM [2+ source into concrete for the microbial self-healing process, as used in selected previous studies, might not be necessary from this point of view.Influential factors in the process of bacteria-induced calcium precipitation such as the carbon source, nitrogen source, pH and Ca studied . Experiman 30 mM , making 3 cannot be precipitated deep inside the crack might be because of the shortage of oxygen. It is well known that both water and O2 need to be present inside the crack to activate the bacterial self-healing concrete. While water is sucked into the tiniest microcracks due to capillary suction, the question may arise as to whether adequate oxygen will be available deep inside the cracks. Given that both bacterial spores and nutrients were provided in the self-healing system, lack of oxygen inside the concrete structure might be a major inhibition factor. It was observed that there exists a major drawback in the current microbial self-healing system, i.e., the calcium precipitation seems to only happen on the concrete surface area nearby the crack. The reason why CaCOTo deal with the problem mentioned above, a pioneer attempt at Shenzhen University has been made to develop a strategy to supply oxygen for the microbial calcium precipitation performance . FirstlyThe calcium precipitation of the binary self-healing system was evaluated in bacteria medium solution, and the result was shown in 3 started to form in the tubes where bacteria were incubated. In the beginning stage of calcium precipitation, no difference in the formation of CaCO3 could be found between the tubes with or without oxygen supply. However, for the tubes with oxygen supply, the amount of insoluble Ca2+ increased sharply from 7 days. After 32 days, insoluble Ca2+ reached 27.5 mM  was very low, which might account for the lower calcium precipitation by the bacteria in the absence of oxygen supply  are able to germinate, grow, and induce calcium precipitation effectively, and the stable oxygen supply is important for microbial calcium precipitation. Further study will be related to the longevity issues of the adopted bacteria/ORT system.Generally speaking, natural polymers are biocompatible while synthetic polymers are deleterious in various degrees for microorganisms like bacteria. That is why most of the existing biological microcapsules are walled by natural polymer materials. However, natural polymers are usually hydrophilic, i.e., they take up water and cause the shell of the microcapsule to swell. It is clear that this is not suitable for self-healing systems for concrete. Various attempts were made at Shenzhen University to find harmless and waterproof microcapsules which are suitable for application in microbial self-healing systems ,41,42,43490 are positively correlated with the bacterial concentration. After encapsulation, the fraction of surviving spores is over 90%, indicating the process has a mild effect on spores’ viability  mixed with spores. The shell material was ethyl cellulose (EC) in the first attempt  and lateThe survivability of the encapsulated bacteria was studied, together verifying the protection functionality of the microcapsule. It was found that pure spore has the highest mineralizing activity. Broken microcapsules have higher mineralizing activity than non-broken ones, which indicates that EC microcapsules can effectively protect microorganisms in this system b,c.The trigger mechanism and the working process was further studied by means of the optical microscopy and the high resolution X-ray Computed Tomography (XCT) . The craThe behavior and performance of the microcapsule based self-resilience system have to be evaluated through effective and meaningful ways . Up to now, various techniques have been applied to appraise the obtained system with success to some extent. In addition to the traditional macro test methods  ,46,47,48The whole healing process and healing effect of a microcapsule based self-healing system in concrete was monitored and qualitatively as well as quantitatively evaluated by means of XCT and an image analysis technique. The experimental procedures are illustrated in The crack-healing process and healing effect were evaluated with the help of the reconstruction of inner microstructures  and quantitative analysis. With the help of an image analysis technique, the total volume of air void (cracks and pores) was labeled and calculated. As shown in The evaluation of performance characterization of self-resilience system remains to be a major challenge. Due to the inhomogeneity and randomness, bulk behavior of the concrete after resilience is difficult to experimentally evaluate at a macro level. The only feasible way to do this is to examining the individual behavior of a single unit in the randomly distributed system on a fundamental level. The relevant material properties are then determined experimentally with the help of specific facilitated test set-ups. Afterwards, numerical methods have to be exploited by means of the obtained key parameters at the micro level to simulate the bulk behavior of the resilient system.The behavior of a single microcapsule in concrete was numerically studied at Shenzhen University . The effIt is evident that the key parameters have to be experimentally determined numerically to obtain a realistic picture. To realize this, the following working activities have to be fulfilled: (a) working out feasible and workable micro testing facilities; (b) quantifying the relevant key parameters for various components  quantifying the physical and chemical trigger mechanism; and (d) examining the healing and recovery performance of a single microcapsule system.An attempt in this direction was made recently at Shenzhen University ,52,53. AThe evaluation of a self-resilience system plays a paramount role as far as the successful application of such a system in concrete structures is concerned. Current approaches and methodologies are strongly based on the traditional macro level methods, which make it difficult to appraise newly created systems in an appropriate way. To deal with this challenge, a new RILIEM TC SHE was set up recently (in June 2016), which aims at solve this problem through international cooperation. (i)establish the relationship between the multi-actions and the action effect for concrete;(ii)link resilience requirements with environmental action and the related degradation mechanisms;(iii)incorporate existing self-healing and self-recovery systems to enhance the long-term performance of concrete structures by creating resilience to physical and chemical attack, through the addition of agents within microcapsules, that are able to mitigate the action effects in a range of aggressive environments;(iv)set up facilities and procedure to evaluate self-healing and self-recovery effects at the micro level;(v)quantify key parameters related to fundamental material properties in order to supply the basic input for numerical simulations;(vi)validate the design procedure and numerical model using data from bespoke tests at multi-scales;(vii)demonstrate the self-resilience system by using experimental and numerical data.(viii)realize applications in practice.A comprehensive self-resilience system was developed protecting against attacks in aggressive environments, resulting in concrete structures with durable and sustainable properties that far exceed those of conventional ones. Multi-scale self-healing and self-recovery systems, embedded in concrete, will provide the required resilience. Research at Shenzhen University will focus on the following key issues to address the major challenges related to the development and application of a microcapsule based on self-resilience systems in concrete structures, namely to:


Previous studies showed that type II cGMP-dependent protein kinase G (PKG II) could inhibit the activation of epidermal growth factor receptor (EGFR). Both c-Met and EGFR belong to family of receptor tyrosine kinases (RTKs) and have high molecular analogy. However, the effect of PKG II on c-Met activation is unclear. This study was designed to investigate the inhibitory effect of PKG II on the activation of c-Met and consequent biological activities. The results from CCK8 assay, Transwell assay and TUNEL assay showed that HGF enhanced cell proliferation and migration, and decreased cell apoptosis. Activated PKG II reversed the above changes caused by HGF. Immunoprecipitation and Western blotting results showed that PKG II could bind with c-Met and phosphorylate its Ser985, and thereby inhibited HGF-induced activation of c-Met and MAPK/ERK and PI3K/Akt/mTOR mediated signal transduction. When Ser985 of c-Met was mutated to Alanine for preventing phosphorylation of this site, the blocking effect of PKG II on c-Met activation was annulled. When Ser985 of c-Met was mutated to Aspartic acid for mimicking phosphorylation of this site, HGF-induced activation of c-Met was prevented. In conclusion, the results indicated that PKG II could block c-Met activation via phosphorylating Ser985 of this RTK. Gastric cancer is a significant public health problem worldwide due to its high incidence and mortality. Despite a universal decline in the incidence over the last several decades, this cancer remains the fourth most common cancer and is the second leading cause of cancer-related death worldwide , 2. Therc-Met, also known as hepatocyte growth factor receptor (HGFR), is another key member of RTK super family and is expressed in epithelial cells of many organs, including the liver, the pancreas and the prostate , 10. c-MType II cGMP dependent protein kinase G (PKG II) is a serine/threonine kinase and has inhibitory effect on cell proliferation and metastasis but promote apoptosis, especially in tumor cells , 19. RecThe expression c-Met is high in majority of tumor cells and the high expression makes it a potential marker for assessment of the degree of malignancy of gastric cancer . To confCell proliferation is an important characteristic of life activity, and excessive proliferation promote tumorigenesis. To investigate the role of HGF/c-Met in regulating proliferation of gastric cancer cells and the effect of PKG II on it, cell proliferation activity was analyzed by CCK8 kit, and the expression of proliferating cell nuclear antigen (PCNA) was detected by Western blotting in AGS and HGC-27 cells. The results from CCK8 kit analysis indicated that the proliferation was increased by stimulation with HGF , while the increase of PKG II activity through infecting the cells with Ad-PKGII and stimulating the cells with cGMP efficiently prevented the HGF-induced proliferation Figure . SimilarCell migration is a central process in the development and maintenance of multicellular organisms, but errors during this process have serious consequences, such as tumor formation and metastasis. Vigorous migration is the deviant tendencies of tumor cells. The results from transwell migration assay showed that HGF treatment increased the migration activity of AGS and HGC-27 cells, and the increased expression and activity of PKG II efficiently inhibited the HGF-induced migration. Figure .Matrix metalloproteinase-7 (MMP-7), as a member of the matrix metalloproteinase (MMP) family, is known to be involved in tumor metastasis and inflammatory processes. Hence, the expression of MMP7 can also reflect the invasion activity . The resApoptosis is the normal death that may occur in multicellular organisms. Apoptosis reduction promote the tumorigenesis and tumor progress . TUNEL mBcl-2 is an important anti-apoptotic protein and Bax is the protein promoting cell apoptosis . The ratThe activation of c-Met can trigger the signal transduction of MAPK signal pathway, initiating cell proliferation and migration . ERK1/2 MEK1/2 is a member of the dual specificity protein kinase family, which acts as a MAPK/ERK kinase , 34. ActThe PI3K/Akt/mTOR mediated pathway is an important intracellular signaling pathway in regulating the cell cycle . In multSince the above results showed that PKG II inhibited HGF/c-Met-triggered multiple signal pathways and cell biological activities, suggesting that the initial inhibition site of PKG II might be at the upstream of the pathways, i.e. the receptor c-Met. We applied Western blotting with the antibody against p-c-Met Tyr1234) to detect the c-Met activation under the HGF and/or PKG II treatments. The results revealed that the level of p-c-Met (Tyr1234) was increased distinctly in cells infected with Ad-LacZ and treated with HGF, and decreased in cells infected with Ad-PKG II, treated with cGMP and HGF, demonstrating that the increase of PKG II activity efficiently blocked the HGF-induced activation of c-Met.  antibody is the only available antibody to detect the serine phosphorylation of c-Met, and the prediction result from the Group-based Prediction System (GPS) software also showed that Ser985 is a potential site that PKG II act on c-Met. So we first analyzed the effect of PKG II on the Ser985 phosphorylation of c-Met. Cells were infected with Ad-PKG II and treated with 8-pCPT-cGMP, and then the total cell lysate was immunoprecipitated with anti-c-Met antibody and the phosphorylation level of c-Met at Ser985 was detected by Western blotting with antibody against p-c-Met (Ser985). It was found that there was an obvious increase of Ser985 phosphorylation of c-Met in the cells, suggesting that high expression and activity of PKG II increased Ser985 phosphorylation of c-Met Figure .On the basis of the above results, c-Met-S985A (Ser985→Ala) mutant, c-Met-S985D (Ser985→Asp) mutant and its wild-type (WT) counterpart in pIRES2-EGFP vector were created. In the c-Met-S985A mutant, 985 site could not be phosphorylated. In c-Met-S985D mutant, changing Serine 985 to Aspartic acid could simulate the phosphorylation of this site Figure To further confirm whether PKG II inhibit c-Met activation through Ser985, the effect of Ser985 mutation on Tyr1234 phosphorylation of c-Met was analyzed. COS-7 cells were sequentially transfected with mutant c-Met-S985A plasmid, infected with Ad-LacZ or Ad-PKG II, stimulated with 8-pCPT-cGMP, and treated with HGF for 10 min. The total cell lysate was harvested and immunoprecipitated with anti-GFP antibody, and then the Tyr1234 phosphorylation level was detected by Western blotting with anti-p-c-Met (Y1234) antibody. The results showed that PKG II had no obvious effect on HGF-induced Tyr1234 phosphorylation of c-Met, suggesting that the mutation of Ser985 to Ala could annul the inhibitory effect PKG II on Tyr1234 phosphorylation/activation of c-Met triggered by HGF Figure .In order to further prove that the phosphorylation of Ser985 inhibited Tyr1234 phosphorylation of c-Met, the Ser985 was mutated to aspartic acid (c-Met-S985D) to simulate the phosphorylation of this site. The wild type c-Met-WT and mutant c-Met-S985A and c-Met-S985D plasmids were transfected into COS-7, and the cells were treated with Ad-PKG II, 8-pCPT-cGMP and HGF as described above. The total cell lysate was collected, ectogenic c-Met protein was precipitated with anti-GFP antibody, and then the Tyr1234 phosphorylation level was detected. The results showed that HGF treatment increased Tyr1234 phosphorylation of c-Met and PKG II inhibited this increase in cells transfected with c-Met-WT (WT). The above inhibition effect was not observed in cells transfected with c-Met-S985A (S985A), indicating that mutating Serine 985 to non-phosphorylated arginine interrupted the inhibitory action of PKG II on activation of c-Met. In addition, in cells un-infected with Ad-PKG II, mutant c-Met-S985D suppressed Tyr1234 phosphorylation of c-Met stimulated by HGF. These results suggested that Ser985 phosphorylation of c-Met could block the Tyr1234 phosphorylation, and PKG II promoted c-Met phosphorylation at Ser985, thereby blocked the tyrosine phosphorylation/activation of c-Met Figure .Our previous study demonstrated that PKG II could inhibit the activation of some key members of the RTK family, including EGFR, VEGFR, PDGFR and IGF-1R, and the consequent signal transduction in gastric cancer cells . As anotIn the experiments, we investigated the effect of PKG II on HGF induced biological activities of gastric cancer cell, including the activation of c-Met, its downstream signaling of MAPK, PI3K/Akt and STAT mediated pathways, and the changes of proliferation, migration and apoptosis. First, we found that the activated PKG II suppressed the HGF-induced increase of proliferation and migration, and reversed the anti-apoptosis effect of HGF, suggesting that PKG II inhibited most HGF/c-Met enhanced biological activities. Further study showed that PKG II inhibited the HGF-induced signaling of MAPK/ERK and PI3K/Akt/mTOR mediated pathways, indicating that PKG II blocked the HGF/c-Met stimulated downstream signal transductions. The above results not only confirmed the inhibitory effect of PKG II on HGF/c-Met induced signaling and biological activities but also suggested that PKG II might inhibit the activation of c-Met.Next, the mechanism through which PKG II blocked the activation of c-Met was explored. To confirm the direct inhibition of PKG II on the activation of c-Met, it was imperative to make clear whether PKG II bound with c-Met and caused phosphorylation of it. The Co-IP result showed that PKG II bound with c-Met. When c-Met was enriched by IP and detected by Western blotting with antibody against pan Serine/Threonine phosphorylation, the results showed that activated PKG II could increase serine/threonine phosphorylation of c-Met. These results indicated that PKG II could directly cause phosphorylation of c-Met.et al. found that PKC induced Ser985 phosphorylation of c-Met could inhibit the kinase activity of this receptor and block HGF-triggered signaling [et al. reported that phosphorylation of Ser985 in c-Met inhibited tyrosine phosphorylation of this receptor [And then, further study was carried out to identify the PKG II specific phosphorylation site of c-Met. In 1994, Gandino ignaling . A few yreceptor . Thus, wIn Conclusion, this study proved the inhibitory effect of PKG II on activation of c-Met and the consequent signal transduction and biology activities, and elucidated the mechanism that PKG II suppressed c-Met activation. Combining our previous research results, we summarize that PKG II exerts a wide inhibitory effect on the activation of key RTK members, including EGFR, VEGFR, PDGFR, IGF-1R and c-Met. Therefore, PKG II may act as a potential wide range inhibitor for the above RTKs. This will throw new train of thought to planning anti-cancer strategy and discovering anti-cancer drugs.™ 2000 and E.coli BL-21DE3 were from Invitrogen . QuikChange Lightning Site-Directed Mutagenesis Kit was from Agilent Technologies . SanPrep Column Plasmid Mini-Preps Kit was from Sangon Biotech . Cell Counting Kit-8 (CCK8) was from R&S Biotechnology . In Situ Cell Death Detection kit (TUNEL Technology) was from Roche Diagnostics . Transwell Permeable Support was from Corning Incorporated . Electrochemiluminescence (ECL) reagents were from Millipore .Human gastric cancer cell line AGS and HGC-27, and African green monkey kidney fibroblast-like cell line, COS-7 were provided by the Institute of Cell Biology . Adenoviral vectors encoding the cDNA β-galactosidase (Ad-LacZ) and PKG II (Ad-PKG II) were kind gifts from Dr Gerry Boss and Dr Renate Pilz, University of California, San Diego, CA, USA. c-Met (NP_000236.2) Wild-type counterpart in pIRES2-EGFP vector were generous gifts from Dr Yi-Ching Wang, National Cheng Kung University, Tainan, Taiwan. Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco . The antibody against PKG II was from Abgent Biotechnology . The antibody against c-Met, p-Met (S985), RhoA, GFP, and horseradish peroxidase (HRP)-conjugated antibody against β-actin were obtained from Santa Cruz . Rabbit anti-p-MEK1/2 (S217/221) was from Cell Signaling Technology . Mouse anti-Rac1 was from BD Biosciences . Rabbit anti-phosphserine/threonine was from Abcam . Rabbit anti-p-Met (Tyr1234), p-ERK1/2 (Thr202/Tyr204), p-Akt (Thr308), p-PI3K p85 (Tyr458), p-mTOR (Ser2448), PCNA, Bax, Bcl-2, MMP-7 were from Bioworld Technology . The horseradish peroxidase (HRP)-conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories . The cellular permeable cGMP analog 8-pCPT-cGMP was from Calbiochem . HGF was from Sigma . The BioepitopeR protein A+G agarose IP was from Bioworld Technology . The cell transfection reagent Lipofectamin2. The medium was changed every two days and the cells were sub-cultured at confluence. On the day before infection, cells were freshly planted at 70–80% confluence, and the infection with Ad-LacZ and Ad-PKG II for 24 h was performed. Then the cells were serum-starved for 12 h, followed by treatment with 8-CPT-cGMP (250 μm/ml) for 1 h and then with HGF (50 ng/ml).Human gastric epithelial cell line GES-1, african green monkey kidney cell line COS-7 and gastric cancer cell lines AGS, HGC-27, SGC-7901 and BGC-823 were cultured in DMEM supplied with 10% FBS and maintained at 37°C in a humidified incubator with 95% air and 5% COP < 0.05 considered to be significant.Ten thousand cells (in 150 μl complete DMEM) were seeded in each well of a 96-well plate. After attachment, the AGS and HGC-27 cells were infected with Ad-LacZ or Ad-PKG II for 24 h. The cells were then washed and serum-starved overnight. Thereafter, the cells were incubated with 8-pCPT-cGMP (250 μΜ) for 1 h, and then treated with HGF (50 ng/ml) for 24 h. Approximately 10 μl of CCK8 dye was added to each well, and the plate was incubated for 30 min. The optical density (OD) at 450 nm was measured using microplate reader. Data were presented as a percentage of the value of control group, with 4 cells were seeded into the upper chamber containing culture medium without FBS. Cell migration to the bottom side of membrane was induced by medium containing 10% FBS in the lower chamber for 12 h at 37°C in a tissue culture incubator. The cells remaining in the upper chamber were carefully removed with cotton swabs. Migrated cells on the bottom side of the membrane were fixed in 4% paraformaldehyde solution for 30 min, stained in Giemsa solution for 10 min, and then rinsed in water. The stained cells were subjected to microscopic examination under a light microscope. Migrated cells were counted in 5 randomly selected fields per insert, and the values were averaged. All experiments were performed with three replicates under each migration condition.Migration activity of AGS and HGC-27 cells were detected by transwell system. After trypsinization, 5 × 10In Situ Cell Death Detection kit, following the manufacturer's instructions. Briefly, cells grown on 24-well plates were fixed and endogenous peroxidase activity was quenched with 2% H2O2 for 5 min. The cells were incubated with TUNEL reaction mixture  in a humidified atmosphere at 37°C for 1 h. The cells were washed with PBS and incubated with anti-fluorescein antibody conjugated with horseradish peroxidase for 30 min. After rinsing with PBS, the cells were stained with DAB solution, washed with PBS and then covered by cover slide. Under microscope, cells exhibiting brown nuclear staining were considered to be apoptotic. For each well, five × 200 fields were randomly selected and the positive cells in the fields were counted. The average of the positive cell numbers was taken as value of one experiment. The assay was repeated 3 times.TUNEL was performed with Point mutations Ser985→Ala (S985A), Ser985→Asp (S985D) were introduced using the QuikChange site-directed mutagenesis kit according to the manufacturer's protocol. The sequences of all generated constructs  were confirmed by sequencing. For transfection, COS-7 cells were sub-cultured the day before the process and the transfection of the cells with plasmids was performed by using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. After the transfection for 6 h, infection with Ad-LacZ or Ad-PKG II was performed.Protein samples were subjected to SDS-PAGE (8–12%) gel according to the molecular size of the protein and transferred onto a PVDF membrane. The PVDF membranes were blocked using 5% (w/v) non-fat milk in TBS-T for 1 h at room temperature. The incubation with the primary antibody was at 4°C overnight, followed by incubation with the corresponding secondary antibodies at room temperature for 1 h, with three washes after each incubation. The signal was visualized using ECL detection reagents.3 VO4) 48 h after transfection and/or infection. The supernatant was obtained by centrifugation . The supernatant was mixed with the antibodies or matched immunoglobulin as a negative control for 8 h rocking at 4°C. Fresh protein G conjugated to agarose was then added, followed by 2 to 3 h rocking at 4°C. The reaction mixtures were centrifuged at 2,000 rpm for 1 min at 4°C. The supernatant was then discarded, and the pellet was washed four times with binding buffer and then resuspended with the same volume of 2 × SDS sample buffer. The precipitates were probed with antibodies against the protein.The cells growing on 100-mm culture plate were washed two times with cold PBS and lysed by RIPA buffer .


Embedded fiber Bragg grating sensors have been extensively used worldwide for health monitoring of smart structures. In civil engineering, they provide a powerful method for monitoring the performance of composite reinforcements used for concrete structure rehabilitation and retrofitting. This paper discusses the problem of investigating the strain transfer mechanism in composite strengthened concrete beams subjected to three-point bending tests. Fiber Bragg grating sensors were embedded both in the concrete tensioned surface and in the woven fiber reinforcement. It has been shown that, if interface decoupling occurs, strain in the concrete can be up to 3.8 times higher than that developed in the reinforcement. A zero friction slipping model was developed which fitted very well the experimental data. The conventional concrete rehabilitation technique by means of betòn-plaques has been proven to be an effective strengthening solution in civil engineering applications but it has many disadvantages such as high weight and corrosion due to exposure to harsh environments that may eventually increase the overall maintenance cost. This has encouraged research on new materials and technologies that allow for a reduction of the installation costs. The use of fiber reinforced plastics (FRPs) or fiber reinforced cementitious matrices (FRCMs) for concrete beam strengthening and column confinement is an example of the results of the research efforts devoted to this important task during the last few years and represents a noteworthy improvement with respect to traditional methods. In this context, deployment of embedded fiber optic sensors (FOSs) as a real-time health monitoring measurement system is very attractive, since it might provide a powerful method for substantially improving the durability and the safety of the retrofitted structure ,2,3,4. TApplication of FOSs in concrete was first suggested by Mèndez et al. in 1989 , while tWhile the use of FOSs embedded in concrete has already been reported in several studies, their application for monitoring composite reinforcements used for concrete structures rehabilitation and retrofitting is much more recent. Sundaram et al.  and WangIn other two studies, Lau et al.  and ChanSo far, the actual strain transfer mechanism between reinforcement and concrete was never evaluated. Indeed, this is thought to be a key factor for forecasting the incipient failure of the strengthened structure, since the knowledge of the interface strain alone might be insufficient in most cases and does not give information of whether the stress limit in the concrete or in the reinforcement is approaching. In this paper the problem of evaluating strain sharing in strengthened concrete beams has been addressed using FBG sensors embedded both into the concrete tensioned surface and into the woven fiber reinforcement. It has been shown that strain in concrete can be much higher than that developed in the reinforcement if relative slipping occurs. A theoretical model which is helpful for interpreting the obtained experimental results is also discussed.BΔλ/Bλ) depends on either the applied strain variation or the temperature difference, as shown by the sensing equation [Bλ is the Bragg wavelength, pe the photo-elastic constant of the optical fiber, ξ the thermo-optic constant, αf the thermal expansion coefficient, ΔT the temperature interval  and Δε the total strain variation along the grating due to the combined action of external forces and apparent strain induced by differential thermal expansion of the optical fiber relative to the host structure. These constants can be condensed into two coefficients, εK and TK, which represent the strain sensitivity and the temperature sensitivity, respectively. If the two wavelength contributions of Equation (1) can be discriminated, it is then possible to measure simultaneously both temperature and strain.In-fiber Bragg gratings exhibit dual sensitivity since their output relative wavelength shift (equation :(1)ΔλBλThree standard laboratory size rectangular concrete beams were prepared for three-point bending tests to be carry out according to the ASTM C293-94 standard. The concrete beams were manufactured using a wooden form with suitable liquid disarming. The final dimensions of the specimens are 400 × 85 × 85 mm (L × W × H). The concrete mixture used for casting was determined in relation to the desired class of strength (C 32/40 MPa), employing aggregates of three different sizes (up to 15 mm). Afterward 24 h, the beams were removed from the forms and cured in water at 20 °C for 28 days.T) operates in a strain free condition and behaves as an optical fiber thermometer, allowing the measurement of the actual temperature, while the embedded grating (FBGε) gives out a signal which depends on either temperature or strain.After the aging, and before applying the reinforcement system, the beam was instrumented with a 1450 nm nominal wavelength FBG sensor  glued with epoxy adhesive into a small groove that had been machined longitudinally into the concrete surface see a. The opThe strengthening system consisted of a fiber reinforced cementitious matrix (FRCM) with polyparaphenylene benzobisoxazole (PBO) bidirectional (0°/90°) mesh woven , Italy) . It makeε. A dummy gauge in half-bridge configuration was used in this case for temperature compensation. The instrumented patch was finally laid-up on the tensile and shear surfaces  of the concrete beam using two layers of cementitious mortar . The PBO fabric was instrumented using two FBG sensors  having different nominal wavelengths (1501 nm and 1547 nm), intended to measure the longitudinal strain and the temperature variation, respectively b. The laWavelength shifts from FBG sensors were measured by a multiwavelength-referenced FBG interrogation system , which provides fiber optic sensors illumination by a broadband light-emitting diode (LED). The wavelength peak information is extracted from the reflected FBG spectrum through a single-fiber Fabry–Perot (FFP) tunable filter that scans the entire wavelength range; then the filtered output is electrically detected, differentiated and zero-crossing processed to achieve subpicometer wavelength measurement resolution. SG output was measured using a multi-channel acquisition system . Both FBG and SG values were recorded every second (1 Hz acquisition rate). −1 cross-head speed. While embedding of FBG sensors into concrete has already been proved to be effective ,2,3,4, m−1 cross-head speed. Three tests were performed to assess repeatability.Three-point bending tests were carried out by mounting the composite-reinforced concrete beams with 300 mm span test fixtures . Three mi)B × ch) is able to withstand the applied load until failure occurs;The whole cross-section of the concrete beam (ii)The tensile strength of the cementitious matrix is negligible with respect to that of the PBO.Then, the concrete beam and the FRCM could be considered as two de-coupled systems, which exchange shear stresses through the bonded interface  and on the homogenization coefficient of the mortar relative to the reinforcing fiber:The maximum tensile stress reached in the cross-section as the beam is loaded with a bending moment rI as:By writing the moment of inertia m acting through the concrete-mortar interface can be computed as:χ (χ = 6/5 for a rectangular cross section) and the homogenized section of the reinforcement rA, defined as:Finally, the average shear stress τcmσ) interface and at the mortar-PBO textile (fmσ) interface can be obtained as:Therefore, the stresses at the concrete-mortar  and (19), the strain components can be evaluated in any section of the beam as a function of the applied external load. PBO. Note the both values are close to that stated by the manufacturer . As load is increased, non-linearity can no longer be associated to beam loading/supporting adjustments. More importantly, results of bending tests underlined a marked difference between the strain values measured in the concrete tensioned surface and those recorded in the reinforcement, highlighting inefficient load transfer. This behavior may be explained by predicting a relative slipping between the concrete and the cementitious matrix. By assuming zero friction between the two materials, a complete decoupling will occur and the deflections of the two beams become independent from each other. Therefore, the bending moments into the two virtual beams are proportional to the respective bending stiffness. The observed experimental trends can be predicted by using the zero friction slipping model developed in The relative slipping between the concrete and the PBO mesh woven is mainly due to the peculiar characteristics of the mortar used as matrix of the FRCM system. Although the bond of a multi-filament yarn in a fine grained cementitious matrix is obviously controlled by the bond properties between filaments and matrix, more detailed information is needed to evaluate the mechanisms of such a complex system under a pull-out load. Recent pull-out tests conducted by Banholzer et al.  have bro1)Strain in concrete can be much higher than that developed in the reinforcement if slipping between the interface surfaces occurs.2)For FRCM strengthened concrete beams, almost complete de-coupling has been observed. A zero friction slipping model was developed which fit very well the experimental data.3)Otherwise, if perfect adhesion occurs, strains in concrete and in reinforcement tend to be similar.This paper discussed the problem of evaluating strain sharing in woven fiber strengthened concrete beams subjected to three-points bending in the linear elastic range. Fiber Bragg grating sensors were embedded both in the concrete and in the reinforcement. A theoretical model was also developed for interpreting the obtained experimental outcomes. The main findings can be summarized as follows:


Antibiotics were most frequently prescribed in the age group of 18–39 years (66.9%), followed by the age group of 65 years and above (59.0%), and the age group of 40–64 years (58.7%), p value < 0.001. Macrolides were significantly more likely to be prescribed for younger adults, while fluoroquinolones were more likely to be prescribed for patients 65 years or older. Duration of antibiotic use was significantly longer in older adults. Sex and race were not associated with antibiotic prescribing. Our findings highlight the urgent need to reduce inappropriate antibiotic use for uncomplicated acute bronchitis, particularly in younger adults.Reducing inappropriate antibiotic prescribing is currently a global health priority. Current guidelines recommend against antibiotic treatment for acute uncomplicated bronchitis. We studied antibiotic prescribing patterns for uncomplicated acute bronchitis and identified predictors of inappropriate antibiotic prescribing. We used the Epic Clarity database  to identify all adult patients with acute bronchitis in family medicine clinics from 2011 to 2016. We excluded factors that could justify antibiotic use, such as suspected pneumonia, COPD or immunocompromising conditions. Of the 3616 visits for uncomplicated acute bronchitis, 2244 (62.1%) resulted in antibiotic treatment. The rates of antibiotic prescribing were similar across the years,  Reducing inappropriate antibiotic prescribing is currently a global health priority. The 2015 United States National Action Plan for Combatting Antibiotic Resistant Bacteria set a goal to reduce inappropriate antibiotic use by 50% in outpatient settings, where the majority of antibiotic prescriptions occur ,4. AdherAs per guidelines from the American College of Physicians and American Academy of Family Physicians, antibiotic treatment is not indicated for uncomplicated acute bronchitis ,6. AntibOur study included 3616 visits representing 3134 unique patients over the period of 2011–2016. Most of the visits for uncomplicated acute bronchitis were made by the age group 40–64 years .Of the 3616 visits, in 2244 (62.1%) an antibiotic was prescribed. Of the 1469 excluded visits with a chest X-ray, 961 (65.4%) resulted in antibiotic treatment. Broad-spectrum antibiotics accounted for 97.8% of all antibiotic prescriptions. Macrolides (mostly azithromycin) were prescribed in 87.0% of all prescriptions, followed by amoxicillin-clavulanate (5%), fluoroquinolones (3.3%), and amoxicillin (2.3%). The remaining 2.3% of antibiotics included trimethoprim-sulfamethoxazole, cephalosporins, and clindamycin. The mean duration in days of prescribed azithromycin was 5.1 (standard deviation (SD) 0.6). The mean duration in days of amoxicillin-clavulanate prescription was 9.8 (SD 1.0).p value for trend = 0.07 .The rates of antibiotic prescribing for bronchitis were similar across the years, d = 0.07 . Antibio < 0.001 . The meap value = 0.6).Macrolides were more likely to be prescribed for younger adults, while fluoroquinolones were more likely to be prescribed for older patients (≥65 years). Duration of antibiotic prescription was significantly longer in older adults Sex was not associated with antibiotic prescribing in the logistic regression analysis . Race was also not associated with antibiotic prescribing in the logistic regression analysis  and by excluding factors that could justify antibiotic use such as suspected pneumonia , presence of immunocompromising conditions, and additional diagnosis for which antibiotics might be indicated. Furthermore, our study adds information on antibiotic choice, and duration of antibiotics prescribed, and the relationship between patient characteristics and antibiotic prescribing. We found that young, healthy adults are more often treated with antibiotics for acute bronchitis, despite the national guidelines recommending against this practice. Therefore, this group represents an important target for antimicrobial stewardship.Our findings call for implementation of strategies to reduce inappropriate antibiotic use in adult patients with acute bronchitis. Implementation of a decision support strategy for acute bronchitis reduced the overuse of antibiotics in a recent cluster-randomized trial in primary care . Other pThe study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Baylor College of Medicine on 20 June 2017 (H-39705).International Classification of Diseases, Ninth Revision (ICD-9) codes for acute bronchitis listed as a diagnosis: 466.0 (acute bronchitis and bronchiolitis) and 490.0 . The study included two private family medicine clinics in a large urban area. Approximately 19,777 patients attend 3248 appointments at these clinics each month. Patients in both clinics are predominantly female (58%) and white (54%). De-identified records were extracted from the Epic Clarity database . We selected all patients 18 years and older during the period of January 2011–August 2016 who had any of the two Patient visits were excluded from analysis if they had any of the following additional diagnosis codes for which antibiotics might be indicated: chronic bronchitis, emphysema or COPD, pneumonia, urinary tract infections, sinusitis, pharyngitis, skin, cutaneous and mucosal infections, acne, gastrointestinal infections, otitis media and miscellaneous bacterial infections, as well as visits with chest X-ray orders or abnormal vital signs  that could be consistent with pneumonia. Presence of immunocompromising conditions  was an additional exclusion criterion. Lastly, follow-up visits for bronchitis within 3 weeks of initial visit were excluded in order to prevent duplicating visits of a single episode.For each eligible visit, we extracted the following variables: patient age, sex, race, date of visit, type of antibiotic prescribed, and duration of treatment. The type of antibiotic was classified as either “narrow-spectrum” or “broad-spectrum”. Amoxicillin, cephalexin, trimethoprim-sulfamethoxazole, lincomycin derivatives (clindamycin) were categorized as “narrow-spectrum”. Amoxicillin-clavulanate, macrolides , 2nd to 4th generation cephalosporins, and fluoroquinolones were categorized as “broad-spectrum”.2 test for trend. We used the χ2 test to compare antibiotic prescribing between different patient age groups. Logistic regression analyses were used to determine independent predictors of antibiotic prescribing for acute bronchitis. Factors were regarded as being significant in the regression analyses when they had a p value of <0.05. Analyses were carried out using SPSS .Descriptive statistics were performed on the general characteristics of acute bronchitis visits, the type of antibiotics prescribed, and duration of treatment in each visit. We studied the trends in the prescribing of antibiotics over the years (2011–2016) using the Mantel–Haenszel χ


The influence factors of inoculum dose, yeast extract concentration and pH regulators  was investigated and discussed. Results showed that W. domercqiae can grow in the rice straw holocellulose hydrolysate, and acquire relative high SL yield of 53.70 ± 2.61 g/L in shake flask culture. Inoculum dose, yeast extract concentration and pH regulator made obvious influence on fermentation parameters, especially on final broth pH and SLs production. Furthermore, there is a strong negative linear correlation existing between final broth pH and lactonic SL or ratio of lac SL/tot SL. Additionally, comparison between SL production and non-glucose carbon sources, culture methods, microbes in previous reports was carried out. These results will be benefit for acquiring SL mixture with suitable lac SL/tot SL ratio for specific purpose and scope economically.A novel lignocellulose material, holocellulose from rice straw via the pretreatment of SO Candida apicola, Rhodotorula bogoriensis, Torulopsis gropengiesseri, Candida bombicola, Wickerhamiella domercqiae, Torulopsis bombicola, Pichia anomala, Candida batistae, Candida rugosa, Candida stellata, Candida floricola etc. were reported to produce SLs , an extracellular biosurfactant, are being applied in fields of detergent  was firstly introduced for SLs production by Ma et al. . Total S3 micro-thermal explosion” was proposed by our group and could be applied to pretreat rice straw, wheat straw and other lignocellulose. Preliminary work has proved that this method has advantages of high lignin removal rate, low consumption, less inhibitors and little wastewater generating  were investigated and discussed. Finally, the relationship between the final broth pH and lactonic SL or lac SL/tot SL ratio was fitted. The current study is an attempt to address substrates and main parameters of SL fermentation to arrive at an economically SL-producing procedure.The aim of the present work was to explore inexpensive substrate for SL production and obtain SL mixture with appropriate lac SL/tot SL ratio for various use. Firstly, rice straw pretreated by the method of SO3 micro-thermal explosion, ammonia wash, alkaline wash, water wash and drying processes, successively  with filter paper activity (FPA) of 67 FPU/mL was used. Before enzymatic hydrolysis, rice straw holocellulose (RSH) was desiccated at room temperature and subsequently milled to power in preparation. RSHH was obtained by hydrolyzing 10 % of RSH with the enzyme dose of 25 FPU/g of dry RSH in 0.2 M sodium acetate buffer (pH 4.8) at 45 °C and 150 rpm for 72 h. After hydrolysis, the liquid fraction was collected by centrifugation and the cellulase was inactivated at 80 °C for 0.5 h. All RSHH was stored at 4 °C prior to use.Wickerhamiella domercqiae var. Sophorolipid CGMCC 1576 was offered by Professor Song of Shandong University and now preserved in China General Microbiological Culture Collection Center (CGMCC). The seed inoculum was prepared by growing the organism in seed medium on a rotary shaker at 220 rpm for 16 h at 30 °C. The seed medium contained glucose 20, peptone 20 and yeast extract 10 (g/L). Then the seed culture was transferred to different fermentation media (50 mL in 300 mL flask) and cultivated for 7 days at 220 rpm at 30 °C.2PO4 1.00, Na2HPO4·12H2O 1.00, MgSO4·7H2O 0.05, and oleic acid 60.00 , was used for SLs production and set as the control group. For hydrolysate fermentation medium, rice straw holocellulose hydrolysate (RSHH) which containing 60.00 g/L of glucose was used to replace glucose as carbon source. Then, effects of different seed inoculum dose, yeast extract concentration and pH regulator on SLs production and lac SL/tot SL ratio in RSHH fermentation medium was investigated. The experiment was carried out in triplicate, respectively. Different seed inoculum dose of 1, 2, 4 %, yeast extract in different concentration  and different pH regulator  were added at the beginning of the cultivation. The other ingredients in RSHH medium were the same as that in the control group.The chemical defined fermentation medium, which containing ingredients  of glucose 60.00, yeast extract 3.00, KHThe chemical composition of rice straw and obtained holocellulose was analyzed by the method of NREL. The elementary analysis was determined by elemental analyzer . The experiments of chemical composition and elementary analysis all were carried out in triplicate and the results reported are mean ± SD of the three independent experimental results.3 micro-thermal explosion. The images were collected under the magnification of 1000 and 200 times, at the accelerating voltage of 20 kV.Scanning electron microscope  instrument was used to observe morphology variation of rice straw and confirm the pretreatment effects of SOn-butanol/ethanol/chloroform (10:10:1), and centrifuged at 8000 rpm for 10 min. The solid residue was washed twice with distilled water, and dried at 50 °C to a constant weight. All data were presented as the mean of three readings. For lactonic SL determination, two volumes of ethyl acetate were added to 0.5 mL fermentation broth firstly, after shaking and extracting, the lactonic SL in organic phase was measured by anthrone method. For total SL determination, 1.0 mL of ethanol was added to 0.5 mL broth to dissolve SLs, then the solution was centrifuged at 8000 rpm for 5 min, and total sugar content in the supernatant was quantified by anthrone method. Total SL production was calculated according to the glucose standard curve with glucose content of total sugar content minus residual glucose content  with an injection volume of 25 μL. Broth pH value was measured by PHS-3C pH meter . For biomass determination, 1.0 mL of culture broth was mixed with two volumes of W. domercqiae was studied. Several parameters including substrates, inoculum dose, yeast extract concentration and pH regulator was optimized to attempt to reduce the cost and make it more economical.In the present study, the composition or structural characteristics of holocellulose have been detected and analyzed firstly. And major fermentation influencing factors of SLs production by The differences in chemical, elemental and SEM analysis of rice straw before/after pretreatment were compared and the results were shown in Tables 3 micro-thermal explosion. As shown in Table We could see that rice straw contained 56.02 % of holocellulose and 14.00 % of lignin and lignin content reduced to 5.77 % in holocellulose after pretreatment. In other words, the delaminating rate of lignin reached as high as 58.79 % with the pretreatment of SONormally, chemical structure of lignocellulose mainly is carbon skeleton and main elements are hydrogen, oxygen, nitrogen and sulfur . Regarding the rice straw  in RSHH was effectively utilized in the early exponential phase and almost used up at the end of the fermentation.The optimum inoculum dose in RSHH medium dose for total and lactonic SL production was 2 and 4 %, respectively. Lactonic SL production and proportion of lactonic SL to total SL increased with increasing inoculum dose, which demonstrated that high inoculum dose is more suitable for lactonic SL production in RSHH medium. The highest total SL production of 42.33 ± 1.65 g/L, 3.90 % higher than the control group, was obtained with the inoculum dose of 2 %. Therefore, the optimal inoculum dose was set as 2 % in the follow-up experiments.Considering the effects of varying YE levels, the growth of yeasts, pH value of final broth and production of SL in RSHH fermentation medium containing different yeast extract (YE) contents were shown in Table Wickerhamiella domercqiae could survive and utilize sugars in RSHH even no extra nitrogen source adding (YE 0.00 %), which might due to the residual cellulase used in hydrolysis process was applied as nitrogen source.As shown in Table Total SL production increased with the increasing yeast extract concentration under 0.15 % and decreased with further increasing up to 0.30 %. The highest total SL yield of 53.70 ± 2.61 g/L and superior lactonic SL yield of 22.06 ± 5.33 g/L (the highest was 22.32 ± 2.37 g/L) was obtained with the YE addition of 0.15 %, respectively. Furthermore, yield of total SL still reached 41.53 ± 3.62 g/L without YE supplement, 1.94 % higher than that of control group. However, more acidic SL was synthesized and low ratio was obtained in this condition.All of the final broth pH of RSHH cultures was higher than that of control group and final broth pH value increased with the YE adding content. Except for the one without YE supplement (YE 0.00 %), maximum final broth pH, minimum total SL, lactonic SL and Lac SL/tot SL ratio of 4.77, 45.83 ± 2.00, 18.12 ± 0.48 g/L, and 39.53 % were obtained with 0.30 % of YE addition, respectively. Interestingly, the results also revealed that there could be certain relation between final broth pH, SL production and ratio within limits.In order to verify above speculation that final broth pH is negative related to lactonic SL and Lac SL/tot SL ratio, experiments on effects of different pH regulator on SLs production in RSHH were developed. Different pH regulators divided into acidifying agent, alkaline and buffer salt with the same addition was selected and applied to the RSHH medium, respectively. The influence of different pH regulator on SLs production was shown in Table 3 and mild acid like C6H8O7 were more suitable for lactonic SL production than alkali like NaOH and Lewis base . Meanwhile, high Lac SL/tot SL ratio of 47.08 and 48.84 % was gained, respectively. It is worth noting that the final broth pH dropped significantly to 3.89 than the others when AlCl3 was added and lactonic and total SL production reached 23.58 ± 4.79 and 50.09 ± 3.64 g/L, with an increase of 25.2 % and 23.0 % compared to the control group, respectively. Besides, lactonic SL production and Lac SL/tot SL ratio of the medium contained K2CO3 and CaO was much lower, in accordance with the trend of final broth pH. The results demonstrated that different pH regulator regulates SL production by changing pH value of the culture again.The results showed that different pH regulator have different effects on final broth pH and SL production. In general, comparable or higher SL production compared to the control group was acquired with different pH regulator. When the content of pH regulator was kept constant, Lewis acid like AlClAll the results above-mentioned suggested that there may be a certain correlation between final broth pH, lactonic SL and Lac SL/tot SL ratio in RSHH. The software of Minitab 17 was used to fit the correlation and the fitting curves and equations were displayed as followed Fig. .Fig. 2CoAs shown in Fig. Generally, glucose is common used as hydrophilic carbon source for SL production. Considering glucose is rather rarely found in larger quantities in wastes, which means non-recyclable and a high costs in SL production. In the present comparison, SL yield in non-glucose medium with different hydrophobic carbon sources, nitrogen sources and culture methods by various microbes are compared and exhibited in Table Candida bombicola ATCC 22214, are the most common strains for SLs production in various fermentation cultures. The production is significantly lower when only one carbon source was supplied than two types of carbon source were provided  and continuous fermentation (CF) are beneficial for SL accumulation than batch. Yeast extract with different contents are common used nitrogen source in the listed references. Currently, lignocellulosic materials are potential substrates for SL production and relative high yields could be obtained. Further optimization on the pretreatment of lignocellulose, culture modes and reactors would improve the fermentation results even more.3 micro-thermal explosion on rice straw, our previous study indicated that the lignin content was dropped dramatically and there are no or little fermentation inhibitors in RSHH (Yao and Li Series of reports showed that blockage of the lignin droplets on the surface of the cellulose was the main cause of cellulase inhibition. Besides, the intensive structure of rice straw also hinders the attack of cellulase (Xing et al. Various factors were reported to affect sophorolipid accumulation, including C/N ratio, nitrogen source addition, temperature and oxygen supply (Stüwer et al. As the increase of inoculum dose, the biomass was increased and the final broth pH was decreased. This phenomenon was usually ascribed to the increasing organic acid amount generated during more prosperous biological metabolism (Gupta and Prabhune Nitrogen type and content were reported to have extraordinary influences on SLs production and composition (Cooper and Paddock C. apicola (Stüwer et al. W. domercqia (Ma et al. 3C6H5O7·2H2O showed a decrease in lactonic SL production by W. domercqia in RSHH. Furthermore, a strong negative correlation existing between final broth pH and lactonic SL or Lac SL/tot SL ratio was found. It demonstrated the feasibility of SLs obtainment with proper Lac SL/tot SL ratio for specific purpose and scope in a simple, cost efficient and sustainable way.Opposite to W. domercqia, biosynthesis of SL production and the enzymes activities in different conditions.Based on the above, inoculum dose, yeast extract addition and pH regulator can be regarded as factors involved in the complex regulation of sophorolipid accumulation, and the adjustment of final broth pH was the ultimate way to achieve suitable sophorolipid mixture. These factors affected the final broth pH may be closely related to biological metabolism of Exploring the cheaper substrate to instead of glucose and optimizing the fermentation process was the main aim of this work, of course, which was the same meaning and purpose to the similar articles. For the overview of SL yield in non-glucose medium with different hydrophobic carbon sources, nitrogen source additions and culture methods, a lot of cheap wastes were studied containing sugars, sugar-producing biomass and lignocellulose wastes from crops. Considering the Sustainable development, harmfulness on environment and economic benefits etc. factors, DCCR, CCR and RSH etc. lignocellulosic materials from crops were regarded the most potential substrates. Besides, we believe that the recycling of crop waste was the inevitable trend in the future.


A 60-year-old African-American male presented to the emergency department with abdominal pain and distention associated with decreased appetite and weight loss for several weeks. A computed tomography (CT) scan of the abdomen and pelvis showed an 8 cm mesenteric mass with surrounding stranding and poorly defined borders. The patient underwent exploratory laparotomy and complete resection of the mass since the frozen section could not give a definite diagnosis. Histopathology showed fibro-adipose tissue with lymphoid hyperplasia, vague nodular collections of foamy histiocytes with giant cell reaction, marked chronic inflammation, fat necrosis, and prominent sclerosis/fibrosis. Methenamine silver and acid-fast stains were negative for fungal and mycobacterial organisms respectively. Examination of tissue with immunohistostains showed increased immunoglobulin G4 (IgG4)-positive plasma cells. Other features observed were scattered areas of phlebitis, pockets of tissue eosinophilia, and focal storiform fibrosis leading to the diagnosis of IgG4-related sclerosing mesenteritis. The patient did not require steroids after the surgical resection and was disease free at six-month follow up. Immunoglobulin G4 (IgG4)-related sclerosing mesenteritis is one of the manifestations of a rather uncommon IgG4-related disease (IgG4-RD). It is characterized by chronic inflammation and fibrosis resulting from deposition of IgG4-positive plasma cells in affected tissues. Other organs commonly involved in IgG4-RD are pancreas, orbit, salivary glands, and retroperitoneal structures. Here, we present a case of IgG4-related sclerosing mesenteritis presenting with a mesenteric mass.A 60-year-old African-American male with a past medical history of a stab wound to the abdomen, post laparotomy years ago, presented to the emergency department with abdominal pain and distention for seven weeks. The pain was characterized as continuous, involving all quadrants, nagging, nonradiating, 7/10 in intensity, associated with decreased appetite, bloating, constipation, and weight loss of 50 lbs over seven weeks. The patient denied fever, chills, night sweats, nausea, vomiting, and blood in the stool. The patient endorsed shortness of breath on exertion and disturbed sleep due to abdominal distention. The patient had colonoscopy 1.5 years ago and reported it as normal. The patient denied tobacco, alcohol, illicit drug abuse, and he worked as a security guard. The patient’s vital signs were within normal limits and his physical examination was remarkable for abdominal distention with mild tenderness on deep palpation in all quadrants with rebound tenderness. Laboratory findings showed mild anemia with hemoglobin of 11.8 g/dL, hematocrit of 37%.The patient first underwent computed tomography (CT) scan of the abdomen and pelvis with intravenous contrast that showed an 8 cm mesenteric mass with surrounding stranding and poorly defined borders, no bowel ileus or obstruction but thickening of the descending colon through the sigmoid, with mild surrounding stranding Figure .Oncology and general surgery services were consulted and the decision was made to proceed with exploration laparotomy and biopsy of mesenteric mass with frozen section. No metastatic disease or ascites were noted. There was a mass at the base of the mesentery involving the transverse mesocolon overlying the middle colic as well as the superior mesenteric artery. The mass area was ligated and wedge resection was performed and sent for frozen section. Tru-Cut biopsies of the retroperitoneal area were also sent. The pathologist was unsuccessful in identifying a diagnosis intraoperatively as what was mostly seen was fibrosis. More specimen was required, hence careful dissection was started. The base of the mesentery was opened and the mass was then carefully dissected. Resection was continued along the mesentery to the small bowel where the mass, as well as the small bowel, was excised. The results of the preliminary biopsy of the mesenteric mass and omentum showed fibro-adipose tissue with lymphoid hyperplasia, vague nodular collections of foamy histiocytes with giant cell reaction, marked chronic inflammation, fat necrosis, and prominent sclerosis/fibrosis Figure .Immunohistochemistry showed CD20 and paired box containing (PAX) 5-stained lymphoid follicles and scattered B cells, CD3-stained interfollicular areas and scattered T cells, and CD56 stained histiocytes (foam cells). Pan-cytokeratin stain was negative. Methenamine silver and acid-fast stains were negative for fungal and mycobacterial organisms respectively. Flow cytometry showed a mixed population of polytypic B-cells (32%) and T lymphocytes (49%) with no pan-T cells, no pan-T cell antigen deletion or B cell light chain restriction detected. Consultation with John Hopkins reference laboratory was obtained and immunohistostains done there showed increased IgG4-positive plasma cells  . The patient had a protracted hospital course requiring two additional laparotomies for resection of the ischemic small bowel.The patient was discharged with oncology outpatient follow up. A CT scan of the abdomen with contrast at three months showed a residual soft tissue mass-like density within the mesenteric fat anterior to the third portion of the duodenum measuring 6.4 x 2.7 cm. A CT scan of the abdomen repeated six months after the surgery showed that the mass-like density in the mid-abdomen had mostly resolved.IgG4-RD represents a relatively recently recognised disease that has several features: a tendency to form tumefactive lesions in multiple sites, a characteristic histopathological appearance, and often, but not always, elevated serum IgG4 concentrations . The diaOur patient represents a case of IgG4-related sclerosing mesenteritis. Pathologic descriptions and case reports of this clinicopathological entity started to appear in the literature in the last decade -8 but stClinically, patients with IgG4-related sclerosing mesenteritis present with similar symptoms as patients with classic sclerosing mesenteritis; chronic abdominal pain -4, 6-8 aUsually, these patients are found to have an abdominal mass during the imaging of the abdomen and subsequently undergo laparotomy as diagnosis which is difficult to establish without pathological examination -4, 6-8.The histologic characteristics of this mass included lymphoid hyperplasia in the mass, the presence of storiform fibrosis, and areas of phlebitis with vessel obliteration. There were, however, focal areas of fat necrosis but overall the tumor meets all three histopathological features for IgG4-RD . There wThe treatment of IgG4-related sclerosing mesenteritis is not well established because of the rarity of the condition. However, there is an increasing data from case reports and case series on the treatment of IgG4-related autoimmune pancreatitis which is the most commonly diagnosed form of IgG4-RD, and also, we have historical data from case reports and case series of patients with “nonspecific” sclerosing mesenteritis. We, however, acknowledge that IgG4-related sclerosing mesenteritis is a separate entity and needs more studies regarding treatment outcomes before evidence-based guidelines can be created. The therapy of choice for IgG4-related disease is corticosteroid treatment, and it was effective in some of the published cases , 8; howeOur case represents a case of IgG4-related sclerosing mesenteritis presenting as a mesenteric mass, which meets the histologic criteria for this rare disease and is compatible with several other cases described in the literature. More observations are needed to better define the natural course and optimize treatment for this illness.


Heroin use in the United States has reached epidemic proportions. The objective of this paper is to estimate the annual societal cost of heroin use disorder in the United States in 2015 US dollars.An analytic model was created that included incarceration and crime; treatment for heroin use disorder; chronic infectious diseases  and their treatments; treatment of neonatal abstinence syndrome; lost productivity; and death by heroin overdose.Using literature-based estimates to populate the model, the cost of heroin use disorder was estimated to be $51.2 billion in 2015 US dollars . One-way sensitivity analyses showed that overall cost estimates were sensitive to the number of heroin users, cost of HCV treatment, and cost of incarcerating heroin users.The annual cost of heroin use disorder to society in the United States emphasizes the need for sustained investment in healthcare and non-healthcare related strategies that reduce the likelihood of abuse and provide care and support for users to overcome the disorder. The widespread availability and use of heroin has created a major public health crisis in the United States. The number of heroin users doubled from 2000 to 2013, rising from 1.0 per 1,000 persons in 2000 to 2.0 per 1,000 persons in 2013; heroin overdose deaths have also more than tripled since 2002. There arThe economic impact of heroin use disorder (the contemporarily accepted term for heroin addiction) to society in the context of the ongoing heroin epidemic is unclear. Characterization of the current economic burden of heroin use disorder is important to understanding the magnitude of its impact, which can subsequently inform the extent to which resources should be directed towards mitigating the devastating impact of heroin use disorder. Thus, the aim of this paper is to estimate the annual societal cost of heroin use disorder in the United States in 2015 US dollars; specifically, we sought to calculate the amount of money society would save if heroin use disorder was eliminated.An analytic model was designed in TreeAge Pro 2017  to calculate the societal cost of prevalent heroin use disorder using the most recently available data . The modIn the model, the overall population with heroin use disorder was stratified into incarcerated population and non-incarcerated populations. Throughout this study, the phrase “heroin user(s)” refers to users with heroin use disorder as we have operationalized it unless as a citation to another study, for which we defer to the original researchers' definitions; the same is also true with the application of “heroin use” in this study. Subsequently, the model was structurally identical and only differentiated by the prevalence rates of health state occurrence. Each incarceration group was then stratified by the treatment of heroin use disorder. The infectious disease branches followed the heroin use disorder treatment branches in a similar arrangement . CertainMutually exclusive and exhaustive events occurred by literature-identified probabilities at each branching point shown in Medline, Google Scholar (each with results limited to English), and US governmental agency websites were searched to identify model inputs. The model inputs included: number of heroin users in the general and prison populations (used to estimate proportion of incarcerated users); prevalence rates, treatment costs, and treatment rates for heroin use disorder, infectious diseases, and NAS associated with heroin use; cost of incarceration; productivity loss due to heroin use or incarceration; crime costs associated with heroin use; cost of heroin to users; mortality due to heroin overdose; and probability and cost of heroin-related overdose .Costs of treatment for heroin use disorder, overdose, NAS, and infectious diseases were assumed to be the same amongst the incarcerated and non-incarcerated populations; these treatment costs were weighted by the rates of treatment for the condition to calculate an expected treatment cost for patients  and 1.5 Using 100,000 samples in a Monte Carlo simulation with the top 9 model inputs that the model results are most sensitive to in one-way sensitivity analyses, the mean per-user cost was $57,115.76 with a standard deviation (SD) of $5,619.45 ; the cost for the overall population was $52.1 billion .The US societal cost of heroin use disorder was estimated to be $51.2 billion for 1,008,000 heroin users  in 2015 US dollars. To put the overall annual economic in context, we present the annual societal costs of some other chronic illnesses. For example, chronic obstructive pulmonary disease (COPD) was estimated to cost $2,567 per patient ($38.50 billion in 2015 dollars for 15 million patients), and diabetes was estimated to cost $11,148 per patient ($248.59 billion in 2015 dollars for 22.3 million patients)., 55 Our A previous study by Mark et al. estimated the societal cost of heroin addiction (the previous term for heroin use disorder) to be $21.9 billion in 1996 dollars  of heroin users abused at least 1 other substance . AdditionAlthough the model was developed while considering both available data and clinical knowledge, some conditional probabilities of disease prevalence were not found in the literature; in the absence of these figures, certain assumptions were made . For exaThe downstream externalities of heroin use disorder, treatment of chronic infection(s), and treatment of heroin use disorder were not captured in this model. Treatment of these patients for their disease(s) may drastically reduce their chances of transmitting the viruses to others , 64, andThe generalizability and reliability of estimates for some model inputs were limited, as the source studies were often conducted in a single location with a small sample size. It is challenging to conduct studies on drug using populations , particularly imprisoned heroin users., 67 For A major strength of this study was the consistent application of a prevalence-based approach to estimating cost of illness and careful choice of inputs based on stated criteria for the population of interest. Additionally, the model-guided calculation of cost of heroin use disorder in this study allowed for the detailed determination of productivity loss and treatment costs by health state. Mark et al. chose to sum relevant costs and applied a different framework than the approach of this study and could then include a wider scope of costs. AlthoughSources vary in the prevalence of heroin use disorder, depending on the definition by the researcher; 200,000 incarcerated heroin users, 324,000 In choosing a base-case value for the number of individuals with heroin use disorder, we expected past-year users/users with heroin use disorder and current/physically dependent users to have similar societal costs, and exclusion of users who were not heroin dependent nor actively currently using heroin from the study would severely underestimate the cost of heroin use disorder. Additionally, defining chronic heroin use as those who used at least 4 times in the past month may be oThe range of 324,000 (people currently using heroin) to 1.5 mThe total cost was also sensitive to the cost of HCV treatment. Because the cost of HCV treatment has recently increased dramatically due to the introduction of several novel HCV treatment options, this aspWithout meaningful public health efforts, the number of heroin users is likely to continue to grow; the downstream effects of heroin use, such as the spread of infectious diseases, 62 and Despite the limitations of this study, many of which are inherent to cost of illness studies, the study provides important evidence to inform policy on combating the heroin epidemic. The societal cost of heroin use disorder has not been characterized since 1996, and the results of this study use the most recently available data and trends to provide a cost estimate of the burden of heroin use disorder to society in the United States. Even with a comparatively narrow perspective, heroin use disorder exacts a tremendous cost to society at $50,799 per user. Possible targets for reduction of the societal cost of heroin use disorder such as reducing the overall number of heroin users, reducing the proportion of users who are incarcerated, and others were also identified. Downstream effects of heroin use such as newly acquired infections due to needle sharing or high-risk sexual behaviors and the effects of heroin use disorder and chronic infectious disease treatments were not captured in this study; additional research is needed for better characterization of these outcomes.S1 Calculations(DOCX)Click here for additional data file.S1 List(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


MET amplification appears to be a predictive biomarker for MET inhibition. Prior studies reported a MET amplification rate of 9–18% in metastatic colorectal cancer (mCRC) but do not differentiate increased gene copy numbers due to chromosomal level aberrations from focal gene amplifications. Validation of MET amplification rate in mCRC is critical to this field.n = 53) was 22.6% (12/53) and was significantly higher compared to patients not exposed to anti-EGFR therapy (p < 0.001).In tumor tissue-based analyses, overall MET amplification rate was 1.7% (10/590). MET amplification was seen in 0/103 (0%), 4/208 (1.9%) and 6/279 (2.2%) cases, in cohorts 1, 2 and 3, respectively. Rate of MET amplification in cfDNA of cohort 4 patients refractory to anti-EGFR therapy (n = 795) using different methods across multiple cohorts. Cohort 1 (n = 103) and 2 (n = 208) included resected liver metastases and tumor biopsies, respectively, tested for MET amplification using fluorescence in-situ hybridization [amplification: MET/CEP7 ratio ≥ 2.0]. Using another tissue-based approach, cohort 3 (n = 279) included tumor biopsies sequenced with HiSeq (Illumina) with full exome coverage for MET [amplification: ≥ 4 copies identified by an in-house algorithm]. Using a blood-based approach by contrast, cohort 4 (n = 205) included patients in whom the full exome of MET in circulating-free DNA (cfDNA) was sequenced with HiSeq.We analyzed MET amplification in mCRC (Contrary to prior reports, in this large cohort, MET amplification was a rare event in mCRC tissues. In plasma by stark contrast, MET amplification identified by cfDNA occurred in a sizable subset of patients that are refractory to anti-EGFR therapy. Due to iin vivo. , 5–7 METin vivo.  Consequein vivo.  The mostastases. .In this study, we examined a large number samples from mCRC cases across multiple cohorts to identify the frequency of MET amplification as determined by different methodologies along with a novel exploratory determination of MET amplifications in circulating cell-free DNA.p = 0.34), FISH and sequencing (p = 0.53) and primary  and metastatic sites  (p = 0.097)  of 590 tumor tissue biopsies tested by both FISH and sequencing. MET amplification using FISH was seen in 0/103  and 4/208  cases in cohorts 1 and 2, respectively (MET/CEP7 ratio: 2.0–7.7). MET amplification using sequencing was seen in 6/279  (MET gene copy numbers (GCN): 4.0–6.7) (Table ) Figure . Mutatio) Figure .p < 0.001)  or RAS wild-type tumors without prior anti-EGFR antibody exposure (p = 0.018)  cases (Table ) Figure . Further) Figure . No diff) Figure .de novo mCRC.In this large cohort of mCRC patients, we failed to validate the high prevalence of MET amplification in tissue samples as reported in prior studies with either FISH or sequencing.  ContraryDiscrepancies between our study and others can be explained by the limitations of PCR-based assays in detecting copy number. Both gene amplification and polysomy can result in increased GCNs in tumors. Experience in breast cancer reveals these two phenomena to be mechanistically distinct. Cases with Chromosome-17 (site of HER2/neu gene) polysomy compared to focal gene amplification of HER2, have lower GCNs, lower level of HER2 protein expression, features more consistent with HER2 negative tumors and do not derive any benefit from HER2-targeted treatment. , 13 Simide novo mCRC and therefore a difficult target for enrichment trials with MET targeted therapy. We also recommend FISH or NGS with adequate coverage as ideal tests for assessment of MET amplification in mCRC.Gain of 7q without any focal amplifications in MET in the TCGA cohort further supports for our findings.  LikewiseDespite the low prevalence of MET amplification in mCRC, we found that a substantial subset (23%) of patients with mCRC, who have been treated and are refractory to anti-EGFR antibodies, acquire MET amplification, as detected in cfDNA. Bardelli et al. showed that acquired MET amplification in tumor tissue is associated with acquired resistance to anti-EGFR antibodies in patients of mCRC.  Our findFuture efforts should focus on refining evaluation of MET amplification. Using FISH for amplification is not without its drawbacks. Since FISH assessed increase in CEP7 copies > 3 may reflect either true polysomy or centromeric amplification, newer methodologies, such as multiplex ligation-dependent probe amplification that allow simultaneous quantification of multiple loci, can help detect focal MET amplifications that are missed by FISH and may be effective adjunct evaluation in clinical trials.  Furthermn = 103) comprised of newly diagnosed mCRC patients with resected liver metastases. Cohorts 2 (n = 208) and 3 (n = 279) contained refractory mCRC patients with pre-treatment tumor tissue biopsies (both primary and metastatic sites). [n = 205) included mCRC patients with prospectively collected plasma after at least one-prior line of therapy . Focal MET amplification was defined as MET/CEP7 ratio ≥ 2.0. In cohort 3, pre-treatment FFPE tumor tissue was sequenced with HiSeq (Illumina) with full exome coverage for 202 genes (average depth 800) including MET. MET amplification was defined as ≥ 4 copies identified by an in-house algorithm, as previously described. [®), using methodologies for amplification determination previously reported. [In cohort 1 and cohort 2, pre-treatment formalin-fixed paraffin embedded (FFPE) tumor tissue was tested using fluorescence scribed.  In cohoreported.  All testThe study was conducted in accordance with the guidelines of the MD Anderson Institutional Review Board.P value of 0.05, when appropriate was considered significant. Statistical analyses were performed using SAS version 9.3 (SAS Institute Inc.).The primary objective was to determine prevalence of MET amplification in mCRC in tissue-based and blood-based biopsies. MET amplification rate was summarized using percentages and 95% confidence intervals (CIs). Categorical variables were compared using Fisher exact tests. All statistical tests were two-sided, and a de novo MET amplification occurs rarely in mCRC, in contrast to previously reported claims. However, acquired MET amplification can be identified by cfDNA in a significant subset of mCRC patients that are refractory to anti-EGFR antibodies. We therefore, conclude that MET amplification appears to play a minor role in de novo colorectal carcinogenesis but may play an important role in acquired anti-EGFR resistance. These findings have clear implications for identifying patient populations and for designing appropriate clinical trials using MET inhibitors in mCRC.In this large cohort of mCRC, we observed that