In the past few decades, due to the exponential increase of the world’s population, the number of discarded waste tires has become a serious ecological and environmental problem. Decomposition of waste tire rubber can take longer than 50 years, and every year the number of discarded tires is rapidly growing. With the inclusion of waste tire rubber into self-compacting concrete this global problem can be reduced. Waste tire rubber can be incorporated in self-compacting concrete by partially replacing the natural fine and coarse aggregate, reducing consumption of sand and gravel and preserving these natural materials. In addition, recycling and reusing waste tire rubber avoids the need for tire landfilling, as one of the major ecological problem of the near future. Replacement of natural aggregate with waste tire rubber can have an undesirable influence on the mechanical properties of self-compacting concrete, i.e., compressive strength, flexural strength, splitting tensile strength, and modulus of elasticity, however. On the other hand, replacing natural gravel or sand with waste tire rubber can improve impact resistance, ductility, and fatigue resistance. This paper presents an overview of the literature investigating recycled waste tire rubber used as a fine and/or coarse aggregate replacement in self-compacting concrete and its influence on several essential fresh and hardened self-compacting concrete properties. The number of unusable waste tires from different kinds of vehicles is rapidly growing and is in fact turning out as one of the major ecological and environmental problems of the present day. Nearly one-billion waste tires are discarded each year , and areFurthermore, common ways of disposing of the scrap tires, such as landfilling and burning, may cause grave ecological problems, either because of the fast depletion of the site or air pollution, respectively ,7,8. AccDue to the fact that the waste tire rubber, as a non-biodegradable material, has a relatively long lifetime, interest in replacing natural river aggregate in concrete mixtures with rubber derived from waste tires, i.e., rubberized concrete (RC), has attracted the attention of civil engineers and building industry to provide environmental-friendly concrete with recycled tire rubber.3, respectively [The concrete industry is one of the biggest industries in the world. According to the European Ready Mixed Concrete Organization statistics (ERMCO), the average concrete production in 2015 in the European Union (EU), Russia, USA, and Japan was 344.3, 62.0, 365.0 and 138.0 million mectively . Overallectively . From anSimilar to traditional concrete, self-compacting rubberized concrete however has lower levels of emitted radiation and thus safer to building users. Because of its high ductility, improved impact resistance, and energy dissipation properties , rubberiThe main objective of this review article is to provide an overview of fresh and hardened properties of self-compacting concrete with partially replaced fine and/or coarse aggregate with waste tire rubber. The fact that fine and coarse aggregate makes approximately 50% of weight of the self-compacting concrete can be used as a motivation to thoroughly study the influence of partially replacing natural aggregate with waste tire rubber, not only to provide lightweight self-compacting concrete (SCC) and to preserve natural resources, but also to maintain or even improve the mechanical properties and durability of SCC.Currently, there is no standardized methodology to design mixes of self-compacting rubberized concrete . Howeverall dry mix components should first be mixed from 1 to 5 min before progressively adding water;after water is added, mixing should continue for another 3 to 5 min;satisfactory results can be obtained by mixing aggregates and powder for 30 s, without adding any water, then adding one-third of the required superplasticizer, and then mixing for an additional 90 s; and,final step is to add the remaining amount of superplasticizer with all other admixtures and mix for an extra 210 s.So far, there is no mixture design method that fully meets all of the usual requirements, i.e., widely applicable, fit a variety of technical requirements, sustainability, strong robustness for variable raw materials, and cost . HoweverChipped/shredded rubber aggregate (coarse rubber), used as coarse aggregate replacement, i.e., replacement for natural gravel; rubber particles are between 13 and 76 mm.Crumb rubber, commonly used as sand replacement; grain size is between 0.425 and 4.75 mm.Ground rubber, used as cement replacement; grain size is less than 0.425 mm.Fibre rubber aggregate; the shredded rubber is in the form of short fibres with an average of 12.5 mm length.Pretreatment of rubber aggregate is also desirable to improve adhesion between waste tire rubber and cement matrix. Waste tires have been used in experimental investigations to partially replace the aggregate in traditional concrete and self-compacting concrete for two and a half decades. Mostly, fine and coarse aggregate were replaced with rubber aggregate from mechanical grinding of end-of-life tires. Rubber particles in concrete are obtained from waste tires. There is a mechanical procedure occurring either at cryogenic or ambient temperatures. As can be seen in Regardless of rubber type, rubber size, rubber replacement level, and replaced material, most authors have reported similar fresh SCC behaviour, i.e., a reduction in flowability, passing ability and segregation resistance, and an increase in viscosity with an increase of rubber content. A few authors have claimed essentially the opposite, i.e., Mishra and Panda  reportedReplacement levels, as given in To improve the bond connections between rubber aggregate and binder, Ganesan et al.  pretreatMost of the test results that are discussed above were performed in accordance with the European Guidelines for Self-Compacting Concrete , which mUygunoğlu and Topçu  reportedFrom the test results that are discussed in Güneyisi et al.  investign (Herschel-Bulkley model) and coefficient c/μ (modified Bingham model) were observed. While considering that values of exponent n and coefficient c/μ for all SCC mixtures with rubber aggregate were greater than one (n > 1) and greater than zero (c/μ > 0), shear thickening behaviour was noticed. Decreasing rubber content in SCC mixture systematically reduced exponent n and coefficient c/μ, which means a greater shear thickening behaviour. The same behaviour occurred with replacement with different types of sizes of rubber aggregate. With rubber aggregate sizes No. 18, No. 5, No. 18 + No. 5, and tire chips, an increase in exponent n was from 0.253 to 0.391, 1.054, 0.876 and 1.788, respectively, while the increase in coefficient c/μ was from 1.135 to 1.205, 1.410, 1.363 and 1.543, with increase in rubber content ranging from 0% to 25%, respectively.Further, Güneyisi et al. found that the rheological properties of fresh SCRC mixtures were interpreted with two types of models, Herschel-Bulkley model and modified Bingham model. Variations in values of the exponent As in Reduction in the compressive strength of SCC is inevitable when different types of rubber aggregate were used as a partial replacement of natural aggregate, regardless of rubber particle size, rubber replacement level, or replaced type of aggregate. In all cases, a reduction in compressive strength was reported and with higher level of rubber replacement, reduction in compressive strength was more pronounced, as shown in However, in several experiments in which supplementary cementing materials (SCMs) were used, enhancements in SCRC compressive strength was reported. Metakaolin (MK) was found to be the best SCM in terms of improvement of SCRC compressive strength ,37. Whenfc) lower than the limit for structural applications, i.e., fc > 17 MPa [According to the test results that are given in > 17 MPa , but theIn most cases, several authors have reported reduction in flexural strength by implementing waste tire rubber as partial replacement of natural fine or coarse aggregate in SCC. For the same rubber replacement level, flexural strength reduction was similar, irrespective of rubber particle size, as shown in From the test results of different authors, it can be undoubtedly noted that there is a reduction in the splitting tensile strength with increased amount of rubber aggregate in test specimens, regardless of rubber particle size, replacement level, or replaced material, as shown in Replacement of natural fine (FA) and coarse aggregate (CA) with rubber aggregate had a negative influence on the static and dynamic modulus of elasticity. The static and dynamic modulus were reduced with an increasing rubber replacement level. This concrete behaviour can be attributed to air entrainment caused by rubber particles and lower modulus of elasticity of rubber aggregate than natural fine or coarse aggregate modulus . Rubber The influence of rubber aggregate on impact resistance of SCC was studied by AbdelAleem et al. . They reKhalil et al.  also invAs a result of an insufficient number of experimental investigations carried out on impact resistance of SCRC, several investigations of impact resistances of rubberized concrete (RC) are given below, in order to compare the two concrete types and conclude whether similarities or between them.Pedro et al.  reportedThus, to summarize, the inclusion of rubber particles in SCC or traditional concrete appears to cause enhancement in impact resistance. This performance can be attributed to the low stiffness of rubber particles, which causes increase in post-cracking resistance and enhances the ductility of the SCC specimens ,33. HoweHilal  reportedDue to lack of experimental results, this hardening property of SCC with waste tire rubber still needs more investigation. However, other authors also investigated the ductility of traditional concrete with waste tire rubber replacing fine or coarse aggregate and results of these authors are given below. Vadivel et al.  reportedZheng et al.  reportedThe test results that are analysed above are summarized in Hilal  reportedGesoğlu et al.  reportedGesoğlu et al.  tested tFrom the above, it can be concluded that test results from both types of rubberized concrete, SCC and traditional concrete, gives scattered results. Increases and decreases in fracture energy were reported, and thus it cannot be clearly concluded whether rubber inclusion improves or reduce fracture energy. However, with a proper amount and rubber particle size, fracture energy can be improved, and because of this, further investigations on fracture energy of SCRC are promising and should be carried out.Uygunoğlu and Topçu  reportedBideci et al.  reportedGesoğlu and Güneyisi  reportedFor comparison purposes, several test results from different authors of the water absorption of traditional concrete with waste tire rubber are given below. Bjegović et al.  investigFrom the above, it can be remarked that the inclusion of waste tire rubber in both types of concrete, SCC and traditional concrete, causes an increase in water absorption. This behaviour can be attributed to entrapment of air of interfacial transition zone (ITZ) between rubber particles and cement paste ,27, and Uygunoğlu and Topçu  reportedAnother investigation on shrinkage of SCC with rubber aggregate was carried out by Zaoiai et al. . They paShrinkage can be crucial factor for the design of structural members because of the length variation in time , and dueTuratsinze et al.  observedFrom the above, it can be noted that shrinkage of SCC and traditional concrete becomes higher as more waste tire rubber was added in concrete, regardless to rubber particle size. Shrinkage is in direct link with water to powder ratio. As the water to powder ratio increases, porosity is also increasing, causing an increase in concrete shrinkage . NeverthS), which ranged from 90% to 60% of the static flexural strength, and number of cycles to failure (N). An increase in fatigue life was reported when a greater amount of rubber content was included in the SCC mixtures. At a stress level S = 0.90, N increased by the same value of 1.44 times for both SCRC mixtures with 15% and 20% rubber content. At a stress level S = 0.60, N increased by approximately the same value, 1.62 and 1.63 times for both SCRC mixtures with 15% and 20% rubber content.Ganesan et al.  studied N with an increase in maximum stress level (S). However, the increase in N was reported when rubber aggregate had been included in concrete with constant stress level (S). At a stress level S = 0.90, N increased by approximately 7, 11, and 32 times with 5%, 10% and 15% rubber aggregate content, respectively. Similar results were obtained with lower level of stress (S), i.e., 0.60, 0.70 and 0.80, as is shown in Due to a lack of experimental data of fatigue behaviour of self-compacting rubberized concrete, several test results of rubberized concrete and its fatigue resistance are given below. Liu et al.  replacedN also increased with inclusion of rubber aggregate content. At a stress ratio S = 0.80, N was 3.15, 2.95 and 1.62 times higher with 5%, 10%, and 15% rubber content, respectively. Mohammadi et al. [N increased by approximately 19% when 40% of natural aggregate was replaced with crumb rubber. In all other replacement cases, with 10%, 20% and 30% inclusion of crumb rubber, they reported negative or no impact on fatigue behaviour, i.e., N.Analogous test results were reported in another experimental investigation that was carried out by Murugan et al. . They pai et al.  investigFrom the above it can be concluded that regardless of type of concrete, replaced aggregate material or rubber particles size, enhancement in fatigue resistance of concrete and self-compacting concrete is inevitable with addition of waste tire rubber. Although, further investigation on self-compacting rubberized concrete should be carried out in order to give a better explanation of influence of rubber particle size and replacement level on fatigue behaviour.Interfacial bonding between cement matrix and rubber aggregate is one of the most important factors that affect almost every SCC hardened state property. Bignozzi and Sandrolini  reportedWhen compared to untreated rubber, an increase in compressive strength and splitting tensile strength was reported by 37 and 19% respectively, when rubber particles were pre-coated with mortar, which caused a significant improvement in the interfacial bonding and hence improved the stress transformation.Only a small number of investigations have focused on the investigation of ITZ of self-compacting rubberized concrete and on investigation of ITZ while using SEM, although it is obvious that bonding between cement paste and rubber particles is a crucial factor to develop reliable self-compacting rubberized concrete with desirable mechanical properties. Therefore, future investigations of SCRC on SEM still needs to be done, all for finding an optimal solution of bonding between rubber particles and cement matrix.In the present study, the influence of recycled waste tire rubber as a fine and/or coarse natural aggregate replacement in SCC on its fresh and hardened properties was reviewed. See Increase of waste tire rubber negatively effects fresh SCC properties. Flowability, passing ability, and segregation resistance were reduced and viscosity was increased, although a small number of authors claim the opposite. This can be attributed to an increase of rough surface and surface friction of rubber particles. Only a few authors replaced natural aggregate with waste tire rubber above level of 40%, but it can be determined that, in order to maintain acceptable fresh SCC properties, replacement level should not exceed 20%. Furthermore, most authors used different kinds of chemical admixtures to maintain or even to improve fresh SCC properties, i.e., superplasticizer (SP), viscosity modified admixture (VMA), or high range water reducer admixture (HRWRA). Different pre-treatment methods were used by several authors in order to improve the bond connections between rubber aggregate and cement matrix. Water soaking method was shown to be a cost-effective method. Most of the test results of flowability, viscosity, passing ability, and segregation resistance were within the limits given by the European Guidelines for Self-Compacting Concrete.An increase in porosity and air content were detected with the increase of rubber aggregate content in SCC mixtures. As for the other fresh concrete properties that are mentioned above, the same explanation can be used, rubber particles have sharp edges and rough surfaces and therefore entrap air in interfacial transition zone (ITZ) between rubber particles and cement paste and increase the porosity of SCC.Due to lack of experimental investigations and test results further investigation still needs to be done to determine rheological properties of SCRC in order to better understand the workability properties of this type of SCC.Reduction in compressive strength, flexural strength (FS) and splitting tensile strength (STS) of SCC was observed when different types of rubber aggregate were used as a partial replacement of natural fine and coarse aggregate, regardless of rubber replacement level or replaced type of material. This effect can be described as being due to poor adhesion and bond strength between rubber particles and binder content, low rubber modulus of elasticity when compared to natural aggregates and a greater amount of air entrapped between rubber particles and cement paste. However, adding different types of supplementary cementing materials (SCMs), these properties of SCRC can be relatively improved. Also, with the rubber pre-treatment methods, compressive strength, flexural strength (FS) and splitting tensile strength (STS) can be improved but on a smaller level.According to test results obtained in this article, SCRC could be used in some structural applications, but the replacement level must be determined rationally in order to preserve acceptable values of mechanical properties, i.e., compressive strength, splitting tensile strength and flexural strength.Replacement of natural fine and coarse aggregate with rubber aggregate had a negative influence on static and dynamic modulus of elasticity. Static and dynamic modulus were reduced with increasing rubber replacement levels. This effect can be explained with air entrainment on interfacial transition zone (ITZ) between rubber particles and cement paste and lower modulus of elasticity of rubber aggregate than natural aggregate modulus. Although, a relative increase in modulus of elasticity was reported when the rubber particles were pre-treated or when supplementary cementing materials (SCMs) were used in mixtures, especially metakaolin (MK).Enhancement in impact resistance of SCC and traditional concrete occurred with inclusion of waste tire rubber. However, further investigation of SCRC needs to be done to understand the possible applications of SCRC in concrete structures.SCRC and due to the fact that increase in ductility of traditional concrete occurs when rubber was added as replacement for natural aggregate, further investigation of SCRC ductility needs to be done.Due to the lack of test results of ductility of Although scattered results were reported, fracture energy of SCC can be improved with the inclusion of rubber aggregate. Thus, further investigation should be carried out.Waste tire rubber increases water absorption of both type of concrete, SCC and traditional concrete, which can be attributed to the rough surface of rubber particles. Furthermore, an increase in water absorption can also be achieved by increasing the w/c ratio.Rubber aggregate increases shrinkage of SCC and traditional concrete. Shrinkage becomes higher with higher natural aggregate replacement level.Enhancement in fatigue resistance of concrete and self-compacting concrete is inevitable with the addition of waste tire rubber, regardless of type of concrete, replaced aggregate material, or rubber particles size. However, further investigation on self-compacting rubberized concrete should be carried out in order to give reliable explanation of influence of rubber particle size and replacement level on fatigue behaviour.Improvement in bonding between cement matrix and rubber particles can be achieved with proper pre-treatment method and can be quantified by using SEM. Still, future investigations of SCRC on SEM needs to be done to find an optimal solution for bonding between rubber particles and the cement matrix.Overviews of each fresh and hardened property of SCRC as tested and analysed in the literature and several general conclusions can be made:Waste tire rubber can be used as a replacement aggregate material in self-compacting concrete.Many scientists conducted their experimental work by replacing fine aggregate with rubber aggregate, likely because of the better results that were obtained from experiments with fine aggregate replacement as compared to results obtained with coarse aggregate replacement.On behalf of future investigations, it can be suggested that experimental investigations of concrete properties should be investigated with fine aggregate replacement, perhaps with even smaller rubber particles such as waste tire powder.From relationship between overviewed fresh and hardened properties of concrete with number of analysed test results for each property depending on concrete type it can be concluded that further experimental work on properties of self-compacting rubberized concrete still needs to be conducted, because of its high potential to be used in structural applications.The main objective of this review article was a literature overview of fresh and hardened properties of self-compacting concrete with partially replaced natural fine and/or coarse aggregate with recycled aggregate material. From this, it can be concluded:


Trans-splicing mechanisms have been documented in many lineages that are widely distributed phylogenetically, including dinoflagellates. The spliced leader (SL) sequence itself is conserved in dinoflagellates, although its gene sequences and arrangements have diversified within or across different species. In this study, we present 18 Fugacium kawagutii SL genes identified from stranded RNA-seq reads. These genes typically have a single SL but can contain several partial SLs with lengths ranging from 103 to 292 bp. Unexpectedly, we find the SL gene transcripts contain sequences upstream of the canonical SL, suggesting that generation of mature transcripts will require additional modifications following trans-splicing. We have also identified 13 SL-like genes whose expression levels and length are comparable to Dino-SL genes. Lastly, introns in these genes were identified and a new site for Sm-protein binding was proposed. Overall, this study provides a strategy for fast identification of SL genes and identifies new sequences of F. kawagutii SL genes to supplement our understanding of trans-splicing. Many members in this lineage play critical roles in marine ecosystems as primary producers, contributors to red tides, and symbionts of reef corals and other invertebrates. Dinoflagellates are also known for their distinct genomic features which include large genome sizes, permanently condensed chromosomes, lack of nucleosomes . Furthersequence  , mRNA stabilization, 5′UTR sanitization, protein retargeting, as well as creating or destroying upstream open reading frames [Prorocentrum minimum, Karenia brevis, Polarella glacialis, Heterocapsa arctica, Karlodinium veneficum, and Pfiesteria piscicida [The mechanism of g frames ,4,5. Sevg frames . On the g frames . Attemptiscicida ,7. DespiThe presence of SL on the 5′ end of all mature mRNAs has greatly advanced research on dinoflagellates, particularly for Symbiodiniaceae, a group of symbiotic dinoflagellates, by facilitating the identification of dinoflagellate transcripts from mixed samples  and idenIn this study, we identified 18 SL and 13 SL-like genes from stranded RNA-seq reads, and found extra sequences upstream of SLs. The SL genes identified in this study are generally longer than those previously reported. Introns from these genes are also identified and a new potential site of Sm-protein binding is proposed. F. kawagutii  [F. kawagutii [The datasets of stranded RNA-seq reads of wagutii)  were dowwagutii) . Previouawagutii  were alsSL-containing reads were identified by searching for SL sequences (CCGTAGCCATTTTGGCTCAAG) in the reverse reads (R2). Clustering and assembly were performed by iAssembler (version v1.3.3) software  with a mF. kawagutii genes were counted and normalized to their expected appearance as a random occurrence . The secondary structure of SL genes was simulated using MFOLD online service  with the folding temperature set at 20 °C [Five U-rich motifs in introns of Dino-SL genes were selected and their appearances in the introns of at 20 °C .trans-splicing, constitutes the 5′ end of all dinoflagellate mRNA, suggesting extra steps are required to remove these sequences before mRNA maturation (We identified SL-containing reads by searching for SL sequences in the reverse reads (R2) in the stranded RNA-seq datasets. In total, we obtained 195 SL-containing reads from 8 libraries constructed for this study and from a previously published dataset [turation B.F. kawagutii. Therefore, these different consensus sequences represent different loci of tandem SL.Among the reads containing both SL and multiple SL relicts (AGCCATTTTGGCTCAAG), 31 have two SLs and 10 have three SLs. Interestingly, reads containing two SLs clustered into one group while reads with three SLs clustered in another . We notiWe identified 18 types of introns from the different SL loci. In the loci of single SL genes, 16 different sequences  were fouF. kawagutii genes and found that CUUUG, CUUUUG, and GUUUUC are more frequent in introns  motifs, are conserved in different organisms ,23,24, a introns A. We the introns B,C. A buU/C) and putative Sm-protein binding sites (GUUUUC) were absent from the sequences downstream of tandem SLs . For example, there was a transcript having three tandem SL relicts but from which no bone fide SL could be recovered. Aligning and assembling of these sequences resulted in 13 consensus sequences . We inspndem SLs . This sutrans-splicing machinery and SL genes are enigmatic. In dinoflagellates, several SL genes have been cloned in species ranging from Polarella and Heterocapsa to Prorocentrum and Pfiesteria [F. kawagutii SL genes identified in this study displayed several features different from those previously reported. Despite being found in numerous species, the evolution of iesteria ,7. As shP. minimum, K. brevis, P. glacialis, H. arctica, K. veneficum, and P. piscicida, which are predominantly 50–60 bp in length [F. kawagutii SL genes excluding these upstream sequences. However, the lengths of F. kawagutii SL gene averaged to 89 bp, which is still longer than those reported. As these F. kawagutii SL genes were identified using single-ended or pair-ended stranded reads from libraries with insertion sizes of ~200 bp [The lengths of SL genes identified in this study range from 103 to 292 bp, with an average of 164 bp , which in length . In prevn length , SL gene ~200 bp , the lentrans-splicing mechanism was thus responsible for providing the cap structure on all other transcripts. However, the presence of sequence upstream of the SL as shown here suggests the generation of mature transcripts may be more complicated than previously thought  was different from those used in previous studies . Another explanation could be the different strategies used for SL gene cloning in different works. In previous studies, SL genes were cloned using RACE techniques from non-poly(A) RNA, which after removal of poly(A) containing transcripts, were ligated with oligo(A) before reverse transcription [The presence of Sm-protein binding sites, which are usually found in introns of genes in various organisms, is in fact a criterion used for the determination of intronic sequences of genes. Sm-protein binding sites were also found in introns of various SL genes in different organisms . Dinofla studies ,7—as a pcription ,7. DurinF. kawagutii. All of them have multiple full or partial SLs. This is due to the fact that candidates with only one SL or its relict were removed to exclude the possibility of false discoveries caused by random appearance of the SL sequence. This would thus lead to a failure to identify any SL-like genes with only one SL unit. These genes recovered are very similar in sequences to the real SL genes, but differ in that the donor sites for intron splicing are lost. Furthermore, they also lack Sm-protein binding sites. We speculate that these genes cannot be accurately spliced and represent pseudo-SL genes. According to a rough estimation of their expression levels based on their read counts in the different libraries, the expression levels of these SL-like genes are comparable to that of real SL genes. We also identified 13 SL-like genes in Perkinsus marinus and Oxyrrhis marina to Alexandrium tamarense [trans-splicing during the evolution of dinoflagellates. It is interesting that all SL relicts found in these transcripts start with “CCA”—they are thus missing the upstream CCGTAG of SL. One possibility to account for this is that CCGTAG may have been spliced off, given that “AG” could provide a site for intron splicing. However, the SL relicts identified in genomes are longer and contain CCGTAG. The sequence of the tandem partial SLs in the SL genes suggests an alternative interpretation of their appearance in transcripts: there was only one trans-splicing event derived from a tandem-SL gene, instead of multiple rounds of trans-splicing from single copy SL transcripts.Multiple tandem SLs or their relicts had been found in transcripts of many genes of various organisms ranging from amarense ,10. The F. kawagutii genome, at least in part because the datasets used were obtained from polyadenylated transcripts which are likely to be underrepresented in SL genes. Nevertheless, the success identification of SL genes suggests stranded RNA sequencing is a feasible and efficient approach for SL gene identification. Overall, we identified 18 SL genes including 16 single and 2 multiple-SL genes, as well as 13 multiple SL-like genes which have degenerated due to the loss of intron splicing donors. This study provides important supplements to our knowledge of SL genes and illustrates how SL genes can be identified from stranded RNA-seq datasets. The number of SL genes identified in this work is rather limited and presumably only represents a small fraction of the SL genes in


A 25-year-old woman had undergone removal of a cryptogenic tumor in the left maxillary sinus 1 year prior to presentation. The patient experienced abdominal pain for 4 days with repeated vomiting episodes; therefore, she was transferred to our hospital by an ambulance. Contrast-enhanced computed tomography revealed a 3-cm tumor in the ileocecal region, which caused small bowel obstruction. Contrast imaging of the ileus tube showed extrinsic compression of the ileocecal region. Forward-viewing linear echoendoscope revealed an irregular hypoechoic tumor measuring 3 cm outside the gastrointestinal tract. Using a 25G needle, endoscopic ultrasound-guided fine needle aspiration (EUS–FNA) was performed. Pathological finding was an inflammatory fibrous tissue with diffuse lymphoplasmacytic infiltration, with more than 10 IgG4-positive cells detected in a high-power field. Re-examination of a pathology specimen of the maxillary sinus tumor provided by the previous attending physician revealed that the inflammatory tissue had diffuse lymphoplasmacytic infiltration, which were accompanied by storiform fibrosis and obliterative phlebitis. Immunostaining revealed more than 50 IgG4-positive cells in a high-power field, a finding suggestive of IgG4-related disease. The serum IgG4 level was 21 mg/dl, which was within the normal range. Treatment was initiated with prednisolone at a dose of 50 mg/day, and the dose was later tapered off. CT and MRI performed 2 months later showed complete disappearance of the ileocecal tumor. The final diagnosis was asynchronously occurring IgG4-related maxillary sinusitis and sclerosing mesenteritis. IgG4-related disease (IgG4-RD) is an idiopathic inflammatory disorder that affects organs throughout the body . It is cSclerosing mesenteritis is also an inflammatory disease of unknown origin . In receμl and 10.33 mg/dl, respectively, indicating increased inflammatory response. Further, biochemical measurements were as follows: IgA, 215 mg/dl; IgM, 175 mg/dl; IgE, 246 IU/ml; IgG, 1275 mg/dl; IgG4, 21 mg/dl; ANA, negative; ds-DNA Ab, negative; PR3-ANCA, negative; and MPO-ANCA, negative. No findings were suggestive of an autoimmune disease or vasculitis. The serum IgG4 level was within the normal range. Tumor marker levels were as follows: sIL-2R, 611 U/ml; CA19-9, 9.6 U/ml; and CEA, 2.4 ng/ml; a slight increase in sIL-2R level was observed.A 25-year-old woman had undergone tumor removal in the left maxillary sinus at the Otolaryngology Department of another hospital 1 year prior to presentation. Pathological diagnosis was inflammatory pseudotumor of unknown origin. The patient had no remarkable family history and did not consume alcohol or smoke. Owing to intermittent abdominal pain for 4 days and frequent vomiting in the morning of the examination day, the patient was transported by ambulance. On arrival, her vital signs were as follows: body temperature, 36.1°C; blood pressure, 115/85 mmHg; and heart rate, 86 beats/min. Physical examination revealed severe tenderness in the right lower abdomen. There were no signs of rebound pain or muscle guarding. Blood test showed WBC count and CRP level of 16740/Contrast-enhanced computed tomography (CECT) showed a 3-cm hypervascular tumor in the ileocecal region, revealing small bowel obstruction . The lesBased on the findings, an extraintestinal tumor of the ileocecal region and small bowel obstruction were diagnosed. Differential diagnoses included gastrointestinal stromal tumor (GIST), schwannoma, malignant lymphoma, adenocarcinoma, and inflammatory pseudotumor. Although we considered performing ileocecal resection, we decided to first implement preoperative histological examination after consultation with the surgical department.A forward-viewing linear echoendoscope  was inserted into the cecum, which revealed a 3-cm hypoechoic tumor with clear boundaries and irregular periphery outside the gastrointestinal tract .Using a 25G needle , two sessions of EUS-FNA were performed  were found –12.We examined a total of 18 cases including our case . The meaIn conclusion, this condition is common among middle-aged men. The serum IgG4 level tends to be within the normal range or slightly increased, and other organ involvements are uncommon. Approximately 90% of the cases were diagnosed by surgical resection, which suggests the difficulty in preoperative diagnosis. Although the first option for the treatment of IgG4-RD is steroid, no cases were treated with steroids other than our case.To date, no cases have been diagnosed using EUS-FNA, possibly because lesions tend to be present in deep parts of the gastrointestinal tract, making paracentesis difficult. At present, oblique-viewing linear echoendoscope is usually employed for EUS-FNA. However, in the present case, EUS-FNA was performed from the cecum. Because inserting an oblique-viewing echoendoscope into the cecum involved the risk of intestinal damage and perforation, a forward-viewing echoendoscope was used instead. The puncture was from the lower gastrointestinal tract and there was a risk of perforation and peritonitis. Therefore, EUS-FNA using a thin needle (25G) was performed. The procedure was performed without complications. Even with a 25G needle, enough specimens were obtained, and immunostaining could be performed easily.In many cases, preoperative diagnoses of malignant diseases, such as GIST, malignant lymphoma, and cancer, are done based on imaging, resulting in surgeries being performed without biopsies. However, in the case of IgG4-RD, steroid is usually effective in shrinking lesions . As suchWhile the serum IgG4 level in our patient was within the normal range, not all patients with IgG4-RD exhibit increased serum IgG4 levels. In fact, an international study revealed that among patients who are histologically diagnosed with type 1 autoimmune pancreatitis (a pancreatic manifestation of IgG4-RD), 37% had normal serum IgG4 levels . In partRecently, cases wherein the serum IgG4 level was normal and IgG4-positive cells were scarcely detected despite showing typical pathological findings of IgG4-RD  have been reported –19. TherThe elevation of WBC and CRP is not a typical findings in IgG4 related disease. The patient had a bowel obstruction due to the lesion, and it took several days to visit our hospital. Inflammation due to intestinal obstruction may have caused a mild increase in inflammatory response (WBC and CRP).The diagnosis of IgG4-related sclerosing mesenteritis is still debatable. Avincsal et al. reported that although sclerosing mesenteritis shares histological characteristics with IgG4-RD, given the scarcity of cases with elevated serum IgG4 and other organ involvements, it is possible that many of them are not IgG4-related . They coAccording to the pathological consensus regarding IgG4-RD published in 2012, a lesion needs to meet the following criteria  to be considered an IgG4-RD in a new organ [We treated a case of IgG4-related maxillary sinusitis and sclerosing mesenteritis occurring asynchronously. Although the serum IgG4 level was within the normal range, histological findings and the patient's good response to steroid treatment were consistent with IgG4-RD. Although extremely rare, IgG4-related sclerosing mesenteritis does appear to exist. Biopsies need to be proactively performed to avoid unnecessary surgical interventions. Forward-viewing echoendoscope is useful for conducting biopsy from the deep gastrointestinal tract.


Streptomyces is a unique subgroup of actinomycetes bacteria that are well-known as prolific producers of antibiotics and many other bioactive secondary metabolites. Various environmental and physiological signals affect the onset and level of production of each antibiotic. Here we highlight recent findings on the regulation of antibiotic biosynthesis in Streptomyces by signaling molecules, with special focus on autoregulators such as hormone-like signaling molecules and antibiotics themselves. Hormone-like signaling molecules are a group of small diffusible signaling molecules that interact with specific receptor proteins to initiate complex regulatory cascades of antibiotic biosynthesis. Antibiotics and their biosynthetic intermediates can also serve as autoregulators to fine-tune their own biosynthesis or cross-regulators of disparate biosynthetic pathways. Advances in understanding of signaling molecules-mediated regulation of antibiotic production in Streptomyces may aid the discovery of new signaling molecules and their use in eliciting silent antibiotic biosynthetic pathways in a wide range of actinomycetes.The genus  Streptomyces, a unique subgroup of actinomycetes bacteria, is best-known for their ability to produce an enormous variety of bioactive secondary metabolites including antibiotics. The onset and production level of each antibiotic is subject to complex control by various environmental and physiological signals  in Gram-negative bacteria, hormone-like signaling molecules in the Gram-positive Streptomyces bacteria are diffusible low molecular weight chemical substances that can elicit antibiotic production and/or induce morphological differentiation at nanomolar concentrations , furans, γ-butenolides as well as PI factor and N-methylphenylalanyl-dehydrobutyrine diketopiperazine (MDD)  from Streptomyces coelicolor, five virginiae butanolides (VBs A–E) from Streptomyces virginiae, IM-2 from Streptomyces lavendulae, factor 1 from Streptomyces viridochromogenes, and three Gräfe’s factors from Streptomyces bikinensis and Streptomyces cyaneofuscatus  for the biosynthesis of actinorhodin (ACT), undecylprodigiosin (RED), calcium-dependent antibiotic (CDA) and coelimycin. It should be noted that the deletion of the coelimycin gene cluster included scbR2, which encodes a repressor of SCBs biosynthetic gene scbA, and thereby contributed to the overproduction of SCBs in S. coelicolor M1152  . GBLs, fne (MDD) . As mostne (MDD) . So far,fuscatus . To gainomycetes . Earlierelicolor . Among the 2000s . Recentls SCB4–8 . The S. or M1152 . In a BL genomes . In anotomycetes . The widompounds . These sStreptomyces rochei  that encode GBL receptor homologs, and two genes (aco and cyp17) that encode an acyl-CoA oxidase and a cytochrome P450 hydroxylase, respectively. Studies suggested that both the acyl-CoA oxidase Aco and the cytochrome P450 hydroxylase Cyp17 were required for the enzymatic production of avenolide. Furthermore, Aco/Cyp17 homologs were also found in the genomes of Streptomyces fradiae, Streptomyces ghanaensis, Streptomyces griseoauranticus, and S. albus J1074, suggesting that they have the capacity to produce avenolide or avenolide-like autoregulators . The four avenolide-like compounds showed different levels of avenolide activity in stimulating avermectin production in S. avermitilis . Within the gene cluster, sabA encodes an AfsA-like enzyme, whereas sabP and sabD encode phosphatase and dehydrogenase enzymes, respectively. Heterologous expression of sabAPD in E. coli and Streptomyces led to the identification of three novel butenolides   from s rochei , the avermitilis , four buus J1074 , and thromogenes . Of specrmitilis . The gengulators . Recentlbus 1074 . In anotactivity . Metabolrmitilis . It is iroducers . In a re, and 3) . Since t species , especiaptomyces . The rolStreptomyces genomes contain multiple genes for ArpA-like GBL receptors. Examples include ScbR, ScbR2, CprA and CprB in S. coelicolor, JadR2 and JadR3 in Streptomyces venezuelae, and AvaR1, AvaR2, and AvaR3 in S. avermitilis, SabR1 and SabR2 in S. ansochromogenes. It is noteworthy that most genes encoding these GBL receptors are closely linked both to each other and to the afsA-like genes, except that cprA and cprB are not linked to an afsA-like gene and therefore referred to as “orphan GBL receptor” genes   . ScbR2, d avaR3) , and thed avaR3) . It is iS. venezuelae. Such interplay makes jadomycin biosynthesis an illuminating model for regulatory studies of antibiotic production mediated by antibiotics and their biosynthetic intermediates  biosynthetic genes in the presence of a low concentration of JdB and its homologs, but high JdB concentrations cause the dissociation of JadR1 from its target promoters. Thus, JdB interacts with JadR1 directly in a dose-dependent manner to control JdB production in a feedback regulatory mechanism  and dehydrorabelomycin (DHR)] and release its repression on jadY transcription to ensure the timely supply of cofactors for JadG to convert DHR to the late intermediate jadomycin A (JdA) in jadomycin biosynthesis  in antibiotic biosynthesis. The CSR activator JadR1, an OmpR-type ARR of echanism . Similarding RED . Other tynthesis .S. venezuelae, more antibiotics and their biosynthetic intermediates have been found to serve as autoregulatory molecules to modulate their own biosynthesis in many other streptomycetes. For illustration purposes, we herein only highlight several recent findings. For example, ChlF1, a TetR family regulator involved in chlorothricin biosynthesis of Streptomyces antibioticus, was found to repress the transcription of chlF1, chlG (encoding a major facilitator superfamily transporter), and chlK (encoding a type II thioesterase) but activates chlJ (encoding acyl-CoA carboxyl transferase) by binding to their promoter regions. However, ChlF1 was disassociated from its target promoters in the presence of chlorothricin and its biosynthetic intermediates , which directly interacted with the regulator in a concentration-dependent manner  by binding to their promoter regions. Similarly, both calcimycin and its biosynthetic intermediate (cezomycin) can act as ligands to dissociate CalR3 from its target promoters in a concentration-dependent manner (Streptomyces antibiotic regulatory proteins (SARPs) family regulator was also found to regulate antibiotic biosynthesis in response to antibiotics and their biosynthetic intermediates. For example, NosP was found to regulate nosiheptide production in response to both the end-product nosiheptide (NOS) and biosynthetic intermediate (NOS-AC)  serves as an extracellular signaling molecule to regulate rifamycin export in a feedback mechanism (Pseudomonas fluorescens (Inspired by studies on the regulation of jadomycin biosynthesis in t manner . In anott manner . Other t(NOS-AC) . It shouechanism . Anotherorescens . These sred gene cluster, modulates the production of RED as well as that of ACT and CDA    . SimilarGMCC0516 , productuligerus , and pronezuelae . Recent ibiotics . In S. v cluster . These sd avaR3) . Interesd avaR3) . It woulStreptomyces. It is reasonable that signaling molecules can be used to improve antibiotic production through exogenous addition. However, due to the fact that natural GBLs are produced in very small quantities, it is impractical to collect sufficient amount of GBLs for the improvement of antibiotic production. Synthetic GBLs and their analogs are then used for this purpose. Early studies showed that addition of chemically synthesized VB-C enhanced virginiamycin production in Streptomyces virginiae  induced early RED production and premature differentiation  in S. coelicolor. Other angucyclines were also found to elicit similar phenotypes  as the best elicitors of a cryptic non-ribosomal peptide synthetase (NRPS) gene cluster (Streptomyces hiroshimensis (It is well-known that the cryptic gene clusters represent a treasure trove of new natural products, and various strategies and methods has been devised for the activation of these gene clusters . Of specibiotics . Furtherus J1074 . HiTES w cluster . A major cluster . Applicahimensis , and manhimensis . These sStreptomyces. The emerging picture shows a complex interplay of various signals and regulatory proteins. It is interesting to note that both hormone-like signaling molecules and antibiotics/their biosynthetic intermediates actively participate in the regulation of antibiotic production. These small molecule regulators exert their function through the modulation of the binding activity of different regulators to their target genes. Further studies are needed to reveal the complex interplay of autoregulators and multiple GBL receptor homologs in antibiotic biosynthesis of different Streptomyces. However, the number of hormone-like signaling molecules identified from Streptomyces is still limited, most likely due to the fact that they are produced in very small quantities. BLAST searches for homologs of AfsA from S. griseus within the actinobacteria genomes available from public databases indicated that AfsA-like proteins are present in most actinomycetes (S. avermitilis (Streptomyces, including deletion of gene encoding repressor of signaling molecule biosynthetic gene (Streptomyces may be used to increase the metabolic titer of industrially and medically important antibiotics as well as activate silent antibiotic BGCs for the discovery of novel bioactive natural products in a wide range of actinomycetes.Over the past several decades, significant advances have been made in understanding the regulation of antibiotic biosynthesis in omycetes . Similarrmitilis . It is stic gene , bioassatic gene , and hettic gene . These aAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The discovery of host–guest complexation has resulted in significant advancements in various facets of materials science, including: drug delivery, tissue engineering, etc. Various host molecules such as cyclodextrins (CD), urea, thiourea, cyclo-triphosphazines, pillar[6]arene, cucurbit[6]uril, and calix[4]arene have been identified as undergoing inclusion complexation with a range of guest molecules. Among these host molecules, cyclodextrins are well-known for their complexation capabilities with a variety of guest molecules. Through such complexation, the stability of the guest against thermo-chemical, environmental, light, and air degradation is ensured. In addition, the widespread availability of CD-derivatives  has partly contributed to their widespread use in applications extending from textiles to pharmacologics to nuclear waste removal. Another benefit of forming such complexes is that they improve the polymeric properties  by coalescing the guest polymers from their CD inclusion complexes.While the host–guest complexation occurs predominantly via non-covalent interactions, the underlying mechanism differs among the studied guest molecules, with predominant complexation occurring via hydrogen and ionic bonding, van der Waals and hydrophobic forces/interactions. Even though the mechanisms differ between the chosen host/guest molecules, a common criterion for successful complexation remains lowered Gibbs free energy, which can occur either by an enthalpy- or an entropy-driven process. The thermodynamics of the host–guest complexation and associated binding constants are typically characterized using spectroscopy-based techniques such as nuclear magnetic resonance, UV-vis, mass spectrometry, fluorescence-based spectroscopy or calorimetric techniques such as differential scanning and isothermal titration calorimetry . With suThis Special Issue consists of 15 articles, including four comprehensive review articles, written by experts in the field. The first eleven articles are dedicated to the synthesis and characterization of host–guest containing compounds for the fabrication of devices, in particular for biomedical applications. The next four articles are comprehensive review articles discussing the state-of-art synthesis, characterization, and fabrication of various host–guest complexes, including CD–polymer, amylose–polymer, polysaccharide–metal ion, and urea–polymer inclusion complexes. In this series, the first research article reports the synthesis of novel host–guest supramolecular hydrogels comprised of pNIPAm microgels bearing poly[acrylic acid] with attached β–CDs as the host and adamantane–dextrans as the guest molecules. By forming in situ complexation, hydrogels were synthesized that showed sol–gel transitions at physiologically relevant temperatures (37 to 41 °C) .One of the advantages of such a system is in the field of the controlled release/retention of small molecules, mediated in part by the concerned host and the guest molecules. Likewise, the second study reports the fabrication of host–guest aerogels containing fluorescently active quantum dots. In the same way that the interaction with CD protects the small molecules from degradation, this study demonstrated the fluorescence-retention potential of quantum dots upon complexation/interaction with β–CD . E. coli and S. aureus, but also resistance to light-induced degradation [A major challenge in tissue engineering remains the lack of oxygen and frequent bacterial infection during tissue regeneration. By complexation, the third and fourth articles in this Special Issue address these two major challenges in tissue regeneration ,5. By enradation .l-Lysine) [The next three articles reported the synthesis and electrostatic-force-induced complexation of anionic polyelectrolytes and dendrigraft poly(-Lysine) , poly (γ-Lysine) , pillar[-Lysine) . As showhttps://textiles.ncsu.edu/blog/team/alan-tonelli/) has reported a facile technique to render incompatible polymers more compatible by threading through the CD cavity and subsequently removing the CD, resulting in improved compatibility between otherwise incompatible blends. In addition, over the years, we have extended this technique to urea–polymer inclusion complexes as well. Similar to this established approach, Liu et al. report an improved ability to crystallize and compatibility between PLLA/PDLA by coalescing from a common inclusion complex [In the past two decades, our group ( complex . In a si complex . The fin complex .The next section of this Special Issue contains review articles from expert researchers on the synthesis, characterization and fabrication of state-of-the-art devices based on inclusion complexation. The first review, from our group, is based on the fabrication of aliphatic polyester nanofibers containing uncomplexed and complexed CDs or inclusion complexes. In this review, we report various possibilities for improving the topographical, mechanical and cell-adhesive properties of aliphatic polyester nanofibers via an electrospinning process . The nexOverall, we anticipate this Special Issue to be a ready reference for those interested in the synthesis, characterization and fabrication of inclusion complexes between guest polymers and host molecules. We hope these reviews will encourage advanced research on host–guest inclusion complexes. As the editors of this Special Issue, we acknowledge and offer our sincere thanks to the authors and the reviewers for their invaluable contribution to this Special issue.


MET amplification has been shown to develop as a resistance mechanism to treatment in NSCLC. The identification of increased MET copy number within tumour cells is increasingly important to stratify those tumours and patients which are susceptible to treatment targetting MET kinase inhibition. Fluorescence in situ hybridisation (FISH) has been successfully employed to identify patients with abnormal MET gene copy number with numerous probes available for use. Here we report a FISH protocol that reduces probe hybridisation time in NSCLC tissue to 1 hour and compare the results with other protocols. MET gene copy number was determined in 20 NSCLC cases using 3 FISH probes: 1. Kreatech FISH, MET (7q31) SE 7 ready to use probes, hybridised using an overnight protocol; 2. Dako MET IQFISH probe with CEP7 ready to use probe, hybridised for 2 hours; 3. Kreatech MET (7q31) SE 7 XL FISH probe, prepared in SwiftFISH buffer and hybridised for 1 hour. The MET gene copy number and MET: centromere 7 gene ratio were determined for each tissue and cases categorised as having MET high or MET low status. All three FISH probes were shown to demonstrate good agreement with each other. Overall percentage agreement between probes was ≥90%. Intraclass correlation showed good agreement (ICC ≥0.80) between all three assays for MET gene copy number and MET: centromere 7 gene ratio. These FISH protocols provide evidence that rapid laboratory developed FISH assays with short turnaround time perform consistently with standard protocols, potentially enabling faster treatment decisions.The c-Met/hepatocyte growth factor receptor pathway is frequently dysregulated in multiple cancer types, including non-small cell lung cancer (NSCLC).  MET expression have been observed in tumour biopsies of most solid tumours and MET signalling has been documented in a wide range of human malignancies, including bladder, breast, cervical, colorectal, gastric, head and neck, liver, lung, ovarian, pancreatic, prostrate, renal and thyroid cancers, as well as in various sarcomas, haematopoietic malignancies and melanoma [MET have been positively identified in patients with a hereditary form of papillary renal cancer, directly implicating MET in human tumourigenesis [MET gene alterations using cBioPortal [MET gene is altered in 3% of patients; 1 percent of patients were recorded with MET amplification. In Non Small Cell Lung cancer ~5% of queried patients showed a MET gene alteration, 1.66% of which were MET amplification. Mutation prevalence data from cBioPortal are presented in supporting information . In several clinical studies, aberrant c-Met overexpression has been correlated with poor clinical outcome, rapid disease progression and short survival [MET pathway can be induced by mutations resulting in exon 14 skipping [MET amplification or other MET-based mechanisms of resistance [MET amplification has been found after acquired resistance to EGFR tyrosine kinase inhibitors such as osimertinib [The c-Met/hepatocyte growth factor receptor (HGFR) pathway is one of the most frequently dysregulated pathways in human cancer. Receptor activation leads to the recruitment and activation of specific downstream signalling partners that participate in the regulation of diverse processes such as tumour cell growth, migration, scattering and metastasis. In the endothelial and stroma cells surrounding tumour tissue, the c-Met/HGFR pathway acts in a proangiogenic manner to stimulate cell proliferation, migration and survival, which can support tumour expansion and progression. Hepatocyte growth factor (HGF), the ligand for the receptor, and melanoma , 2. Actiigenesis . TCGA PaioPortal , 5; irreskipping . Up to 2sistance –10. MET mertinib .MET amplification status in tissue biopsies can be determined using fluorescence in situ hybridisation (FISH), polymerase chain reaction (PCR) based technology and next generation sequencing (NGS) [ALK FISH and HER2 FISH have been developed as companion diagnostic assays, to detect break apart genes and gene amplification respectively, exemplifying their use in clinical trials and for patient selection [MET gene amplification or aneuploidy, where the target may be drugable. Demonstrating fast turnaround time and accurate results across assay platforms is essential for FISH uptake in clinical trials and clinical practice.ng (NGS) , 12. FISng (NGS)  and are election , 15. FISFISH is a molecular cytogenetic technique used to identify specific segments of a chromosome by hybridising a fluorescently labelled probe to nucleic acids; the number of fluorescent signals is correlated with DNA copy-number , 17. FISMET FISH probes in NSCLC. Traditionally, FISH procedures require probe hybridisation to the target sequence for between 12–18 hours, usually carried out overnight. Recently, several groups have reported methods that reduce hybridisation time [FISH probe hybridisation efficiency and therefore signal intensity and analysis are broadly dependent upon tissue pretreatment and hybridisation duration. The variability of probe hybridisation associated with reduced hybridisation time and pretreatment methods to our knowledge has not been extensively examined between vendors of ion time , 23.et al. processed 159 gastro-intestinal cancer FFPE tissue samples using a MET/CEN-7 IQFISH Probe Mix , Agilent Technologies, Glostrup, Denmark). Probes were hybridised to tissue using a short protocol requiring 90 minutes incubation. Samples were categorised as amplified or non amplified by two observers. The investigators showed 100% overall inter-reader agreement between cases using a short FISH protocol.Jorgensen et al. compared two HER2 FISH assays, the PathVysion HER2 DNA Probe Kit  and the HER2 IQ FISH pharmDx Kit , which require 14–18 hours and 90 minutes hybridisation and result in 2 day and 3–4 hour turnaround times respectively in breast and gastro-oesophageal cancer [Furthermore, Tafe l cancer . Cases wl cancer . Ten outMET amplification in NSCLC and reduce probe hybridisation time to as little as 60 minutes, thus shortening assay duration further compared to previously published assays while maintining assay performance.Fast assay turnaround time is important for patient care where assay results define treatment. Reducing FISH assay turnaround time to within 1 day therefore allows patients to access treatment more rapidly. Here, we evaluate the robustness of fast FISH protocols for the detection of MET amplification as seen in NSCLC patients with tyrosine kinase inhibitor resistance. Consecutive tissue sections were cut at 4μm thickness. Tissue sections were probed using FISH assays within 3 months of tissue sectioning [Twenty commercial NSCLC samples  were selected to represent the same proportion of patients with ctioning .MET gene copy number was determined using 3 FISH protocols (summarised in A single section from each case was probed with Kreatech FISH probes for MET (7q31) and SE 7  using a ThermoBrite system. The probe is premixed in ready to use format. The Kreatech FISH, MET (7q31) SE 7 probe was denatured for 5 minutes at 80°C followed by 12–16 hours hybridisation at 37°C. Tissue was washed under high stringency conditions (72°C) for 1 minute with 0.4x SSC/ 0.3% Igepal (according to Leica Biosystems Tissue Digestion Kit 1 ). TissueA second consecutive section from each case was probed with Dako MET IQFISH probe with CEP7  according to the manufacturers instructions . Using aA third consecutive section from each case was also probed using Kreatech MET 7q31) SE 7 XL FISH probe for BOND, supplied in concentrate and diluted to 1x working concentration in SwiftFISH rapid hybridisation buffer . Using a ThermoBrite system the probe was denatured at 83°C for 5 minutes followed by hybridisation for 60 minutes at 43°C  with 4',6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI).MET) was labelled with an orange/red fluorophore and the probe specific to centromere 7 was labelled with a green fluorophore. Four z stack images were taken at 1 μm intervals and amalgamated using Zeiss extended depth of focus (EDF) feature which combines regions of greatest contrast within the Z stack image to maximise the contrast of the final image.Tissue FISH signals were assessed consistently with guidelines described previously for tissue processing, tumour identification and signal enumeration for the assessment of EGFR FISH . Tissue MET and centromere 7 signals were manually enumerated in 50 tumour cell nuclei at x40 magnification. Nuclei with the highest number of FISH signals per field of view were enumerated. The gene copy number and MET:centromere 7 ratio were calculated in 50 tumour cell nuclei per case and the mean score recorded. If the average MET gene copy number was ≥5 or the MET:centromere 7 ratio was ≥2, the tissue section was determined to show MET high gene status in accordance with parameters used previously to enroll patients to clinical trials [Following digitisation, tumour area was identified and tissue assessed to ensure FISH signals were clear and visible. Using low magnification, the entire tumour area was visually evaluated to identify any regions of signal amplification or aneuploidy. The number of l trials .The overall agreement of the classification of the cases for each assay was calculated against the standard Kreatech assay (which requires overnight hybridisation) as the reference assay. Bland Altman plots were used to visualise agreement in individual scores with the reference assay . The agrTwenty commercial NSCLC cases were assessed with each of three FISH assays. All 20 cases were successfully analysed when tested with the standard Kreatech and fast Dako assays. Seventeen cases were successfully tested with the fast Kreatech assay. Three cases were not enumerated due to the absence of clear interpretable FISH signals.MET high status. Three out of five cases were also MET high using the fast Dako assay. Of the seventeen cases successfully tested with the fast Kreatech assay, 5 were determined as MET high using the standard Kreatech assay, four were determined as MET high using the fast Kreatech assay .Using the standard Kreatech assay, 5 of the 20 specimens tested showed ch assay , Table 220 cases were tested using standard Kreatech and fast Dako assays, 17 cases were evaluated using the fast Kreatech assay.MET gene copy number and MET: centromere 7 gene ratio  between the standard Kreatech probe and the fast Kreatech probe was ±1.33 for MET gene copy number and ±0.63 for MET: centromere 7 gene ratio. ICC showed good agreement between all three assays for MET gene copy number  and MET: centromere 7 gene ratio . Representative images of probe hybridisation to tissue with marginally increased and normal average MET gene coopy number are shown in The agreement interval (±2 SD) of the standard Kreatech assay and fast Dako assay was ±1.39 for MET high and MET low was calculated using the standard Kreatech assay as reference. OPA ≥90% was observed between the standard Kreatech assay and the fast Dako assay and between the standard Kreatech assay and the fast Kreatech assay  in classification of cases into ch assay .The standard Kreatech assay was used as a reference assay to determine the OPA with the fast Dako assay or the fast Kreatech assay. Lower 95% confidence intervals are also presented.MET FISH protocol that reduces probe hybridisation time in NSCLC tissue to 1 hour and compare the results with other protocols. We assess the agreement between three FISH probes by measuring the average MET gene copy number and average MET: centromere 7 gene ratio in a cohort of commercial NSCLC cases. We successfully demonstrate equivalence between assays and maintain high quality staining, using just 1 or 2 hours hybridisation, enabling FISH assays to be completed within a standard 7.5 hour laboratory shift.Recently, FISH protocols have emerged which utilise shorter processing time, through a reduction of hybridisation duration , 24. TheFISH signals were enumerated in 20 commercial NSCLC FFPE cases probed with a standard Kreatech assay and a fast Dako assay. Seventeen out of 20 commercial cases probed using the fast Kreatech assay were successfully analysed; 3 cases were void of signal or showed weak signal following the fast Kreatech assay and were not analysed. A universal pretreatment procedure using the Leica BOND Rx instrument was employed prior to application of FISH probes in this study. Cases void of FISH signal were not retested with adapted pretreatment conditions. The practice of marginally adjusting tissue pretreatment is common during FFPE tissue processing prior to FISH probe hybridisation . UniversThe effectiveness of tissue pretreatment prior to probe hybridisation may also be affected by pre analytical tissue processing, such as fixation. All commercial cases were FFPE tissue, the fixation time however was not specified beyond routine processing. Coupled with short hybridisation times, pre analytical variation of tissue processing may have led to the absence of successful probing in 3 cases using the fast Kreatech assay.MET copy number ICC = 0.94 and for average MET: centromere 7 ratio ICC = 0.80. The mean difference between the standard Kreatech assay and the fast Dako assay or fast Kreatech assay showed no systematic differences between assays  out of the 12 specimens were MET amplified; MET amplification in this instance was defined as MET centromere 7 gene ratio ≥2.0 by these authors [Additionally, difficult to analyse cases such as these may harbour genetic heterogeneity. Jørgensen  authors .HER2 [MET ISH and atypical signal patterns will in future require consistent reporting.Atypical FISH signal patterns have been observed in other pathologies such as HER2 . These aHER2 and ALK FISH assays is regularly reviewed and updated [Cases in this study were imaged using a Zeiss Axio Scan.Z1 slide scanner. Z stack images were captured and amalgamated using Zeiss EDF to visualise the maximum projected signal intensity from each Z stack image, thus preventing the perception that FISH signals may have diminished. FISH signals in 50 nuclei of each case were enumerated manually. This approach has been commonly used for signal enumeration and tissue analysis in various FISH probe companion diagnostic kits (and is described in manufacturers instructions). In order to be reproducible between readers and laboratories, clear guidelines and standard instructions for tissue preparation and signal enumeration are required. For example, clear guidelines for the preparation of samples and analysis of  updated , 40. FurMET status. Numerous solutions for automated enumeration of FISH signals in a clinical environment have been developed for probes such as HER2 [HER2 and MET FISH analysis it is conceivable an automated image analysis solution for MET FISH could be developed further for routine diagnostic use. Furthermore, machine learning could potentially be developed to fully automate tumour cell identification and FISH signal enumeration. Combining image analysis with a fast FISH assay could offer diagnostic testing laboratories significant resource savings in addition to reduced turnaround time. Moreover, image analysis platforms may aid analytical reproducibility and inter-laboratory precision in a clinical environment.In addition to clear analysis guidelines, automated image analysis could aid in the determination of  as HER2 , 41, 42.Fast assays may become more important to testing laboratories as treatment decisions are required more quickly and demands on limited resources grow; a larger study to provide evidence FISH assays are interchangeable is required. Different specimen types such as cell blocks derived from fine needle aspirates or needle biopsies were not included here. Fine needle aspirates are a source of biopsy for NSCLC diagnosis  and an eALK gene rearrangements and copy number gains have been detected by FISH on CTCs [ALK copy number gains was shown to be associated with progression free survival and early progression. The authors present these findings as a potential strategy for real time monitoring of patient response using FISH. Additionally, a method has been developed to capture CTCs based on c-Met overexpression [MET FISH status of a small number of patient CTCs in this study were successfully assessed confirming the presence of gene amplification, although the use of such an assay in a clinical setting is yet to be evaluated. Rapid FISH protocols may therefore be applied to a number of alternative sample types and may provide predictive or prognostic information under these circumstances. Further investigation of these sample types using a validated assay is required.In primary or metastatic NSCLC, if resection or surgery is not a component of treatment, tissue samples may be difficult to obtain. Additionally, repeated biopsy in a patient monitoring setting may not be clinically feasible. In these scenarios alternative sample types to FFPE tissue for biomarker identification such as circulating tumour cells (CTCs), may be an alternative . ALK gen on CTCs  and the pression . MET FISMET FISH probes utilising overnight, 2 and 1 hour hybridisation times. All tissue was pretreated using a common automated protocol, followed by manual probe application. OPA between assays was shown to be greater than 90%. Probe hybridisation for 1 or 2 hours offers testing laboratories the opportunity to significantly reduce turnaround time potentially enabling patients faster access to treatment decisions. Probes from different manufacturers may be used interchangeably, enabling consistent results, provided protocols are fully validated. Full automation and image analysis could potentially provide even faster treatment decisions and reduce assay variability, increasing diagnostic accuracy.Feasibility data presented here demonstrate good agreement between three S1 Fig(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


One with amidine functionalization (Amidino POSS®) and the second with amine and lactamide groups functionalization (Lactamide POSS®). Composite membranes were produced by casting a polyvinyl alcohol (PVA) layer, containing either amidine or lactamide functionalized POSS® nanoparticles, on a polysulfone (PSf) porous support. FTIR characterization shows a good compatibility between the nanoparticles and the polymer. Differential scanning calorimetry (DSC) and the dynamic mechanical analysis (DMA) show an increment of the crystalline regions. Both the degree of crystallinity (® shows a higher interaction with PVA, inducing lower values in the CO2 flux. We have concluded that the interaction of the POSS® nanoparticles increased the crystallinity of the composite membranes, thereby playing an important role in the gas separation performance. Moreover, these nanocomposite membranes did not show separation according to a facilitated transport mechanism as expected, based on their functionalized amino-groups, thus, solution-diffusion was the main mechanism responsible for the transport phenomena.In this article, we studied two different types of polyhedral oligomeric silsesquioxanes (POSS CO2 emissions have dramatically increased in the last 50 years, and are still continually increasing each year [2 emissions, and hence, there is a significant interest in developing technologies to reduce CO2 emissions. Carbon capture and storage (CCS) has become an important instrument to reach the goals agreed on the 2015 Paris international convention [2 capture processes in the power and industrial sector has not yet matured. Key challenges are related to the low partial pressure of CO2 and the large scale required for flue gas treatment. For membranes to be cost-effective, further innovations in process design and membrane materials are thus needed. In this respect, the use of hybrid composite membranes that benefit from both an organic and inorganic part to further improve the gas separation performance have been widely studied. Membrane-based separation processes are not only cost effective and environmentally friendly, but also offer much more versatility and simplicity for customizing system designs with many novel polymeric materials now available. Moreover, membrane modularity and easy scale-up opens for retrofitting existing plants, as well as flexibility with respect to the CO2 capture rate. The technology has, as well, a near instantaneous response, and a high turndown is possible that preserves plant operability.Global warming is one of the major problems that face humanity. Global warming is caused by the emission of greenhouse gases, of which the main component is carbon dioxide  use a carrier to increase the transport of the desired gas through a membrane, without having a negative effect on the selectivity. Many researchers have studied this type of membrane ,23,24,252 based on the selected nanoparticles. The manufactured membranes involve the addition of carriers that are expected to improve the gas separation properties. Nanosized particles, such as polyhedral oligomeric silsesquioxanes (POSS®) embedded in a polymer matrix, are reported here. POSS® nanoparticles have a rigid cage-like structure which is an intermediate between silica and siloxane. POSS® materials are well-defined, three-dimensional nano building blocks that can contribute to create unique hybrid materials, with a precise control of nanostructure and properties [® chemical reagents are nanostructured, with sizes of 1–3 nm. However, unlike silica or silicones, POSS® can be produced with nonreactive substituents, to make them more or less compatible with different monomers or polymers, depending on the way they are obtained [® were expected to provide the suitable carriers for CO2 when humidity is present. This feature makes POSS® an attractive candidate for the fabrication of hybrid materials. POSS® has been used in several gas separation studies in recent years [In this study, facilitated transport was expected for COoperties . POSS® cobtained . The amint years ,29,30,31® without achieving any improvement in CO2 separation. In our effort to increase the performance of the membranes, we have modified these amino nanoparticles into the amidino and lactamide particles reported here. The aim of the current work reported here is to investigate the effect on the gas separation performance of the membranes by the addition of two different functionalized POSS® nanoparticles; one with amidine groups (Amidino POSS®) and the other with amino and lactamide functional groups (Lactamide POSS®). Amino groups are versatile and can undergo different chemical reactions. The functionalization of POSS® can thus be tailor-made, depending on the aimed properties. Amidino POSS® has been specially developed to enhance CO2 capture capacity. Amidino POSS® and Lactamide POSS® would be expected to exhibit different mechanisms toward CO2 capture. While Lactamide POSS® would catch CO2 through forming a competition between carbonate and carbamate, Amidino POSS® would only form carbonate which is beneficial in term of cyclic capacity [® with lactic moieties is believed to improve compatibility of Lactamide POSS® with PVA, as it contains secondary hydroxyl groups.In our previous work , we havecapacity . Moreove®) were synthesized. ® was synthesized by classical sol-gel synthesis from the 3-(aminopropyl)-triethoxysilane. Aminopropyl POSS® particles were subsequently functionalized in solvent-free reactions with N,N-dimethylacetamide dimethyl acetal to obtain Amidino POSS® and with ethyl lactate to prepare Lactamide POSS®. Amidino POSS® and Lactamide POSS® are obtained in n-propanol with a solid content of 42% for Amidino POSS® and 32% for Lactamide POSS®.Polyvinyl alcohol (PVA) was provided by Sigma-Aldrich, the molecular weight of 85,000–124,000, 87–89% hydrolyzed. Polysulfone (PSf) porous support flat sheet membranes with a molecular weight cut-off 50,000 Da were purchased from Alfa Laval (Denmark). The PSf support was washed. Two different types of nanoparticles belonging to the class of the polyhedral oligomeric silsesquioxane  was added to distilled water (97 g). The mixture was refluxed for two hours to dissolve the polymer under stirring at 500 rpm at 90 °C. Then, the PVA solution was cooled down to room temperature. Amidino POSSThe thickness and surface of the composite membranes were studied using a Hitachi TM3030 and a Nova NanoSEM650  field emission gun scanning electron microscope . The cross-section of the samples was prepared by freeze-fracturing/cutting in liquid nitrogen. The samples were sputter coated with a thin gold layer (90 s) to provide electronic conductivity to the samples.2 atmosphere. The analysis was then carried out from the second heating scan.The thermal properties of the membrane materials were investigated using a differential scanning calorimeter . A sample of about 10–12 mg was placed in an aluminum pan covered with a proper lid, together with a standard empty pan, into the DSC sample holder. To eliminate the thermal history and remove any residual solvent, the samples were first heated from room temperature to 110 °C at 10 K/min, kept at this temperature for 10 min, and cooled to −50 °C at a rate of 10 K/min. The samples were then heated up from room temperature to 300 °C at a heating rate of 10 K/min, under an NThe thermal stability of the membranes was investigated with a thermogravimetric analyzer . Around 10–12 mg of sample was placed in the sample pan and heated from 30 °C to 800 °C (10 K/min). Nitrogen was used as both the balance and sweep gas, with flow rates of 20 mL/min.® nanoparticles with the composite membranes. The IR spectroscopy experiments were carried out with a FTIR spectrometer  with a smart endurance reflection cell. FTIR was used to investigate the possible interaction between POSSDynamic mechanical analysis was conducted on a DMA 242 E Artemis  in tension mode under the following conditions: frequency 1 Hz, a proportional factor of 1.1, absolute target amplitude of 40 µm, a maximum dynamic force of 2.182 N, and no additional static force. To eliminate the thermal history, the samples were first heated from room temperature to 80 °C at 5 K/min, kept at this temperature for 10 min, and then cooled afterwards to −50 °C at a rate of 2 K/min, to be heated up again to 350 °C at a rate of 2 K/min. The samples were cut with 10 mm length and 5 mm width, with a thickness between 0.020–0.060 mm. With DMA, various transition temperatures can be determined while the mechanical properties are measured under an oscillatory strain, which supplies information about major transitions, as well as secondary and tertiary transitions not identifiable by other methods .2 flux and CO2/N2 selectivity were calculated from the measured permeate CO2 and N2 concentration in the sweep flow gas. The membrane performance was measured in a feed gas mixture at a pressure of 1.3, 2, and 3 bars with a mixed gas permeation test rig, described elsewhere . The exp2 and N2 was calculated assuming perfect mixing through Equation (1).2 or N2), 3(STP)/m2·h), The mixed gas permeability of CO2/N2 selectivity 2) and 2), is given as follows:The CO®/PVA: 5%, and Lactamide POSS®/PVA: 5%. The dense composite membranes have an average thickness of 1–4 µm. The correct thickness of each membrane has been taken into account when the permeability is calculated. After scanning several membranes and places of the membrane, we conclude that a continuous composite PVA layer is formed on the support, and no evidence of pores or defects was observed. The white points in The composite dense layer deposited on the porous support can be seen in ®/PVA and Lactamide POSS®/PVA systems, respectively. The main IR peaks are given in −1, the peaks for residual acetyl groups 1740, 1265 cm−1 decrease with addition of nanoparticles, since ratio PVA/nanoparticle loading is increasing, and thus, less PVA is present, and the residual acetyl peak decreases. The other peaks can be ascribed to either Amidino or Lactamide POSS® functionalities. The peak in the range of 1080 cm−1 corresponds to the asymmetric (Si–O–Si) stretching vibration band, belonging to the silica cage of POSS®. Its intensity increases with the nanoparticle loading [ loading . −1 corresponds to the PVA crystallites [−1 belongs to the azo compound N=N stretching of the Amidino POSS® nanoparticles, and shows a visible increment for the spectra of 25 and 50% ratio of nanoparticles. Finally, the range between 1550–1650 cm−1 belongs to the secondary amides bending, that also increases with the addition of nanoparticles. The Lactamide POSS®/PVA system shows the aliphatic stretch for C–N at 1020 cm−1, the presence of POSS® at 1080 cm−1, and the secondary amines bending, that increases with the addition of nanoparticles between 1550–1650 cm−1. These spectra confirm the interaction between POSS® nanoparticles with the polymer in the composite membranes, which is influenced by the loading concentration of nanoparticles.The absorption peak at approximately 1142 cmtallites , increas®/PVA and Lactamide POSS®/PVA systems, respectively. The pure PVA sample shows a major weight loss around 280 °C, attributed to the start of decomposition of the main polymer chain ending around 500 °C. The Amidino POSS®/PVA system shows two different major weight losses. The first one lies between 220 and 250 °C, attributed to the decomposition of the NH2 groups and the start of the decomposition of the polymer chain, continuing with a second step at 400–450 °C, due to the byproducts generated by PVA during degradation [® nanoparticles present a similar thermal stability as two major weight losses are observed in the range between 400–420 °C. The lactamide groups increase, slightly, the degradation of the polymer chain in this step, to reach a different final residual as well, depending on the concentration of the loading. The number corresponds initially to the residual of the PVA, and then to the increase of residual silicon content by the addition of nanoparticles. The weight loss between 100–200 °C in the samples containing nanoparticles could be due to either condensation of open POSS structures generating water, or due to the release of CO2 already reacted with the amine and amidine groups from Amidino POSS® and Lactamide POSS® [® having the highest final degradation temperature. The thermal stability of the nanocomposite membranes with various nanoparticle loadings was studied by comparing the decomposition onset temperatures presented in radation ,44. The ®/PVA and Lactamide POSS®/PVA. The degree of crystallinity . The melting point (Tm) of the composite membranes is lower than the virgin PVA polymer membrane. Thus, the melting point of the composite material decreases because of the addition of the POSS® nanoparticles. The decrease in Tm with the addition of POSS® indicates that incorporation of POSS® encourages the crystallization process, and results in crystallites with lower thermal stability. This is explained as POSS® acting as a nucleating agent, increasing the degree of crystallinity [allinity ,46. The ® acting as nucleating agent. The difference in the values between the Amidino and Lactamide POSS® can be due to the lactic moieties that are believed to improve compatibility of Lactamide POSS® with PVA, as it contains secondary hydroxyl groups.The composite membrane shows a typical thermoplastic behavior. The storage modulus is higher for the pure polymer, and it decreases slightly with the increase of nanoparticle loading, corresponding to the primary relaxation associated with the glass–rubber transition of amorphous PVA. Then, when the loading reaches 50%, the E’ modulus increases as compared to the virgin PVA. In this case, this increment can be explained by the crystallization occurring by the addition of POSS®/PVA, and Lactamide POSS®/PVA, respectively. ® and Lactamide POSS® nanoparticles show that the tan delta gradually decreases by the loading of the nanoparticles. A second peak is found in the region above the Tg. This peak is also known to be the alpha star transition (Tα*). In semi-crystalline polymers, this transition is associated with the slippage between crystallites [® nanoparticles, these materials produce a different type of crystalline segment, thus inducing a modification of the tan delta (Tand Tg) values, and then, an increase of the alpha start transition is visible induced by the movement (slippage) of the crystalline segments. These changes are confirmed in the DSC measurements, where the melting temperature (Tm) is decreasing as an indication of forming different lamellar conformation. The alpha star transition also increased with the nanoparticles loading, see ®/PVA system shows that the addition of these nanoparticles has a higher effect of interaction since the Tand Tg and the alpha star transition (Tα*) are higher in comparison with those obtained in the Amidino POSS®/PVA system. Moreover, these interactions are also visible in the degree of crystallinity (® loadings. This improved compatibility was expected as the Lactamide groups can interact better with the PVA polymer chain. Similar behavior is observed for the storage modulus (E’).The addition of the Amidino POSStallites ,47. When2, H2O, and the fixed carrier. As a general trend, with increasing feed pressure, the concentration of CO2 dissolved into the membrane, increases. As the CO2 concentration increases, some carriers may be saturated, and fewer sites are available to facilitate the CO2 transport, resulting in a decrease of CO2 permeance [2 permeance may decrease due to a high CO2 partial pressure on the feed side saturating the carriers [2 permeability, while the selectivity increases with the pressure, whereas a bigger change is visible in the loading for lactamide 25% between the pressure range 1.3 to 2 bar; this is possibly due to some inaccuracy during measurement. Unlike CO2 permeance, N2 permeance decreases with increasing feed pressure, which is consistent with the characteristic of ‘‘solution-diffusion’’ mechanism [2 transport to the gas separation in the tested pressure range.The effect of the feed pressure could have an important significance on the gas separation for facilitated transport membranes. The facilitated transport is based on a reversible reaction of COermeance ,49. In ocarriers ,34. To iechanism , resultiechanism . This tr® can give different properties to the polymer, like thermal and chemical stability, and acts as a nucleating agent [2 permeability as function of the degree of crystallinity , and more research about this is needed. olecules . The add® nanoparticles (Amidino POSS® and Lactamide POSS®) were used to understand the effect of the functional groups on the interaction with the polymer, and the gas separation performance. SEM pictures showed that a continuous composite PVA layer was formed on the support, and no evidence for pores or defects was observed. The FTIR spectra confirmed the structure of the resultant membranes, by showing the change by the addition of the nanoparticles. DSC studies indicated that the glass transition (Tg) decreased by the addition of the nanoparticle loading. It indicated an increase of the enthalpy of melting within the increase of the degree of crystallinity  was found using DMA technique. This alpha transition is known to be the transition where the crystallites slip past one another. The value of this alpha transition increased when increasing the loading. In this sense, the Lactamide POSS® system showed a higher interaction with the polymer, as expected, by having a higher alpha star transition Tα*. The gas separation performance showed a decrease for the CO2 permeability, while the selectivity slightly increased with the pressure. This trend shows that the membranes did not follow a facilitated transport mechanism, and the nanoparticle loading did not induce extra CO2 transport to the gas separation. The system is primarily dominated by the solution-diffusion mechanism. The degree of crystallinity indicated a strong effect in the gas separation performance of the composite membranes. Finally, the permeability was lower when the Lactamide POSS® group was added to the system. This is due to the higher interaction with the polymer, which increases the degree of crystallinity and reduces the composite membrane permeability.Two different types of POSS


SL-induced stomatal closure was strongly inhibited by a Ca2+ chelator and Ca2+ channel blockers, indicating that Ca2+ functions in SL promotion of stomatal closure. Through examining a collection of cpk mutants, we identified CPK33, potentially acting as a Ca2+ transducer, which is implicated in guard cell SL signaling. SL- and Ca2+-induced stomatal closure were impaired in cpk33 mutants. CPK33 kinase activity is essential for SL induction of stomatal closure as SL-induced stomatal closure is blocked in the dead kinase mutant of CPK33. The cpk33 mutant is impaired in H2O2-induced stomatal closure, but not in SL-mediated H2O2 production. Our study thus uncovers an important player CPK33 which functions as an essential Ca2+ signals mediator in Arabidopsis guard cell SL signaling.Strigolactones (SLs) are known to mediate plant acclimation to environmental stress. We recently reported that SLs acted as prominent regulators in promotion of stomatal closure. However, the detailed mechanism by which SLs induce stomatal closure requires further investigation. Here we studied the essential role of the calcium (Ca Particu2+) .2O2) and nitric oxide production in an ABA-independent manner, possibly preventing water loss and pathogen invasion and thereby resulting in plant acclimation to environmental stress cyt] that includes Ca2+ influx elevation from extracellular spaces and Ca2+ release from intracellular stores  anion channels and/or the GATED OUTWARDLY-RECTIFYING K+ (GORK) channel ] have long been recognized to be involved in activating ion channels and stimulating stomatal closure  function as Caignaling . Mechaniignaling . To dateignaling . For insignaling . Disruptignaling . CPK3 anignaling . CPK33 signaling , whereasignaling . It was t stress . Altoget closure .2+ in SL-induced stomatal closure was firstly determined by performing pharmacological studies, which indicate that Ca2+ acts as a prominent mediator functions in SL induction of stomatal closure. Through examining a collection of cpk mutants, we identified CPK33 (and possibly CPK10) which is implicated in guard cell SL signaling. SL activation of stomatal closure, as well as Ca2+-induced stomatal closure, was greatly impaired in cpk33 mutants. Additionally, CPK33 kinase activity is essential for SL induction of stomatal closure. We further found that H2O2-induced stomatal closure was moderately impaired in cpk33 mutants whereas SL-mediated H2O2 production was maintained in cpk33 mutants. Taken together, our study uncovers an important player CPK33 that functions as an important mediator in SL signaling in Arabidopsis guard cells.In this study, the importance of CaArabidopsis ecotype Columbia-0 (Col-0) was used as wild-type (WT) plants in this study. The following mutants have been described previously: max1-1, max2-1, max2-2, max3-9, and max4-1  were crossed. Homozygous double mutants were determined by PCRs using a combination of a gene-specific primer and a T-DNA border primer. The primers used were listed in To obtain the double mutant Seeds were sterilized and sown for germination in 1/2 Murashige and Skoog (MS) medium supplemented with 0.8% (w/v) agar and 1% sucrose (w/v). Seedlings were transplanted into pots and subsequently kept in a growth chamber with a 16h/8h (light/darkness) regime at 21°C. Fully expanded rosette leaves detached from 4- to 6-week-old healthy plants were harvested for immediate use.Stomatal apertures were measured as described previously . In brie2DCF-DA; Biotium Hayward, USA) were performed as described previously , aluminum chloride , trifluoperazine (TFP), and trifluoperazine dihydrochloride  were dissolved appropriately according to the supplier information and prepared stock solutions for further use. Other chemicals used in this study, including H2O2, 2-(N-morpholino) ethanesulphonic acid (MES), and calcium chloride (CaCl2), were purchased from Sigma-Aldrich. All chemicals used are of the highest analytical grade.The preparation of synthetic SL analog GR24  and 2′,7′-dichlorofluorescein diacetate . The fluorescence intensity representing the endogenous H2O2 content was determined using ImageJ software. Fluorescence intensities were normalized to those of controls. The data for fluorescence intensities represent the mean ± SE of three replicates.HStatistical analyses were performed using a one-way ANOVA to discriminate significant differences followed by the least significant difference test.2+ is required for SL-triggered stomatal closure. To this end, we examined the effects of ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetra acetic acid , and LaCl3 and AlCl3 (Ca2+ channel blockers) on SL-induced stomatal closure  induced a similar stomatal closure in SL-related mutants as in WT plants, whereas SL-signaling mutants max2 and d14 were insensitive to GR24 as reported previously  that is involved in guard-cell SL signaling. CPKs, as Ca2+ sensors, are important in the regulation of ABA-mediated and Ca2+-mediated stomatal closure  that might be involved in SL signaling in guard cells, the effect of GR24 on stomatal aperture was examined in a collection of 11 cpk23 Figure 22+, we hypothesized that disruption of CPK33 would impair Ca2+ signaling transduction. To test this hypothesis, we investigated the stomatal response following exogenous Ca2+ application in cpk33-1 and cpk33-2 mutants. Exogenous Ca2+ stimulated stomatal closure in WT plants, but not in cpk33-1 and cpk33-2 mutants , and two independent lines expressing a kinase-inactive CPK33K102R construct in cpk33-1 (K102R cpk3335::CPK33 #1 and #2). Two 35::CPK33 cpk33 lines exhibited GR24-sensitive phenotype similar to that of WT plants, while we found that K102R cpk3335::CPK33 plants, as well as cpk33-1 and cpk33-2 mutants, failed to close stomata in response to GR24  that function as signal mediator(s) in guard cells. However, it remains to be explored whether and how CPK33 is activated by SL-induced H2O2To further investigate the position of H2O2 and nitric oxide, eventually promoting plant resilience to environmental stress  is implicated in SL-triggered stomatal closure.It has been shown that SLs were implicated in various developmental processes of plants and mediate their responses to environmental stress . Recentll stress . Howeverl stress . The dis closing . OST1, a closure . Based o2+ chelator EGTA, and Ca2+ channel blockers LaCl3 and AlCl3 significantly suppressed SL-induced stomatal closure  might also required for SL-triggered stomatal closure. In the meanwhile, our results emphasizes the essential role of H2O2 serving as a hub in the complicated hormone-mediated stomatal closure. Altogether, our study reinforces the understanding of the molecular mechanism by which SLs induce stomatal closure and provides new insights to improve stress acclimatization of plants.Integrated previous studies with our present genetic and physiological analyses, we propose that CPK33, as a Caand Ca2+ Figure 6 closure . However closure . CPK33 wt kinase , indicatThe datasets generated for this study are available on request to the corresponding author.YZ and GW designed the experiments. XW, SL, XH, XG, XS, JK, ZL, and YZ performed the experiments. BC assisted in microscope. WZ contributed materials. YZ, CL, and GW wrote the manuscript. All authors reviewed the manuscript.The study is supported by the National Natural Science Foundation of China (31701294 and 31801210), the Fundamental Research Funds for Central Universities (GK201702016), the SNNU Laboratory Technology Research Funds (SYJS201739), the Start-up Foundation of Hubei University of Medicine (2016QDJZR14), and the Natural Science Foundation of Hubei Provincial Department of Education (Q20182104).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.