Rhizoctonia cerealis, is a destructive disease in hexaploid wheat (Triticum aestivum L.). In this study, a wheat COMT gene TaCOMT-3D, is identified to be in response to R. cerealis infection through microarray-based comparative transcriptomics. The TaCOMT-3D gene is localized in the long arm of the chromosome 3D. The transcriptional level of TaCOMT-3D is higher in sharp eyespot-resistant wheat lines than in susceptible wheat lines, and is significantly elevated after R. cerealis inoculation. After R. cerealis inoculation and disease scoring, TaCOMT-3D-silenced wheat plants exhibit greater susceptibility to sharp eyespot compared to unsilenced wheat plants, whereas overexpression of TaCOMT-3D enhances resistance of the transgenic wheat lines to sharp eyespot. Moreover, overexpression of TaCOMT-3D enhances the stem mechanical strength, and lignin (particular syringyl monolignol) accumulation in the transgenic wheat lines. These results suggest that TaCOMT-3D positively contributes to both wheat resistance against sharp eyespot and stem mechanical strength possibly through promoting lignin  accumulation.Plant caffeic acid 3-O-methyltransferase (COMT) has been implicated in the lignin biosynthetic pathway through catalyzing the multi-step methylation reactions of hydroxylated monomeric lignin precursors. However, genetic evidence for its function in plant disease resistance is poor. Sharp eyespot, caused primarily by the necrotrophic fungus  Triticum aestivum L., namely bread wheat or common wheat, genome AABBDD) is one of the most widely cultivated and consumed food crops worldwide. Stem lodging in wheat is a major agronomic problem that has far-reaching economic consequences1. Crop lodging is a complicated phenomenon that is influenced by many factors from physiology and genetics to husbandry and environment3. Green Revolution has effectively increased lodging resistance and the harvest index through the use of semi-dwarf trait. However, a reduction in plant height for lodging resistance may reduce the photosynthetic capacity of a canopy which would then limit the final yield4. A paper reported that the optimum plant height for maximum photosynthetic capacity is between 70 cm and 100 cm in wheat5. Thus, alternative strategies for further improvement in lodging resistance of crops must be developed. Increasing the stem/culm mechanical strength has been shown to be efficient for improving crop lodging resistance6. Sharp eyespot is a devastating soil-borne fungal disease globally, which results in yield losses of 10–40%8. The necrotrophic fungus Rhizoctonia cerealis Van der Hoeven is a major causal pathogen of sharp eyespot9. The pathogen can infect the basal stems and sheath, destroy the transport tissues of infected plants and block transportation of substances required for nutrition, leading to lodging and dead spikes. Isolation and application of important resistance-related genes make it possible to generate wheat materials with resistance to both this disease and lodging.Hexaploid wheat , transient knock-down of monolignol pathway enzymes  individually and pair-wise led to decreased basal immunity or penetration resistance to the fungal pathogens Blumeria graminis f. sp. tritici and Blumeria graminis f. sp. Hordei, respectively11. In rice (Oryza sativa L.), the cinnamoyl-CoA reductase (CCR) contributed to R-mediated immunity against the hemibiotrophic fungal pathogen Magnaporthe grisea21. In Arabidopsis and tobacco, loss or down-regulation of PAL led to decreased basal immunity to the hemibiotrophic bacterial pathogen Pseudomonas syringae22 and to the biotrophic viral pathogen tobacco mosaic virus24, respectively. In Arabidopsis, through assessment on double mutant (cad-C cad-D), the genetic and functional analysis indicated that the CAD-C (At3g19450) and CAD-D (At4g34230) were the primary genes being involved in lignin biosynthesis26, and acted as essential components of defense response against virulent and avirulent strains of the bacterial pathogen Pseudomonas syringae pv. tomato16. Recently, Rong et al. reported that the wheat CAD named TaCAD12 positively contributed to host defense response to a necrotrophic fungal pathogen R. cerealis27. More recent paper reported that a maize (Zea mays L.) CCoAOMT named ZmCCoAOMT2 was isolated18. ZmCCoAOMT2 has been shown to confer quantitative resistance to both southern leaf blight and gray leaf spot through altering lignin level and other metabolites of the phenylpropanoid pathway and regulation of programmed cell death18. However, defense role of COMT in plant species is poorly understood.Lignin is a phenolic cell wall polymer, which is composed of guaiacyl, syringyl and p-hydroxyphenyl units derived from the monolignol precursors , respectively  binding domain in COMT proteins indicates the use of SAM as the methyl group donor to the hydroxyl group of a methyl acceptor molecule30. COMT catalyzes the multi-step methylation reactions of hydroxylated monomeric lignin precursors32. COMT is involved in the methylation of caffeic acid to ferulic acid, which is then hydroxylated at position five by ferulate-5-hydroxylase. The subsequent methylation by COMT at this position yields sinapic acid. Similarly, COMT catalyzes the 3-O-methylation of caffeoyl aldehyde and conifer-aldehyde/coniferyl alcohol in the process of guaiacyl and syringyl monolignol biosyntheses12. For example, Alfalfa COMT (MsCOMT) exhibits the higher catalyzing efficiency towards 5-hydroxyconiferaldehyde and caffeoyl aldehyde rather than caffeic acid33. In Brachypodium distachyon, COMT has high affinity for a variety of substrates including flavonoid compounds, with the greatest activity towards caffeic acid and caffeoyl aldehyde34. Interestingly, TaCM (GenBank accession number EF413031), a wheat COMT gene, was reported to locate on wheat chromosome 3BL and to encode an authentic COMT enzyme TaCM13. Kinetic analysis indicated that the TaCM protein exhibited the highest catalyzing efficiency towards caffeoyl aldehyde and 5-hydroxyconiferaldehyde as substrates, suggesting a pathway leading to syringyl lignin via aldehyde precursors13. Antisense TaCM transgenic tobacco assay indicated that the down-regulation of TaCM resulted in reduction in COMT activity and a sharp reduction in the syringl monolignol13. It has been demonstrated that in maize, rice and sorghum (Sorghum bicolor L.), the O-methyltransferase responsible for the production of syringyl lignin was also involved in the synthesis of lignin-linked tricin35. Several groups aimed to improve the production of biofuel and generated transgenic plants with deficit/reduction of syringyl lignin unit through knock-out/down of the lignin-related COMT genes in plants39. However, no genetic evidence for defense roles of COMT genes has yet been reported in plants.The COMT is an O-methyltransferase that can potentially act in various branches of the phenylpropanoid pathwayR. cerealis R0301 through comparative transcriptomic analysis on the sharp eyespot-resistant wheat line CI12633 and the susceptible wheat line Wenmai 6. Subsequently, this COMT gene located on wheat chromosome 3DL, designated as TaCOMT-3D, was cloned from the resistant wheat line CI12633. Importantly, through generation and evaluation of TaCOMT-3D- silencing and overexpressing wheat plants, we dissected roles of TaCOMT-3D in resistance response to R. cerealis, lignin accumulation, and stem mechanical strength in wheat. The functional characterization proved that TaCOMT-3D positively contributed to syringl monolignol biosynthesis, wheat resistance response to R. cerealis, and the stem mechanical strength.In this study, the probe with Agilent GeneChip number A_99_P198406, being homologous to certain plant COMTs, was identified to be in response to R. cerealis R0301 . Among them, the probe A_99_P198406, 100% matching to 3′-terminal sequence of a wheat cDNA sequence with accession number AK332908, displayed significantly transcriptional increase in sharp eyespot-resistant wheat lines (CI12633 and Shanhongmai) than in the susceptible wheat line Wenmai 6 at 4, 7, and 21 dpi (day post inoculation) with R. cerealis R0301  (IWGSC). The alignment result displayed that this gene sequence shared 95.33%, 95.42%, and 100% identities with the matched sequences of the TGACv1_scaffold_195106_3AL, TGACv1_scaffold_220646_3B, and TGACv1_scaffold_249256_3DL, respectively. The data suggested that the gene should locate on the long arm of the wheat chromosome 3D. To verify this alignment on the chromosomal localization, genomic DNAs extracted from Chinese Spring Nulli-tetrasomic lines (NTs) were used as template of PCR using the gene-specific primers to confirm the chromosome location of the gene. The results proved that the gene was indeed located on the long arm of chromosome 3D (3DL)  genome (DL) Fig. . Blast sTaCOMT-3D was cloned from cDNA and genomic DNA of CI12633 stems inoculated with R. cerealis R0301, respectively. The comparison of the cDNA and genomic sequences indicated that TaCOMT-3D genomic sequence with 1480 bp-length was comprised of one intron (161-bp length) and 2 exons , the 5′-untranslated region (UTR) with 61-bp, and 3′-UTR with 187-bp 13 and flavonoid O-methyltransferase encoding gene TaOMT2 (GenBank accession no. DQ223971)29. A pairwise alignment indicated that the TaCOMT-3D cDNA sequence shared 95.24% and 95.33% identities with TaCM and TaOMT2. The predicted protein TaCOMT-3D is consisted of 356 amino acids with a molecular weight of 38.697 kD and a theoretical iso-electric point of 6.042. As shown in Fig. 13. As shown in Fig. in vitro and in vivo (transgenic tobacco) results revealed that TaCM was an authentic COMT enzyme and participated predominantly in syringl monolignol biosynthesis13. Thus, TaCOMT-3D should be an authentic COMT enzyme, too.The full-length cDNA and genomic sequences of ons Fig. . The TaC-bp Fig. . Blast rTaCOMT-3D in stems of five wheat lines/cultivars with different resistance degrees, including sharp eyespot-resistant lines CI12633 and Shanhongmai, moderately-resistant line Shannong 0431, moderately-susceptible wheat cultivar Yangmai 158, and susceptible cultivar Wenmai 6 at 21 dpi with R. cerealis R0301. The results showed that TaCOMT-3D transcriptional level was the highest in Shanghongmai, slightly decreased in CI12633, gradually declined in Shannong 0431 and Yangmai 158, and reached the lowest in Wenmai 6  increased with the pathogen inoculation duration  technique was used to analyze the transcriptional levels of i 6 Fig. . The dation Fig. , and thelis Fig. . FurtherTaCOMT-3D in wheat. To knockdown TaCOMT-3D expression in the resistant wheat line CI12633 through BSMV-VIGS method, a 3′-terminal fragment (233-bp) specific to TaCOMT-3D was inserted in an antisense orientation into Nhe I restriction site of the BSMV RNAγ chain to generate BSMV:TaCOMT-3D recombinant construct , compared with that on the stems of BSMV:GFP-treated CI12633 plants (average infection type: 2.1) -based virus-induced gene silencing (VIGS) technique was used to analyze rapidly the functions of uct Fig. . When thuct Fig. , and thents Fig. . The resnts Fig. . Then, t.1) Fig. . These rTaCOMT-3D overexpressing transgenic wheat lines were generated and used to further investigate the role of TaCOMT-3D in resistance response to R. cerealis. By using the primers specific to the transformation vector pWMB-TaCOMT-3D  in T0-T2 generations  were markedly elevated than in wild- type (WT) Yangmai 16 (recipient)  generations showed that compared with WT Yangmai 16, all these four TaCOMT-3D-overexpressing wheat lines  displayed significantly enhanced resistance to sharp eyespot caused by R. cerealis R0301  Fig. , and the 16 Fig. . The sha301 Fig. , Table 1TaCOMT-3D affects the lignin biosynthesis in wheat, lignin content and composition in the second basal internode at milky stage were investigated. The examination data showed that the lignin content in TaCOMT-3D-overexpressing transgenic wheat lines was higher than that in WT Yangmai 16 plants (Table TaCOMT-3D-overexpressing wheat lines displayed increased staining (red-brown) relative to the WT samples  strength of the second basal internode of TaCOMT-3D-overexpressing and WT wheat Yangmai 16 lines was measured using a Texture Analyser (TA) with a three-point bend test setup at milky and harvest stages, respectively. As shown in Table TaCOMT-3D-overexpressing wheat lines  exhibited significantly increased mechanical strength of the second basal internode at both milky and harvest stages. These data suggested that overexpression of TaCOMT-3D enhanced the stem mechanical strength of the transgenic wheat.Previous study showed that 13. However, little is known about COMT participating in defense responses to necrotrophic fungal phytopathogens. In this study, the wheat caffeic acid O-methyltransferase encoding gene TaCOMT-3D was identified through microarray-based comparative transcriptomic analysis. The chromosome-based draft sequence alignment and PCR analysis indicated that TaCOMT-3D located on the long arm of wheat chromosome 3D. Previously, two genetic studies reported that two QTLs on 3D locating in the intervals Xgwm61~Xgwm172–240 and Xgwm314~Xgwm38341 for sharp eyespot resistance, respectively, the latter of which explained 7% of the phenotypic variation41. Here, qRT-PCR analysis showed that the transcriptional level of TaCOMT-3D was higher in sharp eyespot-resistance wheat lines than in susceptible wheat lines/cultivars. To explain the differences in the expression profile of TaCOMT-3D between resistant and susceptible wheat, the gene promoter sequences were also cloned from CI12633 and Wenmai 6, respectively. However, no difference existed between the gene promoter sequences from these two wheat lines. Thus, we deduced the different expression profile of TaCOMT-3D between resistant and susceptible wheat may be due to the differences in regulatory pathways that activate/suppress TaCOMT-3D expression in wheat. The gene expression level was obviously elevated after infection of R. ceraelis. These results imply that TaCOMT-3D may participate in defense response of wheat to R. cerealis infection. BSMV-based VIGS technique has been shown to be an effective reverse genetic tool for rapidly investigating the defense functions of interesting genes in barley and wheat46. In this report, the BSMV-VIGS results show that silencing of TaCOMT-3D in the resistant wheat line CI12633 significantly impairs host resistance to R. cerealis. The data suggest that TaCOMT-3D is required for resistance response of wheat, at least the resistant wheat line CI12633, to R. cerealis infection. To further confirm the defense function of TaCOMT-3D, transgenic wheat plants overexpressing TaCOMT-3D were generated and characterized. Taken molecular assay together disease resistance assessments for T0-T2 generations, these results indicate that TaCOMT-3D-overexpressing transgenic wheat lines display significantly enhanced resistance to sharp eyespot during whole growth stages. Interestingly, TaCOMT-3D overexpression in wheat did not obviously affect major agronomic traits  in wheat  of Blumeria graminis f. sp. tritici, suggesting that TaCOMT-3D had no obvious effect on the wheat resistance phenotype to the mixed strains of powdery mildew. It is also important and interesting to further assay responses of TaCOMT-3D silenced and overexpressing wheat plants to infection of additional pathogens in future. Anyway, the current results broaden the current knowledge of plant defenses against pathogens.Plant COMT catalyzes the multi-step methylation reactions of hydroxylated monomeric lignin precursors, consequently is involved in the lignin biosynthesis13. Sequence analyses reveal that SAM binding motif, catalytic residues, and active site substrate binding/positioning residues of TaCOMT-3D are the same as those in TaCM, even though seven amino acid differences exist between the protein sequences of TaCOMT-3D and TaCM. Interestingly, TaCOMT-3D and TaCM proteins shared 100% identity in all the biochemically functional sites, including the SAM binding motif, catalytic residues and active site substrate binding/positioning residues. The previous study showed that TaCM protein exhibited a strong catalyzing activity towards caffeoyl aldehyde, caffeoyl-CoA, 5-hydroxyferuloyl-CoA and 5-hydroxyconiferaldehyde, revealing that TaCM is a typical COMT involved in lignin (syringyl monolignol) biosynthesis13. Additionally, among the functional sites, 13 amino acid residues being involved in the active site substrate binding/positioning residues have only one amino acid difference between TaCOMT-3D  and the alfalfa COMT 47, with V instead of I in the position 312. The crystal structure of COMT in complex with S-adenosyl-L-homocysteine, ferulic acid, and 5-hydroxyconiferaldehyde provide a structural understanding of the observed substrate preferences. And these crystal structures identify residues lining the active site surface that contact the substrates32. These results suggest that TaCOMT-3D should possess the COMT activity and may participate in lignin  biosynthesis.The sequence alignment of amino acid sequences showed that the TaCOMT-3D protein shares a 98.03% identity with the wheat caffeic acid 3-O-methyltransferase TaCM39. For example, the Brachypodium Bd5139 mutant with a single nucleotide mutation in the caffeic acid O-methyltransferase encoding gene BdCOMT6 displayed a moderately altered lignifiation in mature stems38. Ma and Xu reported that the expression of antisense TaCM in transgenic tobacco specifically reduced the COMT enzyme activity and marginally decreased lignin content, especially sharply reduced the syringl lignin monomer13. Ma reported that TaCM expression participated in lignin synthesis and was positively associated with stem strength and the lodging-resistant trait in wheat lodging-resistant line C6001 and lodging-susceptible wheat H45642. However, little has been known about effect of COMT overexpression on lignin biosynthesis and stem mechanical strength in crop plants. Here, our results indicated that overexpression of TaCOMT-3D increased lignin level, especially syringyl monolignol content. Furthermore, TaCOMT-3D overexpression enhanced the basal stem mechanical strength, and the content of lignin and syringyl monolignol had a significantly positive correlation with the culm mechanical strength, which were in line with the effect of TaCM expression on lignin synthesis and stem strength in two wheat cultivars2. Previous studies documented that lignin accumulation and its composition  are important factors influencing the breaking strength of wheat culm3. Zheng et al. showed that syringyl monolignol accumulation provided significant mechanical advantages to angiosperm species3. In this report, these assays suggest that TaCOMT-3D primarily enhances syringyl monolignol accumulation, leading to the increased the basal stem breaking strength. Lignin has been implicated in plant disease resistance19. Recently, the lignin biosynthesis-related enzyme ZmCCoAOMT2 in maize has been reported to confer quantitative resistance to southern leaf blight and gray leaf spot through increasing levels of lignin and other metabolites of the phenylpropanoid pathway18. Taken together, we can deduce that TaCOMT-3D enhances wheat defense response to R. cerealis and stem mechanical strength possibly through promoting lignin, especially syringyl monolignol accumulation.In previous studies, assays of knock-out/down mutants/transgenic plants indicated that COMT participated predominantly in syringl monolignol biosynthesis and can change lignin content and compositionTaCOMT-3D was identified through microarray-based comparative transcriptomics. The transcriptional level of TaCOMT-3D is higher in resistant wheat lines and significantly elevated after R. cerealis infection. TaCOMT-3D positively contributes to both resistance response to R. cerealis and adult stem mechanical strength possibly through promoting accumulation of lignin . This study provides novel insights into the biological roles of COMT members in plants. TaCOMT-3D is a potential gene for improving resistance to both sharp eyespot and lodging.In conclusion, the wheat gene Six wheat lines/cultivars, including sharp eyespot-resistant lines CI12633 and Shanhongmai, moderately-resistant cultivar Shannong 0431, moderately-susceptible cultivars Yangmai 158 and Yangmai16, and susceptible cultivar Wenmai 6, were used in this research.R. cerealis isolate R0301, was kindly provided by Profs. Huaigu Chen and Shibin Cai . A North China high-virulence strain of the pathogen R. cerealis isolate WK207 was provided by Prof. Jinfen Yu .A major Jiangsu virulent strain of the pathogen 48.Genomic DNA for each sample was isolated from the wheat leaves using the CTAB methodTotal RNA was extracted using TRIzol , and then subjected to Rnase-free Dnase I  digestion and purification.The purified RNA sample (2 µg) was reverse-transcribed to cDNA using the FastQuant RT Kit with gDNase .TaCOMT-3D. The 1319-bp full-length cDNA sequence of TaCOMT-3Dwas amplified by nested PCR from cDNA of CI12633 stems inoculated with R. cerealis R0301 for 4 d. The primers for the first round PCR were TaCOMT-3D-F1 (5′-GAAAGTAGGTCATCGCTCAGTC-3′) and AUAP (5′-GGCCACGCGTCG ACTAGTAC-3′). These for the second round PCR were TaCOMT-3D-F2 (5′-CTCGCTCACACTCAACGC-3′) and TaCOMT-3DL (5′-GGTGGATTTTA ATACACACAA-3′). The deduced protein sequence was analyzed by the Compute pI/Mw tool (http://web.expasy.org/compute_pi/) to determine the theoretical iso-electric point and molecular weight, Pfam database (http://pfam.xfam.org) and smart software (http://smart.embl-heidelberg.de/) to predict conserved motifs, DNAMAN software to analyze sequence identity.The microarray analysis using the Agilent wheat microarray indicated that a probe with Agilent GeneChip number A_99_P198406, corresponding to 3′-terminal sequence of a wheat cDNA sequence with accession number AK332908, was expressed at a significantly higher level in sharp eyespot-resistant wheat lines CI12633 and Shanghongmai than in the susceptible wheat Wenmai 6. This gene cloned from stems of the resistant wheat CI12633, based on AK332908 sequence, was designated as TaCOMT-3D (from 1022 to 1254 nucleotides in TaCOMT-3D cDNA sequence) was sub-cloned in an antisense orientation into the Nhe I restriction site of the RNAγ of BSMV /10 h dark (12 °C) regime. To investigate if BSMV successfully infected CI12633 plants, and to test if TaCOMT-3D transcript was down-regulated, at 10 dpi with the BSMV virus, the fourth leaves of the inoculated seedlings were collected and subjected to analysis on the transcription of the BSMV coat protein gene (CP) by RT-PCR and the transcription of TaCOMT-3D by qRT-PCR. At elongation stage, the BSMV-infected CI12633 plants were further inoculated with R. cerealis isolate WK207 mycelia according to Wei et al.50. The infection types (ITs) of these plants were scored at 45 dpi with R. cerealis WK207 following the protocol described by Chen et al.7.To generate the BSMV:TaCOMT-3D recombinant construct, a 233-bp sequence specific to SMV Fig. . FollowiTaCOMT-3D ORF plus a 6 × His epitope tag sequence was sub-cloned into monocot transformation vector pWMB12251. In the resulting overexpression transformation vector pWMB-TaCOMT-3D  promoter, and terminated by 3′-non-transcribed region of Agrobacterium tumefaciens nopaline synthase gene (Tnos). A total of 274 immature embryos of the wheat cultivar Yangmai 16 were transformed with through Agrobacterium harbouring the pWMB-TaCOMT-3D vector according to the protocol described by Wang et al.52.The -3D Fig. , the traTaCOMT-3D-overexpressing transgene in the transformed wheat plants was monitored by PCR using the specific primers, TaCOMT-3D-F  and Nos-R . PCR was performed in a 25 μl volume containing ~200 ng genomic DNA, 12.5 μl 2 × Taq MasterMix , 1 μl each primer (10 mΜ). The amplified product (318-bp size) specific to the introduced TaCOMT-3D-Tnos chimera was resolved on a 1.5% agarose gel and visualized by ethidium bromide staining.The presence of the TaCOMT-3D in various wheat plants. qRT-PCR was performed using SYBR Green I Master Mix  in a volume of 25 μl on an ABI 7500 RT-PCR system (Applied Biosystems). Reactions were set up using the following thermal cycling profile: 95 °C for 15 min, followed by 40 cycles of 95 °C for 10 s, 56 °C for 30 s, and 72 °C for 32 s. The relative transcriptional levels of the target genes was calculated using the 2−ΔΔCT method53, where the wheat Actin gene TaActin was used as the internal reference. The relative transcriptional levels of the tested genes in the TaCOMT-3D-overexpressing wheat lines or in BSMV:TaCOMT-3D-infected wheat plants were relative to those in WT recipient or in BSMV:GFP-infected wheat plants.qRT-PCR analysis with specific primers TaCOMT-3D-QF (5′-AGAAGGTGCCCTCGGGT-3′) and TaCOMT-3D-QR (5′-TGCATTGGCGTA GATGTAAGTG-3′) was used to investigate the relative transcriptional levels of CP gene in wheat plants inoculated with BSMV, an indicator of BSMV infection, was investigated by semi-quantitative RT-PCR method with the BSMV-CP gene-specific primers .The transcriptional level of BSMV R. cerealis R0301 for 20 d by using the tissue protein extraction kit . Total soluble proteins (~20 μg) for each line were separated on 12% SDS-PAGE and transferred to polyvinyl difluoride membranes (Amersham). The blotting membranes were incubated with 2500-fold diluted Anti-His Mouse Monoclonal Antibody  at 4 °C overnight, then incubated with 4, 000-fold diluted Goat Anti-Mouse IgG (H + L), HPR conjugated secondary antibody  at 22–23 °C for 1 h. The TaCOMT-3D-His fusion protein was visualized using the Pro-light HRP Chemiluminescent Kit .The TaCOMT-3D-His fusion protein in the overexpressing wheat lines was visualized via western blotting analysis. Total proteins were extracted from ~0.2 g of stems inoculated with 1 and 110 plants in T2 generations for each line of the TaCOMT-3D-overexpressing wheat lines and non-transgenic wheat Yangmai 16 (recipient) were inoculated with sterilized grains harboring the well-developed mycelia of R. cerealis isolate R0301 following the protocol of Zhu et al.54. According to the modified protocol described by Chen et al.7, at harvest stage (about 60 dpi), the infection type (IT) of each plant was scored based on the disease lesion squares54, and disease index (DI) for each wheat line were categorized.At the active tillering stage, at least 30 plants in Tet al.55. Briefly, the TA was fitted with a load cell with maximum loading capacity of 5 kg. The support stands were set at 2.5 cm apart (across which the 5 cm stem sample was placed) and the testing probe was set to move at a constant speed of 2 mm/s. The TA, connected to a computer, produces a real-time graphical output, representing the mechanical profile of the stem sample being tested. From this graph, F max, the absolute resistance of the stem sample to break under-load, were obtained.Stem strength testing of the material was carried out using a Texture Analyser (TA) with a three-point bend test setup as described by Miller 26. Briefly, Wiesner staining was performed by incubating sections in 1% phloroglucinol in ethanol: water (7:3) with 30% HCl. Mäule staining was performed by first incubating sections in KMnO4. After 10 min, sections were washed and acidified with HCl for 1 min, washed again, and then incubated in NaHCO3. Cross sections were immediately observed under an optical microscope.Lignin content was quantitatively measured by using the lignin ELISA Kit  according to the manufacturer’s protocols. For histochemical analysis, fresh hand-cut sections were prepared from basal second internodes of wheat. Wiesner and Mäule staining methods were performed as previously describedSupplementary Information


We present an X-ray-optical cross-correlator for the soft ( I.Time-resolved X-ray experiments allow to access a wide variety of transient microscopic processes on the relevant time-scales, e.g., in molecular dynamics,zq is given byλ denotes the X-ray wavelength and ϑ the angle between incoming/outgoing X-ray wavevectors In the soft X-ray range, structural dynamics is hardly accessible since here the X-ray wavevector is small compared to the reciprocal lattice constants. In a specular reflection geometry, the momentum transfer πλsin(ϑ),where λ det al. to photo-excite coherent longitudinal acoustic phonons in a superlattice (SL) structure. Here the SL is a mirror for the whole soft X-ray regime for photon energies In this work, we adapt the concept of von Korff Schmising II.n) at In the case presented here, the investigated SL structure consists of 40 bilayers made of 2 between the Mo and Si layers in the SL structureThe dynamical X-ray diffraction simulationThe time-resolved experiments with either 75 ps . The solid line in this panel corresponds to a cosine-fit with an oscillation period of 575 fs and can be obviously linked to the coherent phonon oscillation in the SL structure with the simulated oscillation period of However, as discussed in Refs. For later delay times, after the SL oscillations have damped out due transmission of the coherent phonons to the substrate, the entire SL structure remains in an expanded state until heat diffusion subsequently leads to thermal equilibrium between the Mo and Si layers in the SL and to cooling of the SL due to thermal transport into the substrate. The later thermal process occurs on a ns-timescalet < 0) and 70 ps after photoexcitation. The incident laser fluence was set to approximately The total expansion of the SL can be directly probed by the shift of each SL Bragg peak in time-resolved α mode (approximately 10 ps at BESSY II)The underlying 10 ps time scale renders this transient peak shift an ideal cross-correlator for ps-pulsed X-ray sources like the normal single bunch of 3rd generation synchrotrons (approximately 75 ps at BESSY II),III.We presented a simple and fast cross-correlation method for the soft (


Retroperitoneal fibrosis (RPF) is a progressive fibroinflammatory disease that can be complicated by urinary obstruction. RPF can be the only manifestation of IgG4-related disease (IgG4-RD). Treatment of IgG4-related RPF is challenging and mostly consists of long-term glucocorticoids leading to significant side effects and treatment intolerance. Recent exploration of the role of rituximab as a B-cell depleting therapy in the treatment of IgG4-RD provides therapeutic potential as a well-tolerated alternative to glucocorticoids. We present a case of IgG4-related RPF for which rituximab was instituted as a steroid-sparing treatment strategy. Following 4 doses, kidney function partially recovered, and the disease went into remission. We discuss the potential merit of rituximab for the treatment of patients with IgG4-related RPF.  Retroperitoneal fibrosis (RPF) is a rare fibroinflammatory disorder that affects the soft tissue in the retroperitoneal space, originally described in 1948 by Dr. John Ormond . AlthougIgG4-related disease is an immune-mediated systemic disorder that was first described as a systemic disease in 2003 after recognition of extrapancreatic manifestations , and itsWe present the case of a 64-year-old woman with biopsy-proven isolated IgG4-related RPF, without other systemic manifestations of the disorder. In light of persistent renal dysfunction due to obstructive uropathy, she was treated with a short course of corticosteroids and rituximab, which was followed by improvement in kidney function. We review the existing literature on IgG4-related RPF and the potential role of B-cell depletion with rituximab as primary treatment for this systemic disorder. A 64-year-old woman with a past medical history of hypertension and right hemicolectomy due to colonic adenomas presented to an outside hospital with a 3-month history of dry heaves and malaise. Prior to this illness, the patient had been in her usual state of health. At the outside hospital, she was found to have acute kidney injury with a serum creatinine of 21 mg/dL (baseline of 1.3 mg/dL). She required emergent hemodialysis for kidney failure and hyperkalemia. A CT scan of the abdomen and pelvis without contrast was performed, demonstrating bilateral hydronephrosis with no evidence of a mass or stone. A retrograde pyelogram was performed, demonstrating bilateral ureteral obstruction with medial deviation of the ureters, and she subsequently underwent bilateral ureteral stents placement. She was discharged with a serum creatinine of 2.7 mg/dL. Four weeks later, she presented to our hospital after she had noticed passing one of the stents in the urine. At that time, her serum creatinine was 3.1 mg/dL. A CT scan of the abdomen and pelvis without contrast showed moderately-severe bilateral hydronephrosis, and absence of the right ureteral stent. It also demonstrated nonspecific presacral and retroperitoneal fat stranding . A retroSix days later, she underwent surgical bilateral ureterolysis, right ureteral reimplantation, and lysis of extensive bowel adhesions. During the procedure, the retroperitoneal soft tissue density was biopsied, which revealed fibrous proliferation with hyalinized collagen, focally-forming storiform fascicles, dense lymphoplasmacytic infiltrate with occasional germinal centers A, B, andTwo weeks later, she was started on a course of prednisone 40 mg once daily for 4 weeks followed by a taper. At the time, the serum creatinine was 1.57 mg/dL with an estimated GFR of 34 mL/min, placing her at stage-3b chronic kidney disease. The urinalysis revealed 2+ blood, 3+ protein, and no leukocytes. Her ESR and serum IgG4 level remained persistently elevated at 84 mm/h and 167 mg/dL, respectively. Complement factors C3 and C4 were normal at 140 mg/dL and 39 mg/dL, respectively. Her IgG4-RD responder index, a recently-developed disease activity score [2, with a total cumulative dose of 2 g over 1 month. Prior to her first infusion of rituximab, the serum creatinine was 2.4 mg/dL, the ESR was 110 mm/h, the CRP level was 42 mg/dL , and the serum IgG4 level had dropped to 94 mg/dL. One month after completing the rituximab course, her repeat serum creatinine was 2.3 mg/dL, the CRP normalized to 0.47 mg/dL, the serum IgG4 level further declined to 64 mg/dL, and the random urine albumin-to-creatinine ratio dropped to 370 mg/g. A repeat MRI revealed a substantial decrease in the amount of retroperitoneal soft tissue distal to the aortic bifurcation, which was accompanied by a decrease in the hydronephrosis bilaterally. The patient subsequently underwent the sequential removal of the two ureteral stents. 21 weeks after initiating therapy, the serum creatinine stabilized at 2.8 mg/dL, suggesting residual stage 4 chronic kidney disease. At the time, the serum IgG4 level was 49 mg/dL, the CRP was 0.24 mg/dL, and the ESR was 25 mm/h. The IgG4-RD responder index dropped to 3, consistent with a marked improvement in disease activity. We further assessed for response to B-cell depletion therapy with rituximab by performing peripheral blood flow cytometry, which showed a CD45-positive absolute count of 1.3K cells/µL  with 0% CD19-positive cells , and 0% CD20 positive cells , in support of a favorable treatment response.She received a course of rituximab to abbreviate her course of glucocorticoids to 2 months. The hepatitis B surface antigen and the hepatitis B surface and core antibody titers were nonreactive. Five weeks after initiation of the prednisone, she received her first of 4 weekly infusions of rituximab 375 mg/mHere, we describe a case of IgG4-related RPF presenting with kidney failure due to extraluminal ureteral obstruction requiring ureteral stent insertion and surgical ureterolysis, and treatment with a short course of glucocorticoids and a 4-week course of rituximab. Two months following treatment, there was substantial decrease in the amount of retroperitoneal soft tissue, accompanied by a decrease in the hydronephrosis, and stabilization of the kidney function. A review of the literature identifies 45 cases of RPF in the setting of IgG4-RD, published in case series and individual case reports  9, 10, , 12, 13.In all cases, first-line therapy consisted of oral prednisone. The dose ranged from 10 to 75 mg/day, and the tapering regimen and treatment duration were variable, ranging from 2 months to long-term low-dose corticosteroids after the initial taper. Second-line treatment options used in the described cases of IgG-4 related RPF included mycophenolate mofetil , methotr2 every week for a total of 4 doses. We observed an improvement in the IgG4-RD Responder Index and normalization of the serum IgG4 level and markers of inflammation. While the observed decrease in the amount of retroperitoneal soft tissue and hydronephrosis as well as the improvement in the kidney function are coincidental, we can only speculate whether the rituximab shortened the natural history of the IgG4-related RPF by promoting the removal of B-cells and accelerating the resolution of the retroperitoneal inflammation. To date, there is no consensus regarding the optimal therapy of IgG4-RD, and expert opinion is derived from observational studies and uncontrolled single-arm trials. The mainstay of therapy is the use of glucocorticoids as monotherapy. Clinical response to glucocorticoids is observed in most patients initially with improvement in symptoms, which is accompanied by a reduction in IgG4-related masses and serum IgG4 levels. Due to the heterogeneity of the disease, an IgG4-RD Responder Index was developed to monitor response to therapy. This index incorporates symptoms, organ damage, urgency of care, and the serum IgG4 level. Although most patients initially respond to glucocorticoids, the caveat to monotherapy with glucocorticoids includes relapse and disease progression in most cases, long-term steroid-related toxicity, and heterogeneity of treatment response to glucocorticoids in patients, varying from a few weeks to several months. As such, some experts in the field have advocated for adding a steroid-sparing agent to complement the therapy and avoid these concerns. This includes the use of B-cell depletion therapy with the anti-CD20 antibody, rituximab. The efficacy of rituximab has been evaluated in a prospective open-label trial of 30 patients with IgG4-RD in whom 3 (10%) patients had retroperitoneal fibrosis and 2 (7%) had aortitis . ParticiIn a systematic review, the efficacy of monotherapy with glucocorticoids was examined in 1,220 patients with IgG4-RD, of whom 97% had a therapeutic response. However, relapses were reported in 33% of patients . In a reWhen considering rituximab for IgG4-related RPF where its role has not been established, potential risks of the therapy need to be considered, including bone marrow toxicity leading to immunosuppression, hepatic toxicity, and neuropathy, and as a result, until further informative clinical studies become available, decision making should be carefully individualized. In conclusion, rituximab monotherapy can be used to induce and maintain remission in patients with IgG4-related RPF. In this case report, the use of rituximab provided a successful means of steroid-sparing with maintenance of remission and subsequent improvement in renal function. Whether this remission is long-lasting or temporary requires further follow up. Future studies should explore the degree of suppression of the fibrosis at a molecular level, establish appropriate dosing for the therapy, and examine the role of rituximab in other manifestations of IgG4-RD. The authors received no specific funding for this work. The authors declare no conflict of interest.


Acoustic emission (AE) is capable of monitoring the cracking activities inside materials. In this study, embedded sensors were employed to monitor the AE behavior of early age concrete. Type 1–3 cement-based piezoelectric composites, which had lower mechanical quality factor and acoustic impedance, were fabricated and used to make sensors. Sensors made of the composites illustrated broadband frequency response. In a laboratory, the cracking of early age concrete was monitored to recognize different hydration stages. The sensors were also embedded in a mass concrete foundation to localize the temperature gradient cracks. It is important to have reliable information about the early age properties of the materials. Classical techniques, such as thermal analysis, X-ray diffraction (XRD) and scanning electron microscopy, have been often used . These cydration ,3.in situ characterizing techniques for early age cement-based materials have been developed and some of them were based on ultrasonic wave measurement [et al. [In the past 20 years, surement ,6,7,8,9. [et al.  used acoet al. [in situ application.AE is defined as the class of phenomena whereby transient elastic waves are generated by the rapid release of energy from localized sources within a material or structure. It could provide an instantaneous response to crack growth inside a structure member and it is capable of determining the location of cracking . In civiet al.  had to uIf AE sensors are designed to be easily embedded into fresh concrete, it will be very convenient for early-age concrete. Recently, piezoelectric type sensors suitable to be embedded into concrete were designed and used for monitoring cement hydration, but not used as AE sensors for crack monitoring ,13. The In this study, Type 1–3 composites were fabricated and used for embedded sensors in concrete to detect cracking during hydration and hardening. The properties of the piezoelectric composites and the frequency response of the sensors were tested. In a laboratory, the hydration process was monitored by embedded and surface bonded AE sensors. Embedded AE sensors were also used in a mass concrete foundation of a high-rise building to detect the inside cracking. In mass concrete, a steep thermal gradient within the concrete could arise due to the hydration heat of cement and due to heat transfer, thus, causing the concrete structure to be exposed to the risk of thermal cracking when subjected to external or internal restraints . Therefow/c ratio = 0.45) was cast into the ceramic rods [m could be calculated as follows [sf and pf are series and parallel resonance frequency, which can be approximately replaced by frequency mf and nf, which are the frequency at minimum and maximum electric impedance, respectively. mf and nf were measured using Agilent 4294A Impedance Phase Analyzer . The impedance spectrum was shown in mf and nf. R is the minimum impedance value. The capacitance C was also measured by the Phase Analyzer at 1 kHz.The piezoelectric composites were fabricated by cutting and filling technique. First, a series of piezoelectric ceramic rods were cut along the polarization direction of the piezoelectric ceramic block. During cutting, a common base of the block was left to hold all the ceramic rods in it. According to previous research, the ceramic volume fraction was about 35%. Cement paste Qm=1Z is:v is wave velocity of the composites, which could be calculated as v = 2stf. t is the thickness of the composite plate. Qm and Z for the piezoelectric ceramic used in the test are 27.7 and 35.3 Mrayl. The Qm of the composites is only 7.7, showing a very obvious decrease. The acoustic impedance of the composites is 10.4 Mrayl, near the value of concrete.The acoustic impedance of the composites The coaxial wire was connected to the electrodes of the composite plate and then enclosed by mixture made of epoxy and cement powder. The mixture was tuned to have a similar acoustic impedance to the piezo-composites by adjusting the cement/epoxy ratio to 3.5 (in weight). The sensors made of the cement-based composites (Type 1–3 sensor) in this research were all 20 × 20 × 20 mm.To obtain the frequency response of the sensor, the sensor was bonded onto an aluminum plate (1000 × 1000 × 50 mm) using silica grease, 100 mm away from the AE source. In this characterization, a pencil with the Nielson Shoe was used to break 0.5 mm 2H lead to generate acoustic waves . The wavThe test instrument is shown in 3 respectively. Water reducer was also admixed and the dosage was 0.0215% of cement in weight. P O 32.5 Portland cement was used and the maximum size of the course aggregate was 20 mm. Because the concrete was designed for usage in mass concrete, fly ash was added to reduce the hydration heat. After mixing, the mixture was vibrated to eliminate air bubbles and then the concrete was poured into the plastic mold. During testing, the specimen molds were put into a big moisture plastic box acting as a curing room and the room temperature was about 20 °C.The mixture proportion of fresh concrete and raw materials were all provided by a commercial concrete mixing plant, which supplies concrete for the later mentioned project. The weight of cement, water, sand, aggregate and fly ash were 310, 202, 695, 1157 and 133 kg/mPrior to AE data acquisition, the two sensors were tested to obtain the noise level. During test the threshold was set 15 dB above the noise level. The AE monitoring was carried out continuously. In t represents the difference of arrival time between sensor 1 and 2; sx is the coordinate of the AE source to be determined; x1 and x2 are the coordinates of the sensors; υ is the wave velocity in steel and the value is 5200 m/s. Obtaining the difference of the arrival time, the AE source could be easily determined.The foundation is the base element of a 100-m high-rise building in Jinan city of Shandong province, China. The depth of the foundation is 2.5 m. According to the Chinese code, JGJ3-2010, the foundation belongs to a mass concrete specimen and the temperature of the foundation at early age should be monitored . The conT1 and T2 was very pronounced. The development of the temperature and gradient for temperature sensors 5–8 were very similar.The development of temperature is shown in 4 s (about 28 h), a large amount of cracking was recorded and, after that point, cracking continued, but with a lower amount per unit time. It could also be seen that most cracks occurred at or above the middle of the depth. This could be related to the temperature gradient development shown in 4 s. Thus, the cracks recorded in this case were mainly induced by the temperature gradient.Cracking would occur when the tensile stress caused by the accumulation of temperature gradient exceeds the tensile strength of concrete. The locations of cracking inside the mass of concrete were determined by PCI-2 system using the method illustrated in Equation (3). The coordinates of two AE sensors were 250 and 2250 mm. These AE signals, which were localized between the two sensors, were valid ones. The information of the AE event number and crack location is shown in in situ AE monitoring for concrete material and structures.In order to obtain the cracking information of concrete at early age, a broadband AE sensor was developed based on the Type 1–3 cement-based piezoelectric composites. The composites had a lower mechanical quality factor and acoustic impedance. The sensor made of Type 1–3 composites showed considerable broadband frequency response. In a laboratory, AE sensors were used to record cracking during the hydration process. Three stages could be divided according to the accumulated AE numbers. Most AE signals induced by the shrinkage of specimen were recorded during the intense hydration period. In the mass concrete foundation, the temperature gradient cracking was localized by the embedded AE sensors. The significance of the Type 1–3 sensor was shown in the


IgG4-related disease often forms a mass and the affected lesion is clinically removed because the mass cannot be differentiated from a neoplasm. Affected lesions commonly occur in the pancreas, hepatobiliary tract, kidney, and retroperitoneum. However, the lesion rarely occurs in the thymus. A histological worldwide consensus of IgG4-related disease proposed that pathological diagnosis of IgG4-related disease should meet more than two of three major features: 1) dense lymphoplasmacytic infiltration with greater than 40% IgG4+/IgG+ plasma cells, 2) storiform fibrosis; and 3) obliterative phlebitis. Currently, fibrosis of IgG4-related disease is thought to be induced by profibrotic cytokines such as transforming growth factor beta 1 (TGFB1), interleukin 1 beta (IL1B) and interferon gamma (IFNG), which are secreted by regulatory T cells (Tregs) and CD4-positive cytotoxic T cells. However, it is unclear whether profibrotic cytokines are associated with the fibrosis seen in IgG4-related thymitis. Here we examined whether cytokines in the mass were increased compared with those in the surrounding thymus, and whether Tregs were present in the mass, using reverse transcription absolute quantitative polymerase chain reaction (RT-ab-qPCR) and immunohistochemistry.A 70-year-old Japanese man contracted IgG4-letated thymitis. Histological and immunohistochemical analyses demonstrated his mass had massive fibrosis with a focally storiform pattern and lymphoplasmacytic infiltration with 40% IgG4+/IgG+ plasma cells, but not obliterative phlebitis. The mass was surrounded by atrophic thymus. We diagnosed the mass as IgG4-related thymitis. Immunohistochemically, Tregs were scattered throughout the mass. RT-ab-qPCR showed that messenger RNA expressions of TGFB1, IL1B and IFNG in the mass were 270-, 158- and 5.5- fold higher than in the surrounding thymus. His serum IgG4 level after surgery was within the normal range .Our results suggested the profibrotic cytokines TGFB1, IL1B and IFNG induce fibrosis and that Tregs might produce some of these cytokines in IgG4-related thymitis as well as in the other affected lesions of IgG4-related disease. Sclerosing mediastinitis is a rare disorder and results from many etiologies, e.g. infection, sarcoidosis, radiation and autoimmune conditions . A few cRecent studies have examined the mechanisms through which fibrosis occurs in IgG4-related diseases of the salivary gland, hepato-bilio-pancreatic systems and kidney –10. TheyWe experienced a case histopathologically consistent with IgG4-related thymitis, which occurred in a thymus of a 70-year-old Japanese man. We assumed that fibrosis of the IgG4-related lesion might be caused by the same mechanism involved in IgG4-related disease of previously examined organs. We evaluated whether Tregs were present and whether TGB1, IL1B and IFNG were increased in the mass of our case using immunohistochemical analysis of FoxP3, and reverse transcription- absolute quantitative polymerase chain reaction (RT- ab-qPCR) for TGB1, IL1B and IFNG messenger RNAs (mRNAs) from paraffin-embedded specimens of our case.A 70-year-old Japanese man was admitted to our hospital because of mediastinal mass found during a routine health examination. He had also suffered from diabetes mellitus, but the disease was fully controlled through medication. Computed tomography (CT) scanning revealed a solid mass measuring 3 cm in the maximum diameter, located adjacent to the left side of the left pulmonary artery. Small calcification was found in the mass Fig. . PreoperThe resected specimen was macroscopically a solid and fibrous mass surrounded by fatty tissue Fig. . SectionHematoxylin and eosin staining analyzed with a loupe showed a pinkish mass surrounded by mature fat tissue Fig. . CalcifiImmunohistochemical analyses were performed on paraffin-embedded materials using Bond-Max autostainer . The mass was negative for keratin, signal transducer and activator of transcription 6 (STAT6), desmin, smooth muscle actin and anaplastic lymphoma kinase (ALK), and there were no terminal deoxynucleotidyl transferase (TdT)-positive immature lymphocytes in the mass. We did not consider the mass as a thymic epithelial tumor such as a thymoma, a solitary fibrous tumor and an inflammatory myofibroblastic tumor accordingly. Almost two thirds of plasma cells were positive for IgG and the mean percentage of IgG-positive plasma cells counted in three × 400 high power fields containing the highest number of IgG4 plasma cells was 40% Fig.  and e. TWe used RT- ab-qPCR to compare the relative copy numbers of TGFB1, IL1B and IFNG mRNAs to TATA box-binding protein (TBP) mRNA of the mass with those of the surrounding thymus. We isolated total RNA from paraffin-embedded specimens of the mass and the surrounding thymus, using a miRNeasy FFPE Microkit . Then, the isolated RNAs were reverse-transcribed using PrimeScript II first strand cDNA Synthesis Kit . RT-ab-qPCR primer pair for TBP mRNA was the same as previously reported , and priTwo weeks after surgery, the serum IgG4 of the patient was 89.3 mg/dl and the IgG was 1490 mg/dl. Fractionation of IgG was within the normal range. He was doing well 2 months after surgery.We experienced a rare case of IgG4-related thymitis. In a broad sense, this case is included in sclerosing mediastinitis, because the thymus is located in the anterior mediastinum. However, it is uncertain whether, in the reported cases, the IgG4-related sclerosing mediastinitis occurred in the thymus or not. Our case is thought to be a first case in which IgG4-related lesions undoubtedly occurred in the thymus. Although our patient did not present high IgG4 in his serum, we diagnosed the mass as IgG4-related disease according to a worldwide histopathological consensus reported by Deshpande et al. . Accordiproteins extracted from fresh or snap-frozen tissues in the mass should be compared to those in the surrounding thymus. However, we did not take fresh material from the thymus although we saved snap-frozen material from the mass. Consequently, we measured the profibrotic cytokine mRNA expressions. Although epigenetic modification occurs post-transcriptionally [Currently, the profibrotic cytokines TGFB1, IL1B and IFNG secreted by Tregs and CD4-positive CTLs are thought to induce fibrosis in IgG4-related disease in hepato-bilio-pancreatic systems, salivary glands, retroperitoneum, kidney and lung , 12–15. tionally , our resWe did not assess CD4-positive CTLs in the mass. However, increased IL1B mRNA in the mass might indicate the presence of CD4-positive CTLs in the mass, because CD4-positive CTLs in previously examined lesions of IgG4-related disease were reported to secrete TGFB1, IL1B and IFNG that induced fibrosis –15.In conclusion, we showed that increased profibrotic cytokines, TGFB1, IL1B and IFNG might induce fibrosis in the affected lesion of IgG4-related thymitis. However, future studies of fibrosis of IgG4-related thymitis should measure cytokine proteins and ensure that Tregs as well as CD4-positive CTLs isolated from the affected lesion can produce the cytokines and induce fibrosis in vivo and/or in vitro.


Metastatic disease in a cancer patient still remains a therapeutic challenge. Metastatic process involves many steps, during which malignant cells succeed to activate cellular pathways promoting survival in hostile environment, engraftment and growth at the distant site from the primary tumor. Melanoma is known for its high propensity to produce metastases even at the early stages of the disease. Here we summarize the most important molecular mechanisms which were associated with the melanoma metastasis. Then, we specifically focus on the signaling pathway mediated by hepatocyte growth factor (HGF) and its receptor c-Met, which play an important role during physiological processes and were been associated with tumorigenesis. We also focus on the effect of the small molecule inhibitors of the tyrosine kinase domain of the c-Met receptor and its effects on properties of melanoma cell. We summarize recent studies, which involved inhibition of the HGF/c-Met signaling in order to decrease melanoma growth and metastatic capacity. Metastatic dissemination still represents a major problem in the treatment of cancer and it still remains the most common cause of death in cancer patients. Metastasis is the result of complex multilevel processes. Malignant abnormal cells grow beyond their usual borders, invade adjacent parts surrounding tumor and spread to other organs. Accumulation of alterations in tumor cells leads to appearance of metastatic cancer cells. They become invasive after detaching from the primary tumor, because they acquire ability to penetrate to the bloodstream or the lymphatic system, to grow and thrive in their new location, and to induce angiogenesis. There are some general principles in the metastatic cascade among the various tumor types, although this complex process still remains to be fully understood .Malignant melanoma arises from melanocytic cells and primarily involves a skin, less frequently an eye , meninges and mucosal surfaces . Even thMelanomas metastasize either by direct dissemination, by the lymphatic or the haematogenous route . In melaHigh risk of microscopic metastases in melanoma patients is handled by using adjuvant therapies in order to delay the recurrence of disease. However, a number of controlled trials with adjuvant chemotherapy in stage II and III patients did not demonstrate any therapeutic advantage. Adjuvant immunotherapy by Interferon (IFN)-α is the treatment of melanoma which has shown a significant improvement of disease-free survival, and also impact on overall survival, albeit with significant toxicity. Ongoing clinical trials test the efficiency of immune system-activating monoclonal antibodies Ipilimumab and Pembrolizumab in melanoma treatment , 8, 9. FIn general, there is still limited number of options for systemic therapy in patients with inoperable regional and distant metastases. Most significant effect on tumor responses was achieved by immunotherapy or targeted therapy using small-molecule drugs and monoclonal antibodies so far. Better understanding of the complex metastatic cascade may unravel novel targets to limit the spread of malignant cells. One of the important signaling pathways which was implicated to play role in many cancers including metastatic spread is signaling by hepatocyte growth factor (HGF) via its cognate receptor c-Met with tyrosine kinase activity. Here we focus on the small-molecule inhibitors of this signaling pathway and its effect on metastatic melanoma.Metastatic spread is a multi-step process releasing tumor cells from a primary lesion to a disparate organ or organs within the body. Tumor cells change their characteristics throughout the process, which enable them to proliferate and migrate, invade surrounding tissue, intravasate through the basement membrane into blood or lymphatic vessels, survive during circulation through the blood or lymphatic system, stay at distant sites, extravasate into a new environment tissue, and proliferate by inducing angiogenesis –12.The classical metastatic cascade starts from a primary, epithelial, neoplastic lesion and includes: (1) epithelial – mesenchymal transition (EMT) with a breach of the basement membrane barrier; (2) dissociation of tumor cells from the primary tumor mass, (3) invasion of the neighboring tissue, (4) intravasation into pre-existing and newly formed blood and lymph vessels, (5) transport through the vessels, (6) extravasation from the vessels, (7) establishment of circulating tumor cells (CTCs) and the disseminated cells at a secondary anatomical site, where they can stay dormant for a prolonged period of time, (8) metastatic outgrowth of micrometastases and macrometastases creating clinically detectable secondary tumors and neoplastic process . Each ofTumor cells employ different strategies of migration to invade into the stroma and proceed toward the blood or lymph stream: single-cell migration and collective migration , 15. TheThe microenvironment at the invasive edge of tumors is quite different than that of the tumor core. Tumor microenvironment has diverse capacities to induce both beneficial and adverse consequences for tumorigenesis and the microenvironment supports metastatic dissemination and colonization at secondary sites. Macrophages, platelets, and mesenchymal stem cells contribute to the EMT at primary sites, allowing for tumor cells to separate from neighboring epithelial cell-to-cell contacts and acquire an invasive phenotype. One major mediator of this event is transforming growth factor-beta (TGF-β), which is secreted by the tumor stroma and participates in a paracrine signaling loop with tumor cells . A largeTumor-associated macrophages (TAMs), cancer-associated fibroblasts (CAFs) and myeloid progenitor cells also tend to cluster at the invasive edge of the primary tumor, where they play an immunosuppressive role by interfering with dendritic cell differentiation. During intravasation of tumor cells into circulation, intravital imaging studies have shown that macrophages are localized to perivascular areas within tumors, where they help tumor cells to traverse vessel barriers . In the Factors contained in exosomes have the capacity to direct organ tropism, modulate immune evasion, support mesenchymal-to-epithelial transition (MET), and they are predictive of metastasis and patient outcome. Tumor exosomes could also facilitate organ-specific metastatic behavior by preparing pre-metastatic niches , 21.Stephen Paget more than 120 years ago (in 1889), proposed a hypothesis of “seed and soil” to describe metastatic outgrowth. Paget observed that primary tumor cells of a given type of cancer preferentially metastasized to one or more particular distant organ sites, detectable metastases only developed at those sites (“soils”) where the tumor cells (“seeds”) were adapted for survival and proliferation . HoweverMACC1) gene in colorectal carcinoma. The expression of these metastasis initiation genes and their targets predicts a poor prognosis in particular types of cancer [The genes, which allow transformed cells to invade the surrounding tissue and attract a supportive stroma, can be defined as metastasis initiation genes and could promote cell motility, EMT, extracellular matrix degradation, bone marrow progenitor mobilization, angiogenesis or evasion of the immune system. Other determinants of invasion are components and modulators of the HGF/c-Met pathway, such as metadherin in breast cancer and the metastasis-associated in colon cancer 1 , TGF-β superfamily, WNT, NOTCH, hedgehog pathway and NF-κB . The PI3Signaling events leading to EMT activate mesenchymal state in the cancer cells, which was associated with increased frequency of cancer stem cells (CSCs) sometimes also termed tumor-initiating cells . These tMelanoma stem cells have been identified in both primary tumors and cell lines. There are several key stem cells markers specified for malignant melanoma: CD20, CD133, ABCB5, CD271 and ALDH1A . RecentlTransmembrane RTKs are composed of a single transmembrane domain that separates the intracellular tyrosine kinase region from the extracellular part. RTKs contain tyrosine kinase and they have a high affinity for polypeptides, such as growth factors, cytokines and some hormones . RTKs play an important role in the physiological developmental processes and the development of many cancers . Human RIn various solid tumors, including gastric, breast, thyroid and hepatocellular carcinomas, the HGF/c-Met pathway was detected as a critical in cancer development . In a reThe c-Met receptor and the HGF are found in a many tissues and organs, but their expression is usually restricted to the cells of epithelial and mesenchymal origins. Mouse genetic studies discovered that both c-Met and HGF are important in embryonic development, organ morphogenesis and cell motility. In adults, their activities are more restricted, predominantly in tissue regeneration and damage repair –44.HGF acts on a wide variety of epithelial cells as a mitogen (stimulation of cell growth), a motogen (stimulation of cell motility), and a morphogen (induction of multicellular tissue-like structure). Due to these functions HGF is considered a key molecule for the construction of normal tissue structure during embryogenesis, organogenesis, and organ regeneration . HGF  and consists of 21 exons separated by 20 introns [Its cognate receptor c-Met is a protein that is encoded in humans by the  introns . The ext introns . The int introns . The hum introns . The c-M introns .HGF is a growth factor for various types of cells: acts as a mitogen for renal epithelial cells, epidermal keratinocytes and melanocytes and others; promote the growth of hepatocytes and hematopoietic cells in culture. The c-Met is present in hematopoietic progenitor cells from human bone marrow and peripheral blood and in the presence of erythropoietin, HGF induces proliferation and differentiation of erythroid progenitors .During embryogenesis HGF and c-Met is crucial, as it was shown that c-Met and HGF play an important role in control of growth, survival and migration of distinct embryonal cells . The c-MHGF/c-Met signaling, which is mainly mediated by the RAS–MAPK and PI3K–AKT pathways, affects gene expression and cell cycle progression through the binding of transcription factors, such as the ETS family. Cytoplasmic signaling cascades mediated by PI3K–AKT and the GTPases RAC1 or cell division control protein 42 (CDC42) modulate cell survival and elicit cytoskeletal changes. Signals to the plasma membrane control cell migration and cell adhesion mainly through the RAP1 and RAC1–CDC42 pathways, which affect integrins and cadherins .HGF acts as a pleiotropic factor and cytokine, promoting cell proliferation, survival, motility, scattering, differentiation and morphogenesis. Physiologically, c-Met is responsible for the cell-scattering phenotype, as first demonstrated with MDCK cells treated with HGF. This process involves the disruption of cadherin-based cell-cell contacts and subsequent cell motility , 53. PI3MET and HGF genes were reported to be up-regulated after injury in different epithelial tissues, such as kidney, lung, skeletal muscle, heart, skin, and liver. In the skin, MET was shown to be essential for wound repair [MET ablation resulted in impaired proliferation and incomplete liver [After liver injury, the HGF mRNA is rapidly induced in the lung, spleen and kidney. Therefore, HGF from neighboring cells in the liver and from extrahepatic organs may function as a trigger for liver regeneration by booth paracrine and endocrine mechanisms . MET andd repair . In the te liver .In the skin, stem cell populations generate different epidermal cell types during normal turnover and wound repair . The resLigand-induced c-Met dimerization activates the tyrosine kinase by phosphorylation of tyrosine residues  in the kinase domain. This initial phosphorylation cascade leads to autophosphorylation of the carboxy-terminal bidentate substrate-binding site (Tyr1349 and Tyr1356) of the c-Met and these residues have been shown as docking sites for downstream signaling molecules . HGF indMET gene often overexpressed or mutated. Thus, the c-Met has become a major target for cancer therapy and its inhibition is currently being tested in the clinical trials [The c-Met, and its ligand HGF have been associated with tumor formation and progression to metastasis, with l trials . HGF/c-Ml trials .Dysregulation of c-Met correlated with a poor prognosis. Interestingly, abnormal activation of c-Met signaling is implicated in the acquisition of tumorigenic and metastatic phenotypes in tumors. Examinations indicated that c-Met was expressed and activated in melanoma tissues and cell lines. It was shown, that overexpression of c-Met was associated with melanoma growth and metastasis . High exMET gene is activated by point mutations in small-cell lung cancer (SCLC) and renal papillary carcinomas. c-Met protein is overexpressed in melanoma and musculoskeletal tumors [Hypoxia is a key regulator of c-Met, as it induces the expression of the transcription factor hypoxia inducible factor 1 alfa (HIF-1α). The existence of this correlation is supported by preclinical studies in mouse xenograft models, which showed that the therapeutic inhibition of angiogenesis reduces tumor vascularization and causes hypoxia, and therefore may promote c-Met-mediated invasion of malignant cells . The METl tumors . Cross-tl tumors .MET gene [MET was found and correlated with poor prognosis in patients. Contrastingly, point mutations in the MET gene and correlation with prognosis was not found in the malignant melanoma [It has been shown, that many types of tumors express both the ligand - HGF and receptor - c-Met. Not all of them are associated with a poor prognosis or with mutations in the MET gene . In melaMET gene . Activatmelanoma .The c-Met is not only a marker linked to metastatic properties but is also a suitable drug target and the molecules which inhibit HGF/c-Met signaling are expected to serve for therapeutic intervention . Several50 of SU11274 was between 1 and 2.5 μmol/L and apoptosis was observed in five melanoma cell lines that expressed c-Met [50 of 3.4 μM and 6.5 μM, respectively. SU11274 induces G1 cell cycle arrest with cells in G1 phase increased from 42.4% to 70.6% at 5 μM, and induces caspase-dependent apoptosis by 24% at 1 μM. SU11274 inhibits cell viability in c-Met-expressing non-small cell lung cancer (NSCLC) cells with IC50 values of 0.8–4.4 μM, and abrogates HGF-induced phosphorylation of c-Met and its downstream signaling [SU11274 was identified as a prototype ATP-competitive small molecule inhibitor of the catalytic activity of c-Met . The exped c-Met . It was ed c-Met . SU11274ignaling , 67.The inhibitor SU11274 specifically decreased the phosphotyrosine signal at the focal adhesion sites in multiple myeloma cells, which was accompanied by a decrease in cell proliferation as well as an increase in number of apoptotic cells. Moreover, the SU11274 significantly reduced the in vitro migratory capacity of myeloma cells and treatment by the SU11274 decreased primary tumor growth and the capacity for liver colony formation in SCID mice .50 for c-Met inhibitor SU11274 was 4–5 μM [High level of the c-Met receptor was confirmed in human melanoma cells M14, M4Beu, A375 and Rel3, and the ICs 4–5 μM .Contrarys 4–5 μM . In the s 4–5 μM .50 of 11 nM and 6.1 nM, respectively. It was identified as a potent, orally bioavailable, ATP-competitive small-molecule inhibitor of the catalytic activity of c-Met kinase. Crizotonib was selective for the c-Met (and anaplastic lymphoma kinase) compared with a panel of > 120 diverse tyrosine and serine-threonine kinases. It potently inhibited c-Met phosphorylation and c-Met-dependent proliferation, migration, or invasion of human tumor cells in vitro . In addition, crizotinib potently inhibited HGF-stimulated endothelial cell survival or invasion and serum-stimulated tubulogenesis in vitro, suggesting that this agent also exhibits antiangiogenic properties [50 for crizotinib ranged 1.25–3 μM in standard adherent cultures of melanoma cell lines M14, M4Beu, A375 and Rel3 [https://clinicaltrials.gov/ct2/show/NCT02223819).Crizotinib (PF-02341066) as a potent inhibitor of c-Met and ALK received approval for the treatment of patients with locally advanced or metastatic NSCLC that is ALK-positive in 2013 . Crizotioperties , 72. We and Rel3 . InhibitThere are several other inhibitors available for the inhibition of the c-Met signaling Fig. . AMG-337AMG-458 is a potent inhibitor of the c-Met, VEGFR-2, and IGFR1 receptor signaling pathways, inhibitor with radiosensitizing effects. AMG-458 significantly inhibited tumor growth of the U-87 MG human glioblastoma xenografts and of NIH-3 T3/TPR-MET tumors with constitutive activation of c-Met . The com, and this data demonstrated for the first time that this inhibitor suppresses angiogenesis and lymphangiogenesis by blocking VEGFR-2/3 and TIE-2 signaling [50 of 21 nmol/L). Single-dose oral administration of EXEL-2880 resulted in prolonged inhibition of phosphorylation of constitutively phosphorylated Met in B16F10 solid tumors as well as ligand HGF-stimulated phosphorylation of Met in whole liver [Foretinib (EXEL-2880) is an ATP-competitive multi-kinase inhibitor inhibitor of the c-Met receptor and VEGFR . Orthotoignaling . Foretinle liver .Cabozantinib  is an inhibitor of tyrosine kinases including VEGF receptors, c-Met and AXL. Cabozantinib has clinical activity in patients with metastatic melanoma, including uveal melanoma. The data from the clinical trial point out benefits of cabozantinib on both soft tissue and bone lesions in patients with metastatic melanoma. The data indicate that targeting of the VEGFR, c-Met and AXL pathways with cabozantinib tends to improve outcomes in patients with metastatic melanoma. Treatment with cabozantinib was associated with encouraging progression-free survival, overall survival and reduction in the size of measurable target lesions was observed in the majority of patients with uveal, cutaneous, and mucosal melanoma . XL184 iPHA-665752 is a potent, selective and ATP-competitive c-Met inhibitor, PHA-665752 inhibited c-Met tyrosine phosphorylation at the activation loop (pY1230/34/35), multifunctional docking site (pY1349), and the juxtamembrane domain (pY1003) at 0.1 μM . NRAS-muMK-2461 is another potent, ATP-competitive multi-targeted inhibitor of the c-Met (WT/mutants): c-Met (Y1235D), c-Met (Y1230C) and c-Met (N1100). In tumor cells, MK-2461 effectively restrained constitutive or ligand-induced phosphorylation of the juxtamembrane domain and COOH-terminal docking site of c-Met. In the cell culture, MK-2461 inhibited HGF/c-Met–dependent mitogenesis, migration, cell scatter, and tubulogenesis .50 of 4.8 nM in A549 cell line, sensitive to oncogenic mutations (no activity to Y1230C mutant). PF-04217903 in association with sunitinib radically inhibits endothelial cells, but not the tumor cells B16F1, Tib6, EL4, and LLC. It heftily inhibits c-Met-driven processes like cell growth, motility, invasion, and morphology of a diversity of tumor cells [PF-04217903 is a selective ATP-competitive c-Met inhibitor with ICor cells .Tivantinib (ARQ 197) is a staurosporine derivative and it represents the first non-ATP-competitive c-Met inhibitor that binds to the dephosphorylated c-Met kinase in vitro. It is clinically tested as a highly selective c-Met inhibitor. In all cell models analyzed, tivantinib did not inhibit HGF-dependent or HGF-independent c-Met tyrosine autophosphorylation .EGFR-mutated human NSCLC cell lines both in vitro and in xenograft mouse models [There are several antibodies targeting HGF/c-Met signaling. Rilotumumab (AMG-102) binds the HGF light chain, locking out the HGF/c-Met binding. Ficlatuzumab (AV-299) is a humanized monoclonal anti-HGF IgG1 antibody that enchains to HGF, thereby inhibiting the HGF/c-Met interplay. TAK-701 is a humanized anti-HGF monoclonal antibody that was found to outperform gefitinib defiance in e models .In summary, current strategies in oncology shift towards the targeted treatment exploiting identification of crucial signaling pathways for metastatic spread of tumor cells. The involvement of the HGF/c-Met signaling in solid tumors, including melanoma, prompted development of new drugs, which have already brought benefit in clinical setting. In the treatment of NSLSC, the crizotinib doubled the survival of patients . We suppTaken together, many results of the in vitro experiments and clinical studies show that the most appropriate treatment is a combination of different inhibitors chosen based on the molecular properties of tumor cells. Targeted therapies have a potential of achieving control over the metastatic disease with limited toxicity and controlling the disease with long-term survival of patients.In this review we describe the major factors affecting metastatic process with a focus on the malignant melanoma. As there are many attempts to find targeted therapy to control the metastatic dissemination, we focus here the signaling pathway c-Met/HGF, which is involved in many aspects of tumorigenesis including cell dissemination and metastasis. We also summarized the outcomes of the recent studies using specific inhibitors of this pathway in an attempt to limit metastatic spread, tumor growth with a focus on limiting melanoma proliferation and tumorigenicity. Detailed understanding of the inhibitory action exerted by targeted RTK inhibitors including those affecting the HGF/c-Met signaling is critical for the durable antitumor responses.


Background. The LRRK2 G2019S mutation is the most common genetic determinant of Parkinson's disease (PD) identified to date. This mutation, reported in both familial and sporadic PD, occurs at elevated frequencies in Maghreb population. In the present study, we examined the prevalence of the G2019S mutation in the Moroccan population and we compared the motor and nonmotor phenotype of G2019S carriers to patients with idiopathic Parkinson's disease. Methods. 100 PD patients were assessed for motor and nonmotor symptoms, current medication, and motor complication including motor fluctuations and dyskinesia. The LRRK2 G2019S mutation was investigated by direct sequencing in patients and ethnically matched controls, all of Moroccan origin. Results. Among the 100 PD Moroccan patients, 41 (41%) were carriers of the G2019S mutation. The mutation frequency was higher among probands with autosomal dominant inheritance (76%) than among sporadic ones (28%). Interestingly, G2019S mutation was also found in 5% of control individuals. Clinically, patients carrying the G2019S mutation have more dystonia  and more sleep disorders  than noncarriers. Conclusions. The LRRK2 G2019S prevalence in Morocco is the highest in the world reported to date. Some clinical features in G2019S carriers such as dystonia and sleep disturbances are worth noting. SNCA (PARK1/4), LRRK2 (PARK8), and VPS35 (PARK17).Parkinson's disease (PD) is the second most common neurodegenerative disorder after Alzheimer's disease affecting approximately 1-2% of the population over 60 years and 4% above 85 years . Patholo LRRK2 gene are the most frequently reported monogenic cause of PD and are common in both early and late-onset PD, occurring in both familial and sporadic PD patients with a wide variety of clinical and pathological features and a variable frequency depending on ethnic origin [ LRRK2 consortium to represent 1% of sporadic and 4% of familial PD patients worldwide [Mutations inc origin . Among torldwide . Intriguorldwide , 11. In orldwide –13 suggeorldwide , 11, 14.orldwide , 16. Amoorldwide . This frorldwide  and is rorldwide . Occurreorldwide , 19–22. Clinically, G2019S mutation carriers develop a very similar PD disease to noncarriers, including the development of motor symptoms and cognitive impairment , but som LRRK2 gene in the Moroccan population and to assess the motor and nonmotor phenotype of G2019S mutation carriers and noncarriers.The present study aims to estimate the prevalence of the G2019S mutation of theA total of 100 unrelated PD patients were recruited consecutively from the outpatient clinic of the Neurological Department at University Hospital Ibn Sina of Rabat from October 2013 to June 2015. One hundred healthy individuals were recruited from the National Blood Transfusion Institute of Rabat and were used as controls. Their mean age was 58.59 (±8.65) and 51 of them were males. Patients and controls provided a written informed consent and the study was approved by the ethic committee of the Medical School of Rabat (CERB).Diagnosis of PD was made by the same neurologist using the United Kingdom Parkinson's Disease Society Brain Bank criteria . PatientOtherwise, all control individuals have no family history of neurological disease but have not been clinically assessed for the presence of PD.A pedigree was established for all patients and the mode of inheritance was classified as “familial” if at least one relative was reported with a diagnosis of PD (FPD) and as autosomal recessive or dominant based on the presence or absence of consanguinity. The remaining patients were classified as “sporadic” (SPD). LRRK2 gene was performed by direct sequencing. Briefly, a 378 bp LRRK2 exon 41 fragment was PCR amplified as described previously [Genomic DNA was extracted from peripheral blood leukocytes using Isolate II Genomic DNA kit from Bioline. The G2019S mutation of theeviously . The PCRt-test or Mann–Whitney test. Categorical variables were expressed as numbers and percentages and were compared using Chi-square test. The relationship between G2109S mutation and the clinical symptoms was analyzed by means of logistic regression adjusting for age, sex, and disease duration on univariate and multivariate analysis. In the multivariate analysis model, we introduced variables that had a p ≤ 0.3 and we forced this analysis for Hoehn and Yahr score for its importance in the disease evolution. p value < 0.05 was considered as statistically significant. For multiple testing, we corrected the p value by the Bonferroni method.Demographic and clinical variables between G2019S-carriers and noncarriers were compared using parametric and nonparametric tests as appropriate using SPSS 13.0 software. Quantitative data were expressed in mean ± standard deviation (SD) or median and interquartile range and were compared using p = 0.03).We examined 100 patients with PD, 56 of whom were males and 44 were females . The mea LRRK2 G2019S substitution was found in 41 of 100  PD patients, 37 of whom were heterozygous and 4 were homozygous. The G2019S prevalence increased to 67% (95% CI 48.11–81.45) for FPD patients, with 76% of patients (95% CI 56.07–88.98) having a DFPD, 0% of patients (0 of 2) having RFPD, and 0% of patients (0 of 2) having an unspecified mode of inheritance. The prevalence reaches only 28% (95% CI 18.35–40.88) for SPD patients with 32% of patients (95% CI 20.27–45.38) without consanguinity and 10% of patients (95% CI 1.79–40.41) with consanguinity (Sixty-seven out of 100 patients were sporadic cases (67%) and 33 had a positive family history of PD (33%). Ten of the 67 SPD patients were from consanguineous marriages . Among tnguinity . Interesp = 0.019 and p = 0.012, resp.). LRRK2 G2019S carriers have less tremor than noncarriers do as first symptom  and exhibited less of the tremor-dominant phenotype than noncarriers (corrected-p = 0.009). They also have more dystonia (p = 0.011) and more dyskinesia (p = 0.002) and take a higher dose (up to 200 mg) of dopaminergic drugs (p = 0.002). Gait disturbances, postural instability, motor fluctuations, UPDRS III score, and H&Y scale during ON state are similar between both groups. Concerning nonmotor symptoms, LRRK2 G2019S carriers have more sleep complaints than noncarriers do (p = 0.046), while they show the same rates of psychiatric symptoms, constipation, urinary urgency, and orthostatic vertigo. There is a trend to more cognitive impairment in LRRK2 G2019S carriers than noncarriers but the difference is not significant (p = 0.059).Motor and nonmotor symptoms of all patients are given in  LRRK2 G2019S mutation is associated with more dystonia  and sleep complaints  but less tremor . Nonetheless, while LRRK2 G2019S carriers have more levodopa-induced dyskinesia (p = 0.002), the statistical significance lacked on multivariate analysis .The relationship between G2109S mutation and the clinical symptoms using logistic regression by adjusting for age, sex, and disease duration is shown in  LRRK2 consortium reported a worldwide frequency of LRRK2 G2019S mutation of 1% in sporadic PD and 4% in familial PD [The internationalilial PD , 11, repAmong the 41 patients with G2019S mutation, four were homozygous carriers with different mode of inheritance, including two with autosomal dominant inheritance and two sporadic cases of which one comes from a consanguineous marriage. This could be due to the high prevalence of the mutation in the general population. Indeed, there were five heterozygous G2019S carriers among the 100 control individuals (5%). LRRK2 G2019S carriers and noncarriers [ LRRK2 subjects have less tremor. This finding is in line with some series [ LRRK2 carriers [ LRRK2 G2019S carriers had a similar UPDRS III and H&Y scores for longest disease duration  compared to noncarriers. It can reflect in some extent a slower disease evolution in LRRK2 G2019S patients. In the same line, Healy et al. [Clinically, most authors reported a similar phenotype betweencarriers , 35. In e series  whereas carriers , 36, 37.carriers , which c LRRK2 G2019S carriers in our series with an ODDS ratio of 4.65. Dystonia is a well-defined symptom in early onset Parkinson's disease (EOPD) as reported by Kilarski et al. [ LRRK2 G2019S carriers and noncarriers (p = 0.207).Furthermore, dystonia is the most specific feature that characterizesi et al.  in theiri et al. , but the LRRK2 G2019S patients exhibited more sleep complaints in our series. This feature is in line with other series that reported more sleep onset insomnia, few or no rapid eye behavior disorders, and less nocturia in LRRK2 G2019S carriers [Otherwise,carriers –43. LRRK2 carriers such as a lower limb onset [ LRRK2 G2019S mutation; the presence of Lewy bodies is strongly correlated to some nonmotor symptoms especially cognitive impairment and dementia. In our study, LRRK2 carriers have a trend to more cognitive impairment that was not confirmed in the logistic regression model. Future studies with wider cohorts are required to determine the cognitive profile of LRRK2 carriers.Other reports described more specific clinical features inmb onset , more hamb onset , 38, 44 mb onset , 36, 45.mb onset  explaineOur study showed that Morocco has the highest reported prevalence of the G2019S mutation in the world, with a mutation frequency of 41% overall and 76% for patients with autosomal dominant mode of inheritance. Furthermore, G2019S carrier patients exhibit clinical features quite similar to noncarriers with some mild differences in particular more dystonia and more sleep complaints.


A complete set of elementary logic gates and two multilevel circuits have been constructed on a label-free and enzyme-free biocomputing platform using caged G-quadruplex as the signal transducer. 2+-dependent DNAzyme which can be made to function via the input-guided cooperative conjunction of the DNAzyme subunits. In the presence of hemin, the unblocked G-quadruplex DNAzyme catalyzes the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) by H2O2 to generate a colored readout signal which can be readily distinguished by the naked eye. This strategy is quite versatile and straightforward for logic operations. Two combinatorial gates (XOR + AND and XOR + NOR) are also successfully fabricated to demonstrate the modularity and scalability of the computing elements. The distinctive advantage of this logic system is that molecular events in aqueous solution could be translated into a color change which can be directly observed by the naked eye without resorting to any analytical instrumentation. Moreover, this work reveals a new route for the design of molecular logic gates that can be executed without any labeling and immobilization procedure or separation and washing step, which holds great promise for intelligent point-of-care diagnostics and in-field applications.A complete set of binary basic logic gates  is realized on a label-free and enzyme-free sensing platform using caged G-quadruplex as the signal transducer. In the presence of an appropriate input, the temporarily blocked G-rich sequence in the hairpin DNA is released through cleavage by the synergetically-stabilized Mg Molecular logic gates that are capable of performing computational operations could be of use in chemical/biological sensing, disease diagnostics and intelligent imaging, and thus have attracted significant research interest.2O2 and to induce the change in color from colorless to blue.Peroxidase-mimicking G-quadruplex DNAzyme is an ideal signal transducer for the construction of DNA logic gates and functional DNA devices.2+-dependent DNAzymevia input-guided cooperative hybridization processes. The resulting DNAzyme cleaves a ribonucleobase (rA)-containing DNA substrate in the presence of Mg2+ to generate output signals. 2O2 to generate a colored readout signal which can be readily distinguished by the naked eye. In the presence of both inputs , a duplex between inptut1 and input2 will be formed since they are complementary to each other. In this case, the formation of a catalytically active DNAzyme is prohibited. λ = 650 nm. An absorption intensity of 0.1 is defined as the threshold value for all the logic gates to judge the positive and negative output signals. When the absorption value at λ = 650 nm is lower than the threshold value of 0.1, the output of the computing system is considered to be “0” . If the absorption intensity is higher than 0.1, the output reads “1” .MgThe solution of the system turned blue (output = 1) only upon activation by input1 or input2 alone, which characterizes an XOR gate. The truth table and circuitry are given in λ = 650 nm. The OR logic gate is represented by the situation where the output is 1 if either or both inputs are 1. The truth table and circuitry are given in An OR logic gate is fabricated by using four DNAzyme subunits (DNA1–DNA4) and the hairpin substrate (DNA5) as the computing elements. As depicted in λ = 650 nm. The AND logic gate is represented by the situation where the output is 1 only if both inputs are 1. The truth table and circuitry are given in λ = 650 nm. The combinatorial circuit has an output of 1 if either or both input signals are 1 and so it behaves like the OR gate in operation. The truth table and circuitry are given in The basic logic gates could actually be correlated for demonstrating higher-order functions like combinatorial circuits  and also yields a true output. In the presence of both inputs, the six DNAzyme subunits (DNA2–DNA7) cannot assemble into any active DNAzyme structure due to the preferred inter-input hybridization and the displacement reactions between the template and the inputs. This results in an output of 0. Thus, parallel activation of a combinatorial circuit established from the XOR and NOR gates could be realized in a single system using the same set of inputs. λ = 650 nm. The combinatorial circuit has an output of 0 only if both input signals are 1 and so it behaves like the NAND gate in operation. The truth table and circuitry are given in Another integrated circuit composed of parallel XOR and NOR logic gates to enforce an overall NAND logic behavior is also designed. The principle is illustrated in To demonstrate the feasibility of the logic system for practical applications, the XOR, OR, AND and XOR + AND logic gates were validated in human serum samples. Input1 and input2 in 10% serum were used as the inputs. The results show that the logic system can also execute those computation functions effectively in human serum  has been successfully constructed on a label-free and enzyme-free biocomputing platform. Using caged G-quadruplex as the signal transducer, the output of the computation can be unambiguously read out by the naked eye. The uniqueness of the logic system lies in the input-guided assembly and disassembly of the computing circuits, the modularity of the gate design and the visible output which can be obtained by observing the color change of the solution. The study has demonstrated the versatility and scalability of the computing elements by constructing two combinatorial gates. We connect the XOR and AND gates into a multilevel circuit (XOR + AND) that enforces an overall OR gate behavior. Another integrated circuit composed of parallel XOR and NOR logic gates (XOR + NOR) to perform a final net NAND analysis is also designed. The construction strategy described herein is simple in design and economic in operation without any labeling and immobilization procedure or separation and washing step, requiring only the mixing of several solutions at room temperature to obtain the intuitive and visible outputs and so it holds great promise for intelligent POC diagnostics and on-site monitoring.All the experiments involving human serum were approved by the Ethics Committee of Guangdong Institute of Eco-environmental Science & Technology and performed in compliance with the relevant laws and institutional guidelines. We obtained informed consent for any experimentation with human subjects.The authors declare no competing financial interest.Supplementary informationClick here for additional data file.