Alanine scan of insulin receptor (IR)-B exon 11 and site-directed mutagenesis of amino acid 718 in human IR-A and IR-B were performed. Ligand affinities to wild type and mutated receptors were studied by displacement of radioactive insulin in binding assay on secreted soluble midi receptors or solubilized semi-purified full length receptors stably expressed in Baby Hamster Kidney cells. Phosphorylation of IR in response to insulin, IGF1 and IGF2 was measured using ELISA.Insulin, insulin detemir and insulin glargine maximally showed two fold differences in affinity for human IR-A and IR-B, but IGF1 and IGF2 had up to 10 fold preference for IR-A. Alanine scan of exon 11 revealed that position 718 is important for low IGF1 affinity to IR-B. Mutational analysis of amino acid residue 718 in IR-A and IR-B demonstrated that charge is important for IGF1 and IGF2 affinity but not important for insulin affinity. The affinity of IGF1 and IGF2 for the mutant IR-A P718K was comparable to the wild type IR-B whereas the affinity of IGF1 and IGF2 for the mutant IR-B K718P was comparable to the wild type IR-A. Changes in affinity were also reflected in the IR activation pattern.Mutating position 718 in human IR-B to the proline found at position 718 in human IR-A increased IGF1 and IGF2 affinity to a level comparable to IR-A and mutating position 718 in IR-A to the lysine found at position 718 in IR-B decreased IGF1 and IGF2 affinity to a level comparable to IR-B, whereas a negatively charged glutamate did not. These changes in the affinities were also reflected in the IR phosphorylation pattern, meaning that position 718 is important for both affinity and activation of the receptor. It should be emphasized that none of the mutations affected insulin affinity, indicating that the mutations did not alter the overall receptor structure and that the effect is ligand specific. The human insulin receptor (IR) is a ligand activated transmembrane receptor tyrosine kinase that exists as two isoforms  2]. It . It 2]. The difference in insulin affinity for IR-A and IR-B is at most two fold , but theInsulin belongs to the superfamily of insulin related proteins that include IGF1 and IGF2  19] [20 [2019] . This stMutational analysis of IGF2 suggests that IGF2 and insulin have similar binding surfaces but subtle differences could contribute to IR and IGF1R specificities; however, these binding surfaces do not contribute to the selectivity between IR-A and IR-B . The C-dCrystal structure analysis of truncated IR/IGF1R hybrids in complex with IGF1  suggest The functional binding epitopes of IR-A and IR-B have been studied by mutational analysis such as alanine scanning  but untiIt has been reported that IGF2 binds to IR-A with high affinity and that IGF2 stimulation of IR-A leads to more mitogenic signaling than insulin stimulation  28], ho, ho28], IR activation can be measured as total tyrosine phosphorylation or by site-specific phosphorylations in three different parts of IR β-subunit; juxtamembrane domain, kinase domain and carboxy-terminus. The EC50 value, the maximal response and the kinetics of IR activation are reported to be ligand dependent . Other s6 cells were transfected with 10 μg plasmid by liposome transfer using Lipofectamine  and allowed to recover three days before applying selection pressure in the form of 1 μM methotrexate. After approximately three weeks single cell clones from each transfection appeared and 12 clones from each transfection were picked and subjected to IR expression analysis by Western Blotting. Based on the highest expression level one clone for each transfection was selected and used in the subsequent experiments. Cells were cultured in Dulbecco’s modified Eagle medium containing high glucose, 10% fetal calf serum and 2 mM L-glutamine, 10 mg/ml streptomycin, and 100 units/ml penicillin  at 37°C in a 5% CO2 enriched, humidified atmosphere.Wild type (WT) human IR-A, IR-B and mutated IRs were inserted in pZem vector and stably over-expressed in Baby Hamster Kidney (BHK) cells to achieve the desired receptor expressions. In brief, 1 x 10AvrII and BstBI) were made to frame exon 11 without changing the amino acid sequence. Oligos with the desired mutations were designed to include the AvrII site (upstream of exon 11). An oligo downstream of exon 11 and the BstB1 was used to obtain a PCR product which was subcloned, hereby replacing the AvrII/BstBI fragment in human IR-A or human IR-B. The point mutation at position 718 in full-length receptors included IR-A P718A, P718E and P718K and IR-B K718A, K718E and K718P.Midi receptor mutants of human IR were made in the soluble high affinity midi receptor construct , but wit125I-(Tyr31)-Insulin (125I-Insulin), human insulin, insulin determir, insulin glargine and human IGF1 were from Novo Nordisk A/S . Human IGF2 was from GroPep, Adelaide, Australia. The IR specific antibody 83–7 [ody 83–7  was liceBHK cells over-expressing either WT or mutated human IR were lysed in 50 mM Hepes pH 8.0, 150 mM NaCl, 1% Triton X-100, 2 mM EDTA and 10% glycerol. The cleared cell lysate was batch absorbed with wheat germ agglutinin (WGA)-agarose  for 90 minutes. The receptors were washed with 20 volumes 50 mM Hepes pH 8.0, 150 mM NaCl and 0.1% Triton X-100, where after the receptors were eluted with 50 mM Hepes pH 8.0, 150 mM NaCl, 0.1% Triton X-100, 0.5 M n-Acetyl Glucosamine and 10% glycerol. All buffers contained Complete .2, 0.025% Tween-20 and 0.05% BSA (Sigma A-6003). SPA beads were incubated with the IR-specific antibody 83–7 and either supernatant from BHK cells expressing midi receptors or solubilized semi-purified full length receptors for one hour at room temperature under gentle rocking. Receptor concentrations were adjusted to achieve 10% binding of 125I-Insulin.SPA PVT anti-mouse beads (Perkin Elmer) were diluted in SPA binding buffer, consisting of 100 mM HEPES, pH 8.0, 100 mM NaCl, 10 mM MgCl125I-Insulin, 5000 cpm/well) and lastly receptor/SPA mix. The plates were rocked gently for 22.5 hours at room temperature, centrifuged for 5 minutes at 850 x g and counted in TopCounter (Perkin Elmer).SPA beads/receptor mixture was washed three times to remove IGFBPs. Dilution series of cold ligands in triplicates  were added to 96 well Optiplate, followed by tracer  (Gibco) containing protease and phosphatase-inhibitors to terminate the stimulation. The cells were lysed with lysis buffer  containing protease and phosphatase-inhibitors. The lysates were cleared by spinning 10 minutes at 14000 rpm and 4°C.BHK cells stably over-expressing human IR-A, IR-B, IR-A P718K or IR-B K718P were starved for 1 hour, stimulated with insulin, IGF1 or IGF2 and incubated at 37°C and 5% CO2SO4 (Fisher Scientific) to each well and absorbance at 450 nm were determined using an ELISA reader .Microtiter 96 well plates were coated with 100 μl 2 ug/ml 83–7 antibody per well for 1 hour at room temperature. Plates were washed three times with 200 μl PBS + 0.05% tween, blocked by adding three times 200 μl StartingBlock (Pierce). Cell lysates were added and incubated for 2 hours at 22°C and 400 rpm shake . After 3 washes with PBS + 0.05% Tween 100 μl 4G10 antibody diluted 1:1000 in PBS was added to each well and incubated for two hours at 22°C and 400 rpm shake. After five washes with PBS + 0.05% Tween100 μl TMB substrate (Kem-En-Tec Diagnostics) was added to each well and incubated for 10 minutes, the reaction was stopped by addition of 50 μl 2M HAll IC50 values and EC50 values were calculated using non-linear regression algorithm in GraphPad Prism 5.0 . The geometric means with their corresponding 95% confidence intervals of at least three independent experiments were calculated. Two-tailed Student’s t-test was used for single statistical comparisons and one-way analysis of variance (ANOVA) with Dunnet’s correction for multiple comparisons was used for statistical testing in experiments where multiple treatments were analyzed. For all statistical analysis p<0.05 was considered statistically significant. For the IR displacement and activation curves representative examples are shown as mean with standard error of the mean (SEM) for triplicate measurements.The alignment was done using the Protein Structure Alignment tool in Maestro version 10.6.014 . The cry125I-insulin. The geometric mean and 95% confidence interval were determined for each ligand to IR-A and IR-B, Affinity of insulin, insulin detemir, insulin glargine, IGF1 and IGF2 to solubilized full length human IR-A and IR-B receptors was measured using SPA binding assay with displacement of 125I-insulin displacement of either insulin or IGF1.Aiming to identify the amino acids contributing to the low IGF1 and IGF2 affinity to IR-B, we performed an alanine scan of the amino acids encoded by exon 11. We performed the alanine scan in the midi receptor construct, which is described in . The midNo significant difference in insulin affinity was found between IR-A and IR-B, whereas the IGF1 affinity was significantly higher to IR-A compared to IR-B . MutatinSince we aimed at finding positions important for IGF1 and IGF2 affinity without affecting insulin affinity, we focused on position 718. Four receptors for each isoform were made; WT IR-A, IR-A P718A, IR-A P718E and IR-A P718K and WT IR-B, IR-B K718A, IR-B K718E and IR-B K718P. These mutations were chosen in order to test the effect of introducing neutral, positive or negative amino acids and to test the impact of introducing a structural important amino acid. Stable BHK cell clones over-expressing the relevant receptor subtype were established and the receptors subsequently isolated, solubilized and semi-purified using WGA. Receptor expression levels were similar between clones indicating that none of the mutations changed receptor processing or folding (data not shown).Affinity was measured in SPA binding assay  and the The mutational analysis showed that by changing the amino acid at position 718 between IR-A and IR-B, the IGF1 and IGF2 affinities were changed as well. The IC50 values for WT IR-A was 705 pM and 95 pM for IGF1 and IGF2, respectively, whereas for WT IR-B it was 4360 pM and 477 pM, respectively. By inserting the lysine found at position 718 in IR-B at position 718 in IR-A (IR-A P718K) the IC50 values were increased to 3672 pM for IGF1 and 595 pM for IGF2, which is significant different from WT IR-A but not significantly different from WT IR-B. IR-A P718E also significant decreased the affinity but IR-A P718A did not. By inserting the proline found at position 718 in IR-A at position 718 in IR-B (IR-B K718P) the IC50 values were decreased to 927 pM for IGF1 and 155 pM for IGF2, which is significantly different from WT IR-B, but not significantly different from WT IR-A. A significantly decreased IGF1 IC50 value were observed for IR-B K718E compared to WT IR-B, whereas IR-B K718A was not significantly decreased. All mutated IR-B receptors has significantly decreased IGF2 EC50 values compared to WT IR-B, meaning that all mutations significantly increased IGF2 affinity compared to IR-B .Finally we aimed at measuring whether changes in affinity translate into changes in IR activation, hence whether IR activation is mainly reflected by affinities or whether any of the ligands and/or receptors harbors some intrinsic differences in activation potential. This analysis included four receptors; WT IR-A, WT IR-B, IR-A P718K which is an IR-A receptor with IR-B like IGF1 and IGF2 affinities and IR-B K718P which is an IR-B receptor with IR-A like IGF1 and IGF2 affinities. IR activation was measured as total tyrosine phosphorylation in ELISA and time course studies revealed that IR activation was maximal or close to maximal for all receptors and ligands at 10 minutes stimulation time (data not shown), hence 10 minutes stimulation time was used for full concentration-responses curves. Representative full concentration-response curves are shown in No significant differences in insulin EC50 values were observed between the mutated and the corresponding WT receptor, hence the mutations did not affect IR activation in response to insulin. IGF1 and IGF2 showed a significantly higher EC50 value for IR-A P718K compared to IR-A, meaning that IGF1 and IGF2 activate IR-A P718K significantly less efficiently than they activate IR-A. IGF1 and IGF2 showed a significantly lower EC50 value for IR-B K718P compared to IR-B, showing that IGF1 and IGF2 activate IR-B K718P significantly better than they activate IR-B. The decrease in IGF2 affinity for IR-A with P718K is reflected in decrease in activation of the receptor, likewise the increase in IR-B affinity by the K718P mutation is reflected in the activation of the mutated receptor. The relation between affinity and IR activation does not apply as strictly for IGF1; however, this might be due to the very low affinity of IGF1 or assay-related challenges in measuring very low affinity and activation correctly.The biological function of having two IR isoforms is not fully understood. Insulin and insulin analogues maximally show two fold differences in the affinity for IR-A and IR-B. However, a significant difference between the two IR isoforms is that IGF1 and IGF2 have fivefold higher affinities for IR-A compared to IR-B. The selectivity of IGF2 for IR-A may be important in cancer and cancer treatment since many cancer types overexpress IR-A and secretes IGF2 . The amiPosition 718 is the first amino acid in exon 11 and maps to the IR αCT peptide, which is defined as residues 704–719 in IR-A. The αCT peptide has been shown to be a part of the ligand binding site 1 through mutational analysis, and is proposed to interact directly with insulin, IGF1 or IGF2 , hence iThe crystal structure of IR-B has not been determined, hence the three dimensional position of the 12 amino acids encoded by exon 11 is unknown both in the apo-receptor and in the ligand bound state. Truncated versions of IR-A and IR-A/IGF1R hybrid receptors have been crystalized and from these structures it has been proposed how insulin, IGF1 and IGF2 interacts with the receptors. The αCT is displaced from IR upon ligand binding and insulin, in which the B-chain opens as a hinge upon engagement to IR . The incIt has been speculated that the position of αCT gives lower affinity to IR-B, because the 12 amino acids encoded by exon 11 extend αCT compared to in IR-A and that this extended αCT cannot pass through the loops in IGF1 and IGF2. Here we find that introducing K718P in IR-B results in high affinity and removing a P718 in IR-A results in low affinity. That can be explained by a structural change in the receptor backbone, where proline introduces a kink in the structure, hereby relocating the 12 amino acids so they thread away from the binding site. However, it is not only the backbone which is important. The charge of the amino acid at position 718 is important as well, since introducing a positively charged amino acid at position 718 in IR-A decreased IGF1 and IGF2 affinity whereas introducing a negatively charged amino acid at position 718 in IR-B increased IGF1 and IGF2 affinity. These observations suggest that differential IGF affinity between IR-A and IR-B is not only due to the structural features introduced by proline, but also due to electrostatic repulsion between positive charges in the IGFs C-domain and at position 718 in IR. Since only the amino acid at position 718 was found to influence IGF affinity to IR, these observations question whether the entire αCT protrudes through the loops of IGF1 and IGF2.We found that IR activation mainly reflects affinities and that the specific IR isoform or specific ligand is of less importance. IR-A P718K is an IR-A receptor with decreased IGF1 and IGF2 affinity and this receptor also has decreased activation level in response to IGF1 and IGF2 in the same range as IR-B. Mutating IR-B with K718P is an IR-B receptor with increased IGF1 and IGF2 affinities in the same range as IR-A and this is also reflected in the activation of the receptor. We did not observe any significant differences in either affinity or activation potential between IR-A and IR-B K718P or between IR-B and IR-A P718K. Hence by changing the one amino acid at position 718 the affinity is changed which was reflected in activation potential. This support the hypothesis that IR activation is mainly affinity dependent and no intrinsic differences in activation potential exist between IR-A and IR-B or between insulin, IGF1 and IGF2.


Among other alterations, the evolution of C4 from ancestral C3 photosynthesis requires changes in the expression of a vast number of genes. Differential gene expression analyses between closely related C3 and C4 species have significantly increased our understanding of C4 functioning and evolution. In Chenopodiaceae, a family that is rich in C4 origins and photosynthetic types, the anatomy, physiology and phylogeny of C4, C2, and C3 species of Salsoleae has been studied in great detail, which facilitated the choice of six samples of five representative species with different photosynthetic types for transcriptome comparisons. mRNA from assimilating organs of each species was sequenced in triplicates, and sequence reads were de novo assembled. These novel genetic resources were then analyzed to provide a better understanding of differential gene expression between C3, C2 and C4 species. All three analyzed C4 species belong to the NADP-ME type as most genes encoding core enzymes of this C4 cycle are highly expressed. The abundance of photorespiratory transcripts is decreased compared to the C3 and C2 species. Like in other C4 lineages of Caryophyllales, our results suggest that PEPC1 is the C4-specific isoform in Salsoleae. Two recently identified transporters from the PHT4 protein family may not only be related to the C4 syndrome, but also active in C2 photosynthesis in Salsoleae. In the two populations of the C2 species S. divaricata transcript abundance of several C4 genes are slightly increased, however, a C4 cycle is not detectable in the carbon isotope values. Most of the core enzymes of photorespiration are highly increased in the C2 species compared to both C3 and C4 species, confirming a successful establishment of the C2 photosynthetic pathway. Furthermore, a function of PEP-CK in C2 photosynthesis appears likely, since PEP-CK gene expression is not only increased in S. divaricata but also in C2 species of other groups.C C4 photosynthesis evolved more than 60 times in various angiosperm lineages including monocots and eudicots , yielding a compound consisting of four carbon atoms, the key and name-giving step of C4  in the Calvin-Benson cycle, which results in drastically reduced photorespiration  and serine hydroxymethyltransferase (SHMT) reactions to the bundle sheath—is a major landmark  in some lineages , respectively  is an outstanding system to study Cde novo assemblies and the analyses of differential gene expression of representative species of Salsoleae with different photosynthetic types, including C3, C4, and C2 photosynthesis. In particular, we sequenced the transcriptomes of the main assimilating organs  of the C3 species Salsola webbii, two distinct populations of the C2 species S. divaricata, the C4 species S. oppositifolia, and Hammada scoparia, which conducts C3 in cotyledons, but C4 in assimilating shoots. Additionally, we included publicly available leaf transcriptome data from Salsola soda, which also exhibits C3 in cotyledons and C4 in leaves  located in Lanzarote and population 198 (Pop-198) located in Gran Canaria), S. oppositifolia, and S. webbii were grown from seeds in potting soil  in a glasshouse with an additional light intensity of ca. 300 μmol m−2 s−1. Samples were harvested between 16th April and 16th May 2014 between 10:30 and 13:00, immediately frozen in liquid nitrogen and stored at −80°C for RNA extraction. A highly reduced phylogenetic tree based on the results of Schüssler et al. . Approximately 200 mg of each sample were used to determine stable carbon isotope ratios  relative to the Pee Dee belemnite standard  cotyledons and leaves of S. divaricata Pop-184, (3) cotyledons and leaves of S. divaricata Pop-198, (4) cotyledons and leaves of S. oppositifolia, (5) cotyledons and leaves of S. soda  assimilation shoots of  assimilahttp://www.bioinformatics.babraham.ac.uk/projects/fastqc/), and filtered and trimmed using the tools “fastx_clipper” (option -M 15 -l 20) to remove adapter sequences, “fastx_trimmer” (option -f 15) to remove the first 14 bases of the 5' end of all reads, “fastq_quality_trimmer” (option -t 20 -l 20) to remove low quality bases (below PHRED score of 20) from the 3' end, and “fastq_quality_filter” (option -q 20 -p 80) to remove all reads with an overall PHRED score below 20. All tools are from the FASTX toolkit (http://hannonlab.cshl.edu/fastx_toolkit/) and were used in the same order as listed.Sequence reads were quality controlled with the FASTQC tool  to see how many contigs were most similar to genes with a known function/annotation . Here, all contigs firstly were mapped again Arabidopsis, the remaining unmapped contigs were mapped against B. vulgaris, and again remaining unmapped contigs were mapped against UniProtKB. A. thaliana was chosen because this species has the best-annotated plant genome, and B. vulgaris was chosen because this is the phylogenetically closest species to tribe Salsoleae with a sequenced genome.Initially, we conducted B. vulgaris transcriptomes and the UniProtKB database. Sets of annotated transcripts served as reference transcriptomes for inferring differential gene expression (see below). Contigs from different species producing the same annotation match in either Arabidopsis, B. vulgaris or UniProtKB databases were defined as putative orthologs. As additional assessment for defined putative orthologs, pairwise reciprocal BLAST  between all six de novo assemblies were performed, and read counts especially of genes involved in C4 photosynthesis and photorespiration were manually double-checked. To exclude the presence of multiple putative loci per real locus and due to that erroneous read counts, unique presence of genes encoding proteins involved in C4 photosynthesis and photorespiration were manually checked. Venn diagrams showing the overlap of the putative orthologous contigs between all assemblies were calculated and drawn using the “venn” package  in R , functional annotation of transcripts was performed via BLAST-based comparison (see above) against the Arabidopsis and De novo assemblies were conducted with at least 97.8 million (S. webbii) and up to 205 million (S. oppositifolia) quality-controlled reads. While we only used data from leaf samples for differential gene expression analysis, we included additional reads from cotyledon samples  for the de novo assemblies of S. divaricata Pop-184 and Pop-198, S. oppositifolia, and S. soda transcriptomes (see above).Results of both assemblers, SOAPdenovo-Trans and Trinity, yielded partly different results Table . We decide novo transcriptome reference assemblies using bwa-mem v.0.7.13  method in edgeR, only contigs that had at least five reads per million in at least three samples (including replicates) were considered. Log2 transcript ratios were calculated and a log2 fold change (log2FC) of ≥1 was applied as threshold to call differentially expressed genes. A significance threshold of 0.01 was defined after Benjamini-Hochberg correction to account for multiple testing  were used for hierarchical clustering using Pearson correlation and average linking method  and principal component analysis . Additionally, hierarchical clusterings of transcript abundance of selected genes encoding known C4 cycle proteins  under accession number E-MTAB-5778.In total, RNA sequencing yielded between 29.6 and 39.4 million single-end raw reads per replicate, with at least 97.8% reads remaining after quality filtering , 34,022 had at least five reads per million in at least three samples, and downstream analyses were limited to this subset.After deciding to use the annotated Trinity assemblies as reference transcriptomes, quality-filtered reads of the different RNA-Seq samples were mapped and counted using bwa-mem. 48,818 contigs had a reliable annotation in one of the six samples; of these, 15,064 contigs have an Arabidopsis annotation, 19,108 contigs have a 2 species S. divaricata grouped together, a result that reflects both phylogenetic relationship and photosynthetic type. The same was observed for the three C4 species H. scoparia, S. oppositifolia, and S. soda, which grouped together , and based on their transcript expression profiles they group together   Figure .P-value ≤ 0.01 and log2FC ≥1, were identified by pairwise comparisons between all samples. By comparing differentially expressed transcripts between S. webbii (C3) and all three C4 species H. scoparia, S. oppositifolia and S. soda, 4,436 genes were found significantly up-regulated in S. webbii while 1,907 genes were up-regulated in all three C4 species (Supplementary Table 3 (S. webbii) and C2 photosynthesis (S. divaricata populations), 6,304 genes were up- and 5,320 genes down-regulated in S. webbii , adenosine monophosphate kinase 2 (AMK2), aspartate aminotransferase 5 (Asp-AT5), bile acid:sodium symporter family protein (BASS) 2, BASS4, beta carbonic anhydrase 3 (BCA3), NADP-dependent malate dehydrogenase (NADP-MDH), NADP-malic enzyme (NADP-ME), PEPC1, PEPC kinase (PEPC-K), pyruvate orthophosphate dikinase (PPdK), PEP/phosphate translocator (PPT) 1, and sodium:hydrogen antiporter 2 (NHD2) were significantly  more abundant in all three C4 species compared to C3S. webbii  as compared to S. webbii (C3). No known C4 cycle gene was significantly up-regulated in the C3 species S. webbii when compared to all other samples.Transcript abundances of 21 genes encoding known Cs Figure . As expeall three C4 species when compared to C3S. webbii (see above), several C4 related genes were increased in one or two of the three C4 species only: Transcripts of dicarboxylate transporter (DiT) 2 and pyrophosphatase 6 (PPase6) were more abundant in S. oppositifolia and S. soda (log2FC between 1.08 and 2.35), and transcripts of PPdK regulatory protein (PPdK-RP) were more abundant in H. scoparia and S. oppositifolia . As expected in C4 species, many of the genes encoding C4 cycle proteins were among the most highly expressed transcripts in the three C4 species. For example, PPdK was the most highly expressed gene in S. oppositifolia and the second most highly expressed gene in H. scoparia. Other highly expressed genes in the three C4 species were Ala-AT, Asp-AT, NADP-ME, NADP-MDH, and PEPC. The NADP-ME subtype of C4 photosynthesis is known from C4 species of the Salsoleae  and PEP-carboxykinase (PEP-CK) were low.In addition to the genes that were increased in 2 species S. dicaricata, AMK2, BASS4, NHD2, PEPC1, PEPC-K, and PPT1 were significantly more abundant compared to S. webbii (log2FC between 1.12 and 4.43). Interestingly, PEP-CK was more abundant in S. divaricata compared to all other species (log2FC between 2.72 and 7.26). Additionally, in S. divaricata Pop-184, transcripts of NAD-MDH, PPase6, and triose phosphate transclocator (TPT) were significantly up-regulated when compared to S. webbii (log2FC ≥1.1), while NADP-ME and PPdK were up-regulated in S. divaricata Pop-198 in comparison to S. webbii (log2FC ≥1.5) .In the C4 photosynthesis (see above), transcripts of genes currently described to have an alleged function related to C4 metabolism  when compared to S. webbii, but still abundant in all other species, including C3S. webbii. Phosphate transporter4.1 (PHT4.1) was significantly more abundant in all species (in S. soda with log2FC of 0.8) compared to S. webbii. The gene encoding PHT4.4 was highly upregulated in all three C4 species compared to S. webbii (log2FC ≥1.85). Levels of mRNA coding for asparagine synthetase (ASN) were increased in S. oppositifolia and S. soda when compared to S. webbii . Also, ASN was the 14th highest expressed gene in S. soda , and gene expression of seven genes were increased in S. webbii in comparison to all three C4 species: serine:glyoxylate aminotransferase (AGT/SGT), GDC P-protein (GDC-P), GDC L-protein (GDC-L), GDC T-protein (GDC-T), glutamate:glyoxylate aminotransferase (GGT), NADP-dependent hydroxypyruvate reductase (HPR), and SHMT (Supplementary Table S. divaricata compared to S. webbii (log2FC ≥1.96), the former also significantly up-regulated when compared to the three C4 species (log2FC ≥1.01). Further, S. divaricata Pop-184 showed an increase of transcripts encoding GDC-H, glycerate 3-kinase (GLYK) and PGP when compared to S. divaricata Pop-198 and S. webbii (Supplementary Table S. divaricata (Pop-184 and 198) and S. webbii. Interestingly, transcripts of GDC-P were not only highly abundant in the C2 and C3 species, but in all six samples.Since photorespiration has been reported as strongly modified between closely related C2 and C4 photosynthesis are complex and require many changes in gene expression patterns compared to the ancestral C3 photosynthesis , which includes, besides C3 species, many C2 and C4 species  or more contigs . Interestingly, we obtained more than twice as many contigs from S. oppositifolia compared to S. soda. This huge difference could potentially have been caused by different ploidy levels in these species: Salsola oppositifolia is octoploid  has been available and large-scale gene expression studies based on RNA sequencing were for the most part absent in this group  were measured since stable carbon isotopes can be used as an indicator of C4 metabolism  or sampled organ , seemed to be of minor importance for differences in gene expression, whereas photosynthesis type was the dominant factor. By comparison, in a PCA of gene expression data of several closely related species of Flaveria with different photosynthesis types, the first three components explained only 27% of the total variance  anatomy, which is the typical C4 anatomy in this group  and phenylalanine to valine at position 794  a different PEPC isoform was recruited into the C4 cycle . Intriguingly, PHT4.1 transcripts were highly and PHT4.4 transcripts slightly more abundant in C2S. divaricata, too. Thus, the results of the current study support a proposed function of the two PHT4 family proteins related to both C4 and C2 photosynthesis.Based on gene expression data, Lauterbach et al.  speculat4 leaves of S. soda when compared to C3 cotyledons of the same species  compared to closely related C3Tarenaya hassleriana  when compared to S. webbii. It is tempting to speculate on a possible function of high PEP-CK abundance in a low level C4 cycle in the C2 species Moricandia arvensis, M. suffruticosa, and S. divaricata. One would expect, however, that genes encoding other proteins involved in the PEP-CK type C4 cycle, such as Ala-AT and Asp-AT, were also highly abundant, which at least in case of S. divaricata was not observed. High abundance of PEP-CK is inconsistent with a study investigating the decarboxylation enzymes of several plant groups, including few species of Salsoleae (Koteyeva et al., S. divaricata (Koteyeva et al., Megathyrsus maximum, anti-Oryza sativa, anti-Ananas comosus; Koteyeva et al., S. divaricata could occur. The latter explanation is at least partly supported because in the two studied populations transcript abundance of PEP-CK differed three-fold. Also, plasticity in the decarboxylation biochemistry of a supplemental PEP-CK pathway has been hypothesized (Furbank, 4 species maize (Pick et al., 4G. gynandra (Sommer et al., S. divaricata, and also in C3-C4Moricandia species.CFurbank,  and predFurbank,  as well 2 type S. divaricata was intermediate between C3S. webbii and the three C4 species. Despite major differences in life cycle, habit and photosynthesis during seedling stage, the C4 species showed similar gene expression profiles of C4 genes and photorespiratory genes. Most C4 genes were highly abundant in all three C4 species. Further, our results suggested that PEPC1 was the C4-specific isoform in Salsoleae, as found for other C4 lineages of Caryophyllales. The protein, however, lacks C4 typical amino acid changes in S. soda. Two recently proposed transporters from the PHT4 protein family might not only be involved in C4 photosynthesis, but also be active in C2 photosynthesis in Salsoleae. Here, they might be involved in protection against reactive oxygen species resulting from the absence of most photorespiratory reactions in the mesophyll. The transcript profile of the C2 species S. divaricata was mostly comparable with that observed in C2 species of Flaveria or Moricandia. Moreover, in one population of S. divaricata, Pop-198, the transcript pattern of C4 genes implied a slightly more advanced C4 cycle than in Pop-184. However, a C4 cycle is not detectable in the carbon isotope values, and this species is not functioning as a C4 plant physiologically (Voznesenskaya et al., 2 photosynthesis in S. divaricata is likely, because PEP-CK was highly increased compared to C3 and C4 species of Salsoleae, a result that was also observed in C2Moricandia, but needs further investigations.The overall gene expression pattern, as visualized by PCA, showed photosynthetic type as the main factor separating the different Salsoleae species. CML designed the research, performed experiments, analyzed and interpreted the data, and wrote the manuscript. HS analyzed and interpreted the data, and critically revised the manuscript. KB performed experiments. TH designed the research and critically revised the manuscript. PW designed the research and critically revised the manuscript. UG designed the research, analyzed and interpreted the data, and critically revised the manuscript. GK conceived and designed the research, interpreted the data, and critically revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


DM remains a risk factor for poor outcome after stent-implantation, but little is known if and how DM affects the vascular response to BVS.The aim of our study was to examine coronary responses to bioresorbable vascular scaffolds (BVS) in swine with and without diabetes mellitus fed a ‘fast-food’ diet  by sequential optical coherence tomography (OCT)-imaging and histology.Fifteen male swine were evaluated. Eight received streptozotocin-injection to induce DM. After 9 months (M), 32 single BVS were implanted in epicardial arteries with a stent to artery (S/A)-ratio of 1.1:1 under quantitative coronary angiography (QCA) and OCT guidance. Lumen, scaffold, neointimal coverage and composition were assessed by QCA, OCT and near-infrared spectroscopy (NIRS) pre- and/or post-procedure, at 3M and 6M. Additionally, polarization-sensitive (PS)-OCT was performed in 7 swine at 6M. After sacrifice at 3M and 6M, histology and polymer degradation analysis were performed.Late lumen loss was high (~60%) within the first 3M after BVS-implantation (P<0.01 FF-DM vs. FF-NDM) and stabilized between 3M and 6M . Neointimal coverage was highly heterogeneous in all swine (DM vs. NDM P>0.05), with focal lipid accumulation, irregular collagen distribution and neointimal calcification. Likewise, polymer mass loss was low  and not associated with DM or inflammation.Scaffold coverage showed signs of neo-atherosclerosis in all FF-DM and FF-NDM swine, scaffold polymer was preserved and the vascular response to BVS was not influenced by diabetes. Patients with diabetes mellitus (DM) are generally at risk for worse outcome after stent-implantation than patients without DM. Due to tThe everolimus-eluting bioresorbable vascular scaffold (BVS) may offer advantages. It elutes everolimus in the first 3 to 6 months after implantation, inhibiting excessive neointimal growth  and starHistology in healthy swine demonstrated that struts are covered at 28 days and resorbed around 3 years with minimal calcification and inflammation., 8 In seAnimal models reflecting the impact of atherosclerosis and DM can be useful, as they allow us to study vascular responses and scaffold degradation in a more complex setting. MoreoverWe examined the mechanistic and morphological aspects of the coronary response to BVS1.1 in DM and non-DM swine fed a fast-food diet  using longitudinal intracoronary imaging and histology.The Erasmus MC Animal Ethics committee approved the study, performed in accordance with the Guide for Care and Use of Laboratory Animals. Fifteen After 9 months, all 8 FF-DM and 5 FF-NDM received single 3.0x18.0mm Absorb BVS1.1 implants in 2, and 2 FF-NDM received single Absorb BVS1.1 implants in all 3 coronary arteries to ensure an even amount of scaffolds in FF-DM (N = 16) and FF-NDM (N = 16)  post-implantation and NIRS pre-, 3M and 6M post-implantation. PS-OCT was performed in N = 3 FF-DM and N = 4 FF-NDM BVS at 6M. 3M imaging was included when pre and/or post-implantation imaging were available and 6M imaging was included when pre- and/or post—and 3M imaging were available. After the 3M imaging assessment, 3 FF-DM and 2 FF-NDM were sacrificed and after the 6M imaging assessment the remaining swine were sacrificed. After sacrifice, hearts were removed, the coronary tree dissected free and coronary arteries containing BVS randomized to histological  or GPC analysis  .Fasting blood samples were obtained at baseline, 3M and 6M to measure glucose, total, low- and high-density lipoprotein cholesterol  and triglyceride levels. Furthermore, in the FF-DM swine, glucose levels were assessed weekly by 24-hour urine samples. When glucose appeared in undiluted urine samples, venous glucose and ketone levels were checked via ear vein puncture and a handheld reader. When glucose levels were high (>20 mmol/L), in combination with ketone production, subcutaneous insulin (approx. 5–15 units once daily) was given to eliminate detectable ketone production while maintaining hyperglycemic state.See OCT parameters for vascular reaction, including lumen and scaffold dimensions and scaffold strut appearance, apposition, neointimal coverage and coverage morphology were assessed in 1-mm intervals using off-line OCT analysis software according to previously published methodology. In pre-iPS-OCT was performed using a prototype imaging system. PS-OCT provides a measure of tissue birefringence, an optical tissue property that describes the interaction with polarized light. It grossly relates to microscopic tissue organization, and enables characterization of collagen content and smooth muscle cell (SMC) density in atherosclerotic plaques.NIRS analysis  was used for lipid core plaque (LCP) characterization. Lipid-coGPC was performed as described previously see .7] Degr Degr7] DSee Histological analysis included neointimal healing and organization, collagen distribution, injury and inflammation score, lipid accumulation and presence of calcium classified as subluminal or surrounding struts .Statistical analysis (SPSS 20.0) entailed the Kolmogorov-Smirnov test for normality of the data. Normal distribution was expressed as mean ± standard deviation. Non-normally distributed data were presented as median with interquartile range. Comparison of in-vivo imaging between FF-DM and FF-NDM was performed by generalized estimating equations (GEE) modeling. GEE is a statistical method that accounts for the clustered nature of >1 scaffold analyzed from one swine, which might result in unknown correlations among measurements within these scaffold clusters. A linear response model was applied with an exchangeable structure for the within-cluster correlation matrix. For repeated measures, GEE modeling was performed using a linear response model with an autoregressive (AR(1)) structure for the within-cluster correlation matrix. Comparison of ex-vivo GPC between FF-DM and FF-NDM swine was performed by independent samples t-test. To assess variable relations, the Spearman correlation coefficient was computed. All statistical tests were 2 tailed, and P<0.05 was considered statistically significant.Average TC, LDL and HDL were similar between FF-DM and FF-NDM (TC 18.7±5.0mmol/l and 19.0±5.8mmol/l (P = 0.86); LDL 15.1±5.1mmol/l and 15.6±5.1mmol/l (P = 0.75); HDL 5.4±0.8mmol/l and 5.7±0.7mmol/l (P = 0.13) in FF-DM and FF-NDM respectively). In the 8 swine that received a steptozotocin-injection, DM was successfully induced. Average plasma glucose and triglyceride levels were elevated in FF-DM compared to FF-NDM (15.0±7.8mmol/l vs. 4.5±1.0 mmol/l (P<0.01), 1.1±0.8mmol/l vs. 0.6±0.5mmol/l (P = 0.02), respectively). All FF-DM swine received insulin in the first 10 weeks after streptozotocin injection and 2 FF-DM swine received insulin throughout the entire study based on detectable venous ketone body production.”All 32 BVS were successfully implanted with a mean S/A-ratio of 1.1±0.1 in FF-DM and FF-NDM (P = 0.20).QCA findings are presented in 2) and 1 FF-NDM BVS . From post-implantation to 3M, mean LD, LA and MLA decreased (~60%). At 3M, all ISA resolved, no late acquired ISA developed and all struts were covered. Restenosis, however, hampered OCT imaging at 3M in one FF-NDM. At 6M, OCT demonstrated a substantial neointima with a highly heterogeneous morphology, which was confirmed by histology  but became stronger at 6M .See See There was no relationship between scaffold degradation and DM (P>0.10), or with OCT-derived pre-implantation %PB (P = 0.22), scaffold recoil (P = 0.59), preserved (P = 0.92), open (P = 0.45) or dissolved black box appearance (P = 0.99) at 3M, or at 6M .See Collagen poor regions were observed within the neointima. They contained leukocytes, were evident at sites with extracellular lipids and often coincided with calcifications . Collage(P = 0.04) at 3M and 6M  indicates the guide wire artifact. The arrowheads indicate the region containing lipid and the drawn white lines indicate calcified regions.(TIF)Click here for additional data file.S2 FigWithin the neointima, 2 specific regions were discerned: para-strut neointima, defined as in contact with the struts, and subluminal: located near the lumen.(TIF)Click here for additional data file.S3 FigRestenosis of a BVS implanted in an FF-NDM swine at 3M. Coronary angiography post-implantation (A) and at 3M (B) demonstrates a significant lumen loss, with a percentage diameter stenosis (%DS) of 70% which persisted at 6M, (C). At 3M OCT was not performed as the lesion was considered too tight to allow passage of an OCT catheter without risk of causing ischemia and all the potential sequelae thereof. Therefore OCT was restricted to 6M follow-up, the scheduled sacrifice time point. OCT demonstrated a highly heterogeneous neointima (D), which is confirmed by histology demonstrating a large neointimal burden with calcification subluminal and surrounding the struts . D Lesion = Diameter of the lesion, Ref D Lesion = Reference diameter.(TIF)Click here for additional data file.S4 FigA-D) showed dense elastic fibers (B) with 3 or more layers of αSMA positive cells (C) without lipid accumulation (D). The unorganized neointima showed myxoid degeneration (double arrow in E-H) with disarray and low density of αSMA positive cells (G). In the same area, lipid accumulation was clearly seen (H). αSMA: alpha smooth muscle cell actin, L: lumen, A-D: 3 months DM, E-H: 6 months non-DM, A and E: H&E, B and F: Resorcin-Fuchsin, C and G: αSMA, E and H: Oil red O, Scale bar in A -H: 100 μm, insert bar of A and E: 1000 μm.The vessels with well-organized neointimal layers (two arrows in (TIF)Click here for additional data file.S1 TableNormally distributed data are presented as mean ± SD, non-normally distributed data as median (interquartile range). FF-DM = fast-food fed diabetic swine, FF-NDM = fast-food fed non-diabetic swine, QCA = Quantitative coronary angiography, OCT = optical coherence tomography, BVS = bioresorbable vascular scaffold, post = post-implantation, 3M = 3 months follow-up, 6M = 6 months follow-up. *P-value for the comparison between FF-DM and FF-NDM swine, †P-value for the difference between post-procedure and 3M, ‡P-value for the difference between 3M and 6M.(DOCX)Click here for additional data file.S2 TableNIRS = Near-infrared spectroscopy, LCBI = lipid core burden index. §P-value for the difference between pre-procedure and 3M. Remaining footnotes and abbreviations are as listed in (DOCX)Click here for additional data file.S3 TablePercentages are calculated as mean from the total (100%). Footnotes and abbreviations are as listed in (DOCX)Click here for additional data file.S1 VideoOCT imaging at 6M in a FF-DM swine demonstrates a highly heterogeneous appearance of the coverage. The red line in the longitudinal view of the OCT pullback (middle panel) corresponds to the location of the OCT catheter in the 2D pullback (left panel) and 3D pullback (right panel). The asterisk (*) indicates the guidewire artefact, the green line delineates the contour of the lumen area, the stars indicate scaffold struts, the arrowheads lipid, the white circles calcium and the red circle indicates the marker of the scaffold. Word did not find any entries for your table of contents.(MP4)Click here for additional data file.S2 VideoOCT imaging at 6M in an FF-NDM swine demonstrates a highly heterogeneous appearance of the coverage. The red line in the longitudinal view of the OCT pullback (middle panel) corresponds to the location of the OCT catheter in the 2D pullback (left panel) and 3D pullback (right panel). The green line delineates the contour of the lumen area, the stars indicate scaffold struts, the arrowheads lipid and the white circles indicate calcium.(MP4)Click here for additional data file.S1 FileSupporting information accompanying the manuscript titled: “Neoatherosclerosis development following bioresorbable vascular scaffold implantation in diabetic and non-diabetic swine coronary arteries”.(DOC)Click here for additional data file.


MicroRNAs (miRNAs) are highly conserved elements in mammals, and exert key regulatory functions. Growing evidence shows that miRNAs can interact with another class of non-coding RNAs, so-called transcribed ultraconserved regions (T-UCRs), which take part in transcriptional, post-transcriptional and epigenetic regulation processes. We report here the interaction of miRNAs and T-UCRs as a network modulating the availability of these non-coding RNAs in bladder cancer cells. In our cell system, antagomiR-596 increased the expression of T-UCR 201+. Moreover, T-UCR 8+ silencing increased miR-596 expression, which in turn reduced total T-UCR 283+, showing that the perturbation of one element in this network changes the expression of other interactors. In addition, we identify the polycomb protein Yin Yang 1 (YY1) as mediator of binding between miR-596 and T-UCR 8+. These new findings describe for the first time a network between T-UCRs, miRNAs and YY1 protein, highlighting the existence of an additional layer of gene expression regulation. Long non-coding RNAs (lncRNAs) have been shown to play crucial roles in a variety of biological processes such as epigenetic control of gene expression, promoter-specific gene regulation ,2,3, X-cTranscribed ultraconserved regions (T-UCRs) are a particular class of lncRNAs. They include a group of 481 highly conserved sequences located in both intra- and intergenic regions. Little is known about their function, but their exact conservation in human, rat and mouse genome suggests an important regulatory role in gene expression . Since TPolycomb group (PcG) proteins are chromatin modifiers that have key roles in the silencing of developmental genes controlling the balance between differentiation and stem cell self-renewal. More recently, they have been found to have important functions in development and progression of several human cancers ,28,29. Ghttp://bibiserv.techfak.uni-bielefeld.de/rnahybrid/ [p value lower than 0.05 [T-UCR sequences were obtained from supplementary data by Bejerano et al.  and convahybrid/ ,40 as taahybrid/ . T-UCRs:Human BlCa J28 cell lines (American Type Culture Collection) were cultured as a monolayer in Minimal Essential Medium supplemented with 10% foetal bovine serum. Cells were grown in a humidified incubator in a 5% carbon dioxide atmosphere at 37 °C.For the transient silencing of T-UCR 8+, J82 cells were transfected with small interference RNAs (siRNAs) using HiPerFect transfection reagent , according to the manufacturer’s instructions. siRNAs were designed using siDirect software  with inp6 cells/well) and incubated for about 3 h at 37 °C. After incubation, cells were transfected with 1 μg of empty vector and 0.01, 0.2, 1, and 5 μg of shYY1 vector using Lipofectamine 2000  transfection reagent, according to the manufacturer’s instructions. Cells were harvested 48 h after transfection and were subjected to RNA extraction and Western blotting analysis.For the transient silencing of YY1 in J82 cells, we used pBLOCK-iT RNAi entry Vector kit  and the following oligonucleotide: TGCTGTATGAGGGCAAGCTATTGTTCGTTTTGGTTTTGGCCACTGACTGACTGACGAACAATAGCTTGCCCTCATAC or Scramble oligos. J82 cells were plated in six-well plates , and the integrity of RNA was checked by gel electrophoresis. Total RNA (1 μg) was then reverse-transcribed using the QuantiTect Reverse Transcription Kit (Qiagen). Quantitative real time PCR (qRT-PCR) was performed using strand-specific primers for T-UCR analysis . A miRCUd change .Total cellular proteins were size-fractionated on Bis-acrylamide 12% gels  using Mini-Cell (Biorad) and transferred onto 0.45-μm nitrocellulose membranes using Immobilon-P  according to the manufacturer’s recommendations. For this experiment we used YY1  as primary antibody. Signal quantification was performed using Amersham Hyperfilm ECL  and normalized to loading control . A dilution of 1:5000 of anti-rabbit IgG  was used as secondary antibody.7 cells), were fixed with 1% formaldehyde for 10 min at room temperature. Cells were washed extensively with phosphate-buffered saline (PBS), and the chromatin was sheared by sonication . The cross-linked RNA-protein complexes were immunoprecipitated with YY1  antibody at 4 °C overnight. Normal mouse IgG was used as negative controls. Immunocomplexes were pulled down through A/G plus resin (Santa Cruz Biotechnology) incubation and RNAs obtained from the decross-linking reaction were retro-transcribed and amplified by quantitative PCR (qPCR). Real time PCRs were performed with T-UCR 8+ and T-UCR 201+ primers  extracted from J82 cell line was incubated in an appropriate buffer with 100 pmol of 5′-biotinylated oligonucleotides T-UCR 8+, T-UCR 201+, T-UCR 128+ and one oligonucleotide scramble in miR-596 binding site overnight at 4 °C with rotation. After complex formation, 100 μL of μMACS Streptavidin MicroBeads  were added and incubated for 30 min at 4 °C. μMACS columns were equilibrated with 100 μL of Equilibration buffer  and rinsed with the same buffer used for the binding reaction. Labelled complex was applied onto the top of the column matrix. Columns were washed with 4 × 100 μL of washing buffer (supplied with the kit) to remove non-specifically binding molecules. RNA labelled to biotinylated oligonucleotides was eluted with 150 μL of elution buffer (supplied with the kit) according to manufacturer’s instructions μMACSTM Streptavidin Kit).t-test with GraphPad Prism software . Comparisons were considered statistically significant when p < 0.05.Results are reported as mean ± standard deviation. Statistical comparisons of all data were performed using the Student n = 962) previously selected from those described [p < 0.05, q < 0.025) in BlCa tissues. From this analysis, we found that ≈ 11% of T-UCRs are interconnected by a single miRNA binding site, 83% of T-UCRs bind multiple miRNAs, and ≈ 6% have no binding sites for miRNAs (p < 0.001), completely abrogated T-UCR 283+ transcription (p < 0.01) (p < 0.001), resulting in increased expression of both T-UCR 201+ and T-UCR 283+ mRNAs by twofold (p < 0.001) compared to the control To predict the extent of miRNA-mediated regulation by T-UCRs in bladder carcinogenesis, we first scanned all sense and antisense T-UCRs  D. We the < 0.01) E by usinp < 0.001) with both T-UCR 201+ and T-UCR 8+. Validation of the binding specificity of these two T-UCRs and miR-596 in vitro support the hypothesis of the existence of a complex mechanism of regulation.In order to validate T-UCR network accuracy, we performed three RNA fishing experiments to demonstrate the effective interaction of these macromolecules. We identified a single-strand region of T-UCR 8+, T-UCR 128+ and T-UCR 201+, and designed specific antisense, complementary and biotinylated peptide nucleic acid (PNA) probes as baits . T-UCR 8http://www.genomatix.de) [Since YY1 proteins have already been reported to interact with multiple DNA and/or RNA sequences , we scanatix.de) . The proDifferential expression of T-UCRs has been reported in BlCa tissues, although the mechanism remains unknown . Here, w


It also displayed 15∼400-fold selectivity over other primary mutants such as L576P and secondary mutants including T670I, V654A (ATP binding pocket) as well as N822K and D816V (activation loop). In addition, it exhibited a selectivity S score (1) of 0.01 among 468 kinases/mutants in the KINOMEScan™ assay. CHMFL-KIT-031 showed potent inhibitory efficacy for KIT V559D mediated signaling pathways in cell and anti-tumor activity in vivo (Tumor Growth Inhibition: 68.5%). Its superior selectivity would make it a good pharmacological tool for further dissection of KIT V559D mediated pathology in the GISTs.KIT kinase V559D mutation is the most prevalent primary gain-of-function mutation in Gastrointestinal Stromal Tumors (GISTs). Here we reported a highly selective KIT V559D inhibitor CHMFL-KIT-031, which displayed about 10-20 fold selectivity over KIT wt in the biochemical assay (IC Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. About 80% GIST patients harbor KIT kinase primary gain of function mutations , 2. The Currently there are a number of KIT kinase inhibitors such as Imatinib , Sunitin50: 0.025 μM) (Table 50: >10 μM) which indicated that this compound displayed KIT V559D on-target anti-proliferative effect. Surprisingly, it did not affect the proliferation of KIT wt transformed BaF3 cells either (GI50: >10 μM) which indicated that it also achieved selectivity over KIT wt kinase. This encouraged us to expand the testing to other mutant isoforms. Interestingly, CHMFL-KIT-031 also did not affect L576P mutant occurred in the JM domain (GI50: 2.2 μM). It showed no apparent inhibitory activity against gatekeeper T670I mutant (ATP binding pocket), T670I/V559D (JM/ATP binding pocket) and activation loops mutants N822K, D816V . It only exhibited moderated inhibitory activity against ATP binding pocket mutant V654A (GI50: 0.4 μM) and V654A/V559D mixed mutant (GI50: 0.81 μM). Comparably, Imatinib was more sensitive against JM domain mutant V559D and L576P than KIT wt. In addition, Imatinib displayed similar sensitivities among KIT wt, V654A, V654A/V559D and N822K but resistant to KIT T670I, T670I/V559D and D816V, while Sunitinib exhibited similar potent activities against all of the tested mutants except that it was relatively resistant to KIT wt, N822K and D816V. Furthermore, Sunitinib also displayed inhibitory activity against parental BaF3 cells which again reflected its multi-target profiles.Through screening a panel of in-house made structure focused type II lead compounds prepared during development of KIT kinase inhibitors with KIT-V559D permanently transformed BaF3 cells , we founM) Table . FurtherIn order to confirm the selectivity observed in the anti-proliferation assay of the transformed BaF3 cells, we then examined the inhibitory effect of CHMFL-KIT-031 for the KIT wt/mutant auto-phosphorylation at Y703, Y719 and Y823 sites to further confirm the on-target efficacy Figure . The res50: 2.4 μM). For NEK3 kinase there is no available biochemical assay yet, therefore we did not confirm it. Together, these results suggested that CHMFL-KIT-031 is a highly selective KIT V559D mutant inhibitor which exhibited a much better selectivity spectrum than Imatinib and Sunitinib  and displayed about 6-fold selectivity over KIT wt (IC50: 168.4 nM). In addition, we also examined its binding Kd using MST technology  in which width (W) is defined as the smaller of the two measurements and length (L) is defined as the larger of the two measurements.Five-week-old female nu/nu mice were purchased from Nanjing Biomedical Research Institute of Nanjing University . All animals were housed in a specific pathogen-free facility and used according to the animal care regulations of Hefei Institutes of Physical Science, Chinese Academy of Sciences . Prior to implantation, cells were harvested during exponential growth. One million BaF3-TEL-KIT-V559D cells in PBS were formulated as a 1:1 mixture with Matrigel (BD Biosciences) and injected into the subcutaneous space on the right flank of nu/nu mice. Daily ip injection was initiated when BaF3-TEL-KIT-V559D tumors had reached a size of 200−400 mm2O for 1 min, it was stained with 1% eosin Y solution for 10−30 s with agitation. Subsequently, the sections were dehydrated with two changes of 95% alcohol and two changes of 100% alcohol for 30 s each, and then the alcohol was extracted with two changes of xylene. Finally, one or two drops of mounting medium was added and covered with a coverslip.HE staining was carried out according to previous publication. First, the sections were hydrated and then the slide was dipped into a Coplin jar containing Mayer’s hematoxylin and agitated for 30 s. After rinsing the slide in Hi-67, tissue sections were quenched for endogenous peroxides and placed in an antigen retrieval solution  for 15 min in a microwave oven at 100°C at 600 W. After incubation in the casein block, mouse mAb anti-Ki-67  was applied to the sections at dilutions of 1:50. Incubations with primary antibodies lasted overnight at 4°C. The secondary detection system was used to visualize antibody binding. Staining was developed with DAB, and the slides were counterstained with hematoxylin, dehydrated and mounted.For IHC demonstration of K2O2 in methanol. Terminal deoxynucleotidyl transferase (TdT) in reaction buffer was applied to sections for 1 h at 37°C. Following washes, the slides were covered by converter-POD solution for 30 min at 37°C. Apoptotic cells were detected after incubation in 3,3’-diaminobenzidine (DAB) chromogen  for approximately 8 min, and the slides were counterstained with hematoxylin.TUNEL staining was performed using the POD in situ cell death detection kit . Briefly, sections were deparaffinized in xylene, rehydrated in decreasing concentration of ethanol, and then treated by nuclease free proteinase K for 15 min at room temperature before endogenous peroxidase was blocked in 3% H


Pancreatic cancer (PC), as the leading cause of cancer death worldwide, is one of the deadliest tumors with a very low 5-year survival rate. Therefore, it is urgent to seek new biomarkers of PC for more accurate and reliable treatments. To identify the differentially expressed miRNAs (DEM) in PC tissues, we performed the systematic microarray and qRT-PCR analyses. We found miR-196b was the top dysregulated DEM in PC tissues as compared with the corresponding adjacent tissues, and positively correlated with poor differentiation, tumor size, lymphatic invasion and TNM stage. Furthermore, the late apoptosis rate was significantly reduced, while the cell proliferation was increased in PANC-1 and ASPC-1 cell-lines after treatment with miR-196b mimics. The qRT-PCR and Western blot analysis demonstrated that the level of CADM1 in PANC-1 cells response to the alteration of miR-196b. Moreover, blockade of CADM1 could decrease the late apoptosis in PANC-1 cells as up-regulated by inhibition of miR-196b. Finally, luciferase report assay confirmed that CADM1 was the direct target gene of miR-196b. Overexpression of miR-196b in PC tissues can increase the late apoptosis of pancreatic cancer cells by targeting CADM1. These findings suggested miR-196b is a potential target for diagnosis and therapeutics of human pancreatic cancer. The incidence of PC in China has been increasing rapidly during past decades. The understanding of PC has been improved, but the prognosis is still very poor, which mainly results from delayed diagnosis, lack of curative treatments, and non-specific symptoms7. Therefore, it is urgent to seek new biomarkers of PC for more accurate and reliable treatments.Pancreatic cancer (PC), the 78. However, the exact causes of PC remain unclear. MicroRNAs (miRNAs), a group of non-coding RNAs, are involved in several biological behaviors  through inhibiting the translation or degrading the target mRNAs10. Moreover, miRNAs have been reported to be involved in various diseases, especially cancers13. Several studies have reported many miRNAs  were dysregulated in PC, but the clinical value and the exact role in pathogenesis of PC were still not clear15. Therefore, systematically identifying differentially expressed miRNAs, to account for the pathogenesis of PC and be associated with clinical characteristics of PC, will provide more evidence for the diagnosis and treatment of PC.Recently, there are several elements such as smoking, genetics, and environment factors have been identified as risk factors of PCIn this study, we identified the differentially expressed miRNAs in PC tissues, find out the correlation of these miRNAs with characteristics of PC patients, and investigated the role of miR-196b in regulating apoptosis and proliferation in PANC-1 and ASPC-1 cells. Our results indicated miR-196b is a potential target to diagnosis and therapeutics of pancreatic cancer.To find out candidate miRNAs associated with PC, 20 paired pancreatic cancer tissues and adjacent tissues were collected during resections, and were assessed by NanoString nCounter Human miRNA assay. The demographic characteristics of these patients were shown in Table Next, we identified the common character among these differently expressed miRNAs by enrichment analysis. According to the database of miRNA related diseases, Human microRNA Disease Database 2 (HMDD2), we found that these DEMs were mainly associated with cancers, such as lung neoplasms, and pancreatic neoplasms Fig. . BesidesAs shown in Table Next, we investigated whether the levels of these differentially expressed miRNAs were associated with characteristics of PC patients. As shown in Table As MiR-196b has been observed to be a candidate biomarker in diagnosis of PC and correlated with clinicopathological features, but the function of miR-196b in PC was still unknown. Therefore, we investigated the function of miR-196b in PC cells by interference experiment. Firstly, we confirmed that the expression of miR-196b was significantly decreased after transfection with miR-196b inhibitor, and was increased after transfection with miR-196b mimics compared with each negative controls Fig. . These rNext, we assessed cell apoptosis in PANC-1 cells by two markers, FITC annexin V and propidium iodide (PI). We found that late apoptosis rate was reduced in PANC-1 cells with treatment with miR-196b mimics, and increased with miR-196b inhibitors compared with controls Fig. . While tThen, We investigated whether miR-196b could regulate proliferation in PANC-1 cells, and found that the percentage of EdU positive cells was decreased in cells transfected with miR-196b inhibitors, while increased after transfected with miR-196b mimics compared with negative controls Fig. , indicathttp://www.targetscan.org/)  Table , We foun/) Table . These r/) Table . Consist/) Table . These rTo verify that CADM1 was the direct target gene of miR-196b, luciferase report assay was performed. By RNAhybrid software, we confirmed that there are two significant binding sites of miR-196b in 3′UTR of CADM1 Fig. . Then, wTo find out whether miR-196b regulated PANC-1 cell apoptosis through CADM1, the loss-of-function experiment for CADM1 by shRNA was performed. As shown in Fig. 6. Therefore, finding new indicators for helping the diagnosis, treatment, and prognosis of PC, is necessary. Besides, miRNAs have been found to be as tumor suppressors or promoters through regulating the expression of target genes12. In our study, we demonstrated that miR-196b expression is significantly up-regulated in PC tissues, positively correlated with differentiation grades and metastasis, and miR-196b regulates apoptosis and proliferation of PANT-1 and ASPC-1 cells via targeting CADM1.PC has been recognized as one of the most malignant cancers worldwide, as the annual mortality of PC is almost same as the morbidity18. Moreover, the level of miR-196b has been demonstrated to be increased in serum of PC patients, but the exact functions of miR-196b in PC was still unknown. In our study, we demonstrated that miR-196b expression was up-regulated in PC tissues compared with adjacent tissues, which was consistent with other cancer tissues and serum of PC patients in previous studies.The miR-196 family, including miR-196a and miR-196b, has been reported to be up-regulated in many cancers, such as breast cancer, leukemia, and colonic cancer12. In this end, we next questioned whether miR-196b was correlated with characteristics of PC patients. We found miR-196b was positively associated with differentiation grades and metastasis in PC patients. Previous studies have shown that miR-196b could regulate differentiation and migration of gastric cancer cells, which may account for the results in this study19. The functions of miR-196b in cancers were also studied, and miR-196b was found to be regulating proliferation, apoptosis, differentiation and migration in different cancer cells21. In current study, miR-196b was found to promote proliferation, but inhibit apoptosis of PANC-1 and ASPC-1 cells, which was consistent with the results in other types of cancer cell in previous studies.Accumulating studies have shown that miRNAs could act as biomarkers for different diseases, especially cancers, and be correlated with clinical characteristics of those patients24. Besides, previous study has found that CADM1 expression was down-regulated in different types of cancers, including gastric cancer, breast cancer, and lung carcinoma27. In this study, we found that miR-196b could reduce the expression of CADM1 in PANC-1 cells. Furthermore, the late apoptosis rate was decreased after transfection of shRNA targeting CADM1, and blockade of CADM1 could decrease the late apoptosis up-regulated by inhibition of miR-196b. Finally, luciferase assay confirmed that CADM1 was the target gene of miR-196b in PANC-1 cells.Our finding demonstrated that CADM1 was a target gene of miR-196b in PANC-1 cells. CADM1, which is also called as TSCL1, has been reported to be involved in cell adhesion, migration, and apoptosisIn summary, our data showed that miR-196b expression is up-regulated in PC tissues, and miR-196b regulate apoptosis and proliferation of PANT-1 cells via targeting CADM1. Besides, the level of miR-196b was positively correlated with differentiation grades and metastasis in PC patients. Therefore, miR-196b might be an important biomarker in PC patients.Paired cancer tissues and adjacent tissues were obtained from patients recruited at Department of Gastroenterology, Zhongnan Hospital of Wuhan University, from August 2014 to July 2016, and all the patients provided written informed consent before the study protocol, with Ethical Approval provided by the Ethics Review Board of Zhongnan Hospital of Wuhan University. This study was carried out in accordance with declaration of Helsinki.Two patient tissue cohorts were used in this study. One cohort consisted of 20 paired cancer tissues and adjacent tissues from patients with PC, which were used for miRNA microarray profiling. The other cohort with 60 paired cancer tissues and adjacent tissues from PC patients was added to the former cohort to further validate the differentially expressed miRNAs in RT-PCR. The demographic characteristics of the two groups of PC patients were described in Table RNA was extracted from tissue samples using TRIzol , and 100ng total RNA of each samples was used for analysis the expression of miRNAs using NanoString nCounter Human miRNA assay (v1) , which contains 654 probes for human miRNAs, according to manufacturer’s instructions. Global miRNA array analysis was performed by importing all data into nSolver Analysis Software v1.0 (Nanostring Technologies) and normalized to the geometric mean of the 100 miRNAs with the highest expression values. All analysis was performed using R/Bioconductor, and filtering of low-intensity signals were then conducted by requiring the mean signal intensity for tissue samples to be larger than 500.3d PCA  and volcano plot were applied to describe the pattern of miRNA expression between pancreatic cancer tissues and adjacent tissues. R package limma was used to compare miRNA expression between pancreatic cancer tissues and adjacent tissues, and the differentially expressed miRNAs were required to meet criteria (absolute (log (fold change)) >1 and adjust p value < 0.05). Human microRNA Disease Database 2 (HMDD2) and Gene Ontology (GO) annotation were used to identify the relationship of the differentially expressed miRNAs with diseases and functions. The enrichment analysis of differentially expressed miRNAs was competed by R, based on the hypergeometric distribution.For analyzing the expressions of candidate miRNAs (miR-200c (Sense: CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCCAAACAC Anti-sense: ACACTCCAGCTGGGCGTCTTACCCAGCAGTGTTTGG), miR-196b (Sense: CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCCCAACAA Anti-sense: ACACTCCAGCTGGGTAGGTAGTTTCCTGTTGTTGGG), miR-1 (Sense: CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGATACATAC Anti-sense: ACACTCCAGCTGGGTGGAATGTAAAGAAGTATGTAT), miR-200a (Sense: CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGACATCGT Anti-sense: ACACTCCAGCTGGGTAACACTGTCTGGTAACGATGT), let-7b (Sense: CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGAACCACA Anti-sense: ACACTCCAGCTGGGTGAGGTAGTAGGTTGTGTGGTT), RNA was reverse transcribed into cDNA using the All-in-One™ miRNA First-Strand cDNA Synthesis Kit (GeneCopoeia), and the miRNA expression was then determined by real-time PCR using the All-in-One™ miRNA qPCR Kit (GeneCopoeia). As for measurement of the expressions of mRNAs (CADM1 (Sense: TGCTGTGCTTGCTCATCATTCT Anti-sense: TCTGCGTCTGCTGCGTCAT), HOXA13 (Sense: CGCTTCAGAACTCGTTGCTTTG Anti-sense: CGGAAGAACTGGCAGTCTTTACCT), IER3 (Sense: CCAGATCCTGATGGCTGAAGAG Anti-sense: TGAGGTCCAGAGCGTAGTCC)-ΔΔCT method with normalization to internal control, U6 (Sense: CTCGCTTCGGCAGCACA Anti-sense: AACGCTTCACGAATTTGCGT) and β-actin (Sense: ATCATGTTTGAGACCTTCAACA Anti-sense: CATCTCTTGGTCGAAGTCCA), respectively.MEF2C (Sense: TCTGTCTGGCTTCAACACTG Anti-sense: TGGTGGTACGGTCTCTAGGA), PTGS2 (Sense: TGACCAGAGCAGGCAGATGA Anti-sense: CCAGTAGGCAGGAGAACATATAACA)), RNA was subjected to reverse transcription with 5 × All-In-One RT MasterMix kit , and quantitative real-time PCR was conducted using a SYBR Green PCR kit (TaKaRa). miRNA and mRNA expression were evaluated by using the 2PANC-1 and ASPC-1 cells were cultured in Dulbecco’s modified Eagle medium containing 10% FBS, 1% penicillin/streptomycin, and 1% glutamine. Here, 5 nmol/L of miR-196b mimic, 50 nmol/l of miR-196b inhibitor and each negative control  were transfected into PANC-1 by using Lipofectamine RNAiMAX (Life Technologies) according to manufacturer’s instructions. The shRNA targeting CADM1 was from position + 1225 to + 1274, relative to the transcription start site, while its negative control did not match any known mammalian genomic sequence.After culture in serum-free medium for 24 hours to induce apoptosis, cells were harvested and stained with FITC annexin V and propidium iodide (PI). Cells fixed by 4% paraformaldehyde for 15 mins were used as positive control for cell apoptosis assay. Subsequently, apoptosis rate was analyzed by cytometry.Cell-light 5-ethynyl-20-deoxyuridine (EdU) Apollo Imaging Kit  was used for measuring the cell proliferation according to manufacturer’s instructions. The percentage of EdU cells was assessed using a fluorescence microscope.Cells were harvested, fixed in 70% ethanol overnight, and incubated with RNaseA, followed by staining with PI in the dark for 30 minutes. Flow cytometry was used to analyze the results.https://bibiserv.cebitec.uni-bielefeld.de/download/tools/rnahybrid.html).The candidate target genes of miR-196b were predicted using TargetScan (http://www.targetscan.org/) and the miRNA-3′UTR binding sites were calculated by RNAhybrid (Cell lysates (20 g) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, and transferred to polyvinylidene fluoride membrane. Subsequently, the membrane was blocked, and incubated with primary and secondary antibodies. Signals were detected using the Amersham ECL Western Blotting detection system. The beta-actin and CADM1 antibodies were purchased from Abcam.3′UTR luciferase reporter assay was conducted to validate the target gene of miR-196b using the wide type and mutant 3′UTR of CADM1. The sequences of Renilla luciferase and firefly luciferase were constructed for reporter fluorescence (Rluc) and calibration fluorescence (Luc), respectively. Cells, miRNA mimic, and vectors were mixed, and cultured for 24 hours, and the luciferase activity was examined by a dual-luciferase reporter assay system.p value < 0.05 (*), p value < 0.01(**).The continuous data following normal distribution (or not) were expressed as mean ± SEM (or medium (IQR)) in this study, while categorical variables were descripted by count (percentage). Student t test and Wilcoxon sum test were used to compare two mean or median of two independent groups, while paired t test and Wilcoxon signed-rank test for paired groups. Besides, the spearman coefficients were used to identify the correlation between miRNAs and characteristics of PC patients. All statistical analysis was conducted using R, and statistical difference was set at Figure S1- Figure S4Table S1Table S2