Discovery of novel pyrazoline derivatives containing methyl-1H-indole moiety as potential inhibitors for blocking APC-Asef interactions.

A series of novel pyrazoline derivatives containing methyl-1H-indole moiety were discovered as potential inhibitors for blocking APC-Asef interactions. The top hit Q19 suggested potency of inhibiting APC-Asef interactions and attractive preference for human-sourced colorectal cells. It was already comparable with the previous representative and the positive control Regorafenib before further pharmacokinetic optimization. The introduction of methyl-1H-indole moiety realized the Mitochondrial affection thus might connect the impact on the protein-interaction level with the apoptosis events. The molecular docking simulation inferred that bringing trifluoromethyl groups seemed a promising approach for causing more key interactions such as H-bonds. This work raised referable information for further discovery of inhibitors for blocking APC-Asef interactions.

Transcriptional reference map of hormone responses in wheat spikes.

BACKGROUND: Phytohormones are key regulators of plant growth, development, and signalling networks involved in responses to diverse biotic and abiotic stresses. Transcriptional reference maps of hormone responses have been reported for several model plant species such as Arabidopsis thaliana, Oryza sativa, and Brachypodium distachyon. However, because of species differences and the complexity of the wheat genome, these transcriptome data are not appropriate reference material for wheat studies. RESULTS: We comprehensively analysed the transcriptomic responses in wheat spikes to seven phytohormones, including indole acetic acid (IAA), gibberellic acid (GA), abscisic acid (ABA), ethylene (ET), cytokinin (CK), salicylic acid (SA), and methyl jasmonic acid (MeJA). A total of 3386 genes were differentially expressed at 24 h after the hormone treatments. Furthermore, 22.7% of these genes exhibited overlapping transcriptional responses for at least two hormones, implying there is crosstalk among phytohormones. We subsequently identified genes with expression levels that were significantly and differentially induced by a specific phytohormone (i.e., hormone-specific responses). The data for these hormone-responsive genes were then compared with the transcriptome data for wheat spikes exposed to biotic (Fusarium head blight) and abiotic (water deficit) stresses. CONCLUSION: Our data were used to develop a transcriptional reference map of hormone responses in wheat spikes.

Re-acquisition of the brittle rachis trait via a transposon insertion in domestication gene Q during wheat de-domestication.

De-domestication is a unique evolutionary process during which crops re-acquire wild-like traits to survive and persist in agricultural fields without the need for human cultivation. The re-acquisition of seed dispersal mechanisms is crucial for crop de-domestication. Common wheat is an important cereal crop worldwide. Tibetan semi-wild wheat is a potential de-domesticated common wheat subspecies. However, the crucial genes responsible for its brittle rachis trait have not been identified. Genetic mapping, functional analyses and phylogenetic analyses were completed to identify the gene associated with Qbr.sau-5A, which is a major locus for the brittle rachis trait of Tibetan semi-wild wheat. The cloned Qbr.sau-5A gene is a new Q allele (Qt ) with a 161-bp transposon insertion in exon 5. Although Qt is expressed normally, its encoded peptide lacks some key features of the APETALA2 family. The abnormal functions of Qt in developing wheat spikes result in brittle rachises. Phylogenetic and genotyping analyses confirmed that Qt originated from Q in common wheat and is naturally distributed only in Tibetan semi-wild wheat populations. The identification of Qt provides new evidence regarding the origin of Tibetan semi-wild wheat, and new insights into the re-acquisition of wild traits during crop de-domestication.

Dihydropyrazole Derivatives Containing Benzo Oxygen Heterocycle and Sulfonamide Moieties Selectively and Potently Inhibit COX-2: Design, Synthesis, and Anti-Colon Cancer Activity Evaluation.

Cyclooxygenase-2 (COX-2) as a rate-limiting metabolism enzyme of arachidonic acid has been found to be implicated in tumor occurrence, angiogenesis, metastasis as well as apoptosis inhibition, regarded as an attractive therapeutic target for cancer therapy. In our research, a series of dihydropyrazole derivatives containing benzo oxygen heterocycle and sulfonamide moieties were designed as highly potent and selective COX-2 inhibitors by computer-aided drug analysis of known COX-2 inhibitors. A total of 26 compounds were synthesized and evaluated COX-2 inhibition and pharmacological efficiency both in vitro and in vivo with multi-angle of view. Among them, compound 4b exhibited most excellent anti-proliferation activities against SW620 cells with IC50 of 0.86 ± 0.02 µM than Celecoxib (IC50 = 1.29 ± 0.04 µM). The results favored our rational design intention and provides compound 4b as an effective COX-2 inhibitor available for the development of colon tumor therapeutics.

Alternative splicing results in a lack of starch synthase IIa-D in Chinese wheat landrace.

We evaluated the SGP-1 protein composition of 368 Chinese wheat landraces using SDS-PAGE. The SGP-D1 null type was identified in three accessions (Xiaoqingmang, Pushanbamai, and P119). An 18-bp deletion and 9-bp variation were found at the junction region of the 7th intron and 8th exon, leading to deletion of the intron-exon junction recognition site AG when aligned the 8261-bp DNA sequence of TaSSIIa-D in Pushanbamai with that of Chinese Spring. Four cDNA types with mis-spliced isoforms were subsequently detected through amplification of TaSSIIa-D cDNAs. Among these, nine type II cDNAs with a 16-bp deletion in the 8th exon were detected, indicating that the major transcriptional pattern of TaSSIIa in Pushanbamai is type II. In the type IV cDNA, a 97-bp sequence remains undeleted in the end of the 5th exon. The amylose content in Pushanbamai was significantly higher than that in all control lines under field conditions, which suggested that deletion of SGP-D1 has an efficient impact on amylose content. As the TaSSIIa gene plays an important role in regulating the content of amylose, it is anticipated that these natural variants of TaSSIIa-D will provide useful resources for quality improvement in wheat.

Fusarium graminearum ATP-Binding Cassette Transporter Gene FgABCC9 Is Required for Its Transportation of Salicylic Acid, Fungicide Resistance, Mycelial Growth and Pathogenicity towards Wheat.

ATP-binding cassette (ABC) transporters hydrolyze ATP to transport a wide range of substrates. Fusarium graminearum is a major causal agent of Fusarium head blight, which is a severe disease in wheat worldwide. FgABCC9 (FG05_07325) encodes an ABC-C (ABC transporter family C) transporter in F. graminearum, which was highly expressed during the infection in wheat and was up-regulated by the plant defense hormone salicylic acid (SA) and the fungicide tebuconazole. The predicted tertiary structure of the FgABCC9 protein was consistent with the schematic of the ABC exporter. Deletion of FgABCC9 resulted in decreased mycelial growth, increased sensitivity to SA and tebuconazole, reduced accumulation of deoxynivalenol (DON), and less pathogenicity towards wheat. Re-introduction of a functional FgABCC9 gene into ΔFgABCC9 recovered the phenotypes of the wild type strain. Transgenic expression of FgABCC9 in Arabidopsis thaliana increased the accumulation of SA in its leaves without activating SA signaling, which suggests that FgABCC9 functions as an SA exporter. Taken together, FgABCC9 encodes an ABC exporter, which is critical for fungal exportation of SA, response to tebuconazole, mycelial growth, and pathogenicity towards wheat.

Transposon insertion resulted in the silencing of Wx-B1n in Chinese wheat landraces.

KEY MESSAGE: A novel Wx-B1 allele was characterized; a transposon insertion resulted in the loss of its function, which is different from the previously reported gene silencing mechanisms at the Wx-B1 locus. The waxy protein composition of 53 Chinese wheat landraces was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional gel electrophoresis; of these, 10 did not show the expression of Wx-A1 (four accession) or Wx-B1 (six accessions) protein. The results of molecular marker detection revealed that the Wx-B1 allele (Wx-B1n) showed normal expression, inconsistent with the findings of SDS-PAGE for the Xiaobaipi accession. Further cloning of the 9160-bp region covering the Wx-B1 coding region and 3'-downstream region revealed that a 2178-bp transposon fragment had been inserted at 2462 bp within the tenth exon of Wx-B1n ORF, leading to the absence of Wx-B1 protein. Sequence analysis indicated that the insertion possessed the structural features of invert repeat and target repeat elements, we deduced that it was a transposon. Further PCR analysis revealed that this fragment had moved, but not copied itself, from 3B chromosome to the current location in Wx-B1n. Therefore, the reason for the inactivation of Wx-B1n was considerably different from those for the inactivation of Wx-B1b, Wx-B1k, and Wx-B1m; to our knowledge, this kind of structural mutation has never been reported in Wx-B1 alleles. This novel allele is interesting, because it was not associated with the deletion of other quality-related genes included in the 67 kb region lost with the common null allele Wx-B1b. The null Wx-B1n might be useful for investigating gene inactivation and expression as well as for enriching the genetic resource pool for the modification of the amylose/amylopectin ratio, thereby improving wheat quality.

A super twin T-DNA vector that allows independent gene expression during Agrobacterium-mediated transformation.

In this study, we designed and constructed a super twin T-DNA vector (pTRIDT313-g) containing two independent T-DNA cassettes-one for the selection gene Hyg and the other for the target gene Gus-to produce marker-free transgenic lines. The resulting vector was transformed into tobacco, and polymerase chain reaction (PCR) analysis showed four types of gene combinations in the T1 and T2 generations: Gus only, Hyg only, Gus+Hyg, and untransformed lines. The intermediate region from the T-DNA of the right border of Hyg to the left border of Gus in the Hyg and Gus lines was not amplified. Genome walking confirmed that the Hyg and Gus T-DNA cassettes were independently inserted in different regions of the tobacco genome. Thus, the two T-DNA cassettes were integrated randomly as independent loci into the tobacco genome. The results of reverse transcription-PCR indicated that Hyg could normally be expressed in the roots, stems, and leaves of transgenic lines, and the resistance test showed that all Hyg transgenic lines could grow in the presence of 50mg/L hygromycin. All Gus transgenic lines showed obvious blue coloration in enzyme activity tests, indicating that the Gus gene could be normally expressed in all the lines. Therefore, the super twin T-DNA vector (pTRIDT313-g) exhibits independent integration, heredity, and normal gene function from two T-DNA cassettes. This vector could be a useful and valuable tool in the production of marker-free transgenic lines.

Structure and expression of phosphoglucan phosphatase genes of Like Sex Four1 and Like Sex Four2 in barley.

Phosphoglucan phosphatases (Like-SEX4 1 and 2; LSF1 and LSF2) were reported to play roles in starch metabolism in leaves of Arabidopsis. In this study, we identified and mapped the LSF1 and LSF2 genes in barley (HvLSF1 and HvLSF2), characterized their gene and protein structures, predicted the cis-elements of their promoters, and analysed their expression patterns. HvLSF1 and HvLSF2 were mapped on the long arm of chromosome 1H (1HL) and 5H (5HL), respectively. Our results revealed varied exon-intron structures and conserved exon-intron junctions in both LSF1 and LSF2 from a range of analysed species. Alignment of protein sequences indicated that cTP and CT domains are much less varied than the functional domains (PDZ, DPS and CBM48). LSF2 was mainly expressed in anthers of barley and rice, and in leaf of Arabidopsis. LSF1 was mainly expressed in endosperm of barley and leaf of Arabidopsis and rice. The expression of LSF1 exhibited a diurnal pattern in rice only and that of LSF2 in both rice and Arabidopsis. Of the investigated stresses, only cold stress significantly reduced expression level of LSF1 and LSF2 in barley and LSF2 in Arabidopsis at late stages of the treatments. While heat treatment significantly decreased expression levels of LSF1 at middle stage (4 h) of a treatment in Arabidopsis only. The strong relationships detected between LSF2 and starch excess4 (SEX4), glucan, water dikinases or phosphoglucan, water dikinases were identified and discussed. Taken together, these results provide information of genetic manipulation of LSF1 and LSF2, especially in monocotyledon and further elucidate their regulatory mechanism in plant development.

Structure and expression analysis of genes encoding ADP-glucose pyrophosphorylase large subunit in wheat and its relatives.

ADP-glucose pyrophosphorylase (AGP), which consists of two large subunits (AGP-L) and two small subunits (AGP-S), controls the rate-limiting step in the starch biosynthetic pathway. In this study, a full-length open reading frame (ORF) of AGP-L gene (named as Agp2) in wheat and a series of Agp2 gene sequences in wheat relatives were isolated. The coding region of Agp2 contained 15 exons and 14 introns including a full-length ORF of 1566 nucleotides, and the deduced protein contained 522 amino acids (57.8 kDa). Generally, the phylogenetic tree of Agp2 indicated that sequences from A- and D-genome donor species were most similar to each other and sequences from B-genome donor species contained more variation. Starch accumulation and Agp2 expression in wheat grains reached their peak at 21 and 15 days post anthesis (DPA), respectively.

Characterization and expression analysis of WOX2 homeodomain transcription factor in Aegilops tauschii.

The WUSCHEL (WUS)-related homeobox (WOX) gene family coordinates transcription during the early phases of embryogenesis. In this study, a putative WOX2 homolog was isolated and characterized from Aegilops tauschii, the donor of D genome of Triticum aestivum. The sequence consisted of 2045 bp, and contained an open reading frame (ORF), encoded 322 amino acids. The predicted protein sequence contained a highly conserved homeodomain and the WUS-box domain, which is present in some members of the WOX protein family. The full-length ORF was subcloned into prokaryotic expression vector pET-30a, and an approximately 34-kDa protein was expressed in Escherichia coli BL21 (DE3) cells with IPTG induction. The molecular mass of the expressed protein was identical to that predicted by the cDNA sequence. Phylogenetic analysis suggested that Ae. tauschii WOX2 is closely related to the rice and maize orthologs. Quantitative PCR analysis showed that WOX2 from Ae. tauschii was primarily expressed in the seeds; transcription increased during seed development and declined after the embryos matured, suggesting that WOX2 is associated with embryo development in Ae. tauschii.

Characterization of high-molecular-weight glutenin subunits from Eremopyrum bonaepartis and identification of a novel variant with unusual high molecular weight and altered cysteine residues.

We characterized two high-molecular-weight glutenin subunit (HMW-GS) variants from Eremopyrum bonaepartis, determined their complete open reading frames, and further expressed them in a bacterial system. The variants have many novel structural features compared with typical subunits encoded by Glu-1 loci: 1Fx3.7 and 1Fy1.5 exhibit hybrid properties of x- and y-type subunits. In addition, unusual molecular mass and altered number and distribution of cysteine residues were unique features of HMW-GSs encoded by Glu-F1 from E. bonaepartis. The mature 1Fx3.7 subunit has a full length of 1,223 amino acid residues, making it the largest subunit found thus far, while 1Fy1.5 is just 496 residues. In addition, the mutated PGQQ repeat motif was found in the repetitive region of 1Fx3.7. Although it has a similar molecular mass to that previously reported for 1Dx2.2, 1Dx2.2* and 1S(sh)x2.9 subunits, 1Fx3.7 appears to have had a different evolutionary history. The N-terminal and repetitive regions have a total of four additional cysteine residues, giving 1Fx3.7 a total of eight cysteines, while 1Fy1.5 has only six cysteines because the GHCPTSPQQ nonapeptide at the end of the repetitive region is deleted. With its extra cysteine residues and the longest repetitive region, features that are relevant to good wheat quality, the 1Fx3.7 subunit gene could be an excellent candidate for applications in wheat quality improvement.

The molecular diversity of α-gliadin genes in the tribe Triticeae.

Many of the unique properties of wheat flour are derived from seed storage proteins such as the α-gliadins. In this study these α-gliadin genes from diploid Triticeae species were systemically characterized, and divided into 3 classes according to the distinct organization of their protein domains. Our analyses indicated that these α-gliadins varied in the number of cysteine residues they contained. Most of the α-gliadin genes were grouped according to their genomic origins within the phylogenetic tree. As expected, sequence alignments suggested that the repetitive domain and the two polyglutamine regions were responsible for length variations of α-gliadins as were the insertion/deletion of structural domains within the three different classes (I, II, and III) of α-gliadins. A screening of celiac disease toxic epitopes indicated that the α-gliadins of the class II, derived from the Ns genome, contain no epitope, and that some other genomes contain much fewer epitopes than the A, S(B) and D genomes of wheat. Our results suggest that the observed genetic differences in α-gliadins of Triticeae might indicate their use as a fertile ground for the breeding of less CD-toxic wheat varieties.

[Imaging findings of non-contrast CT scan, enhancement and HRCT of pulmonary mucosa-associated lymphoid tissue lymphoma].

OBJECTIVE: To explore the imaging findings of non-contrast CT scan, enhancement and HRCT of pulmonary mucosa-associated lymphoid tissue (MALT) lymphoma. METHODS: All the patients with pathologically proven pulmonary MALT lymphoma underwent non-contrast CT scan, of which 7 underwent CT enhancement and 9 had HRCT. CT features of 16 patients were retrospectively reviewed. RESULTS: Major CT signs of 16 cases were consolidations, nodules and masses, of which 11 had two or more signs and 5 had only one. Together with them, normal or dilated air bronchograms were seen in 13 cases, ground-glass opacity (GGO) in 10, interstitial changes and cysts in respective 5, partial atelectasis and mediastinal and hilar lymphoadenopathy in respective 4, pleural effusion in 2 and MALT lymphoma of tracheobronchial tree in 1. Multiple lesions were found in 12 cases and solitary lesion in 4 cases. Consolidations, greater tubercles and masses mostly had a peribronchovascular distribution. Micronodules occurred in a perilymphatic distribution, of which 8 accompanied other signs.On CT enhancement scans, greater tubercles in the trachea had mild enhancement; most of the consolidations and masses had obvious enhancement, while greater tubercles mostly mild enhancement. CT angiogram sign was found in 3 cases. CONCLUSION: Most cases with pulmonary MALT lymphoma have consolidations, greater tubercles and masses with normal or dilated air bronchograms which have a peribronchovascular distribution, together with GGO and micronodules in a perilymphatic distribution; the distinctive manifestations are obvious enhancement with CT angiogram sign. Minority cases have interstitial changes, cysts, partial atelectasis, mediastinal and hilar lymphoadenopathy, pleural effusion and MALT lymphoma of tracheobronchial tree.

Identification of a recombinant equine coronavirus in donkey, China.

Equine coronavirus (ECoV) was first identified in the USA and has been previously described in several countries. In order to test the presence of ECoV in China, we collected 51 small intestinal samples from donkey foals with diarrhoea from a donkey farm in Shandong Province, China between August 2020 and April 2021. Two samples tested positive for ECoV and full-length genome sequences were successfully obtained using next-generation sequencing, one of which was further confirmed by Sanger sequencing. The two strains shared 100% sequence identity at the scale of whole genome. Bioinformatics analyses further showed that the two Chinese strains represent a novel genetic variant of ECoV and shared the highest sequence identity of 97.05% with the first identified ECoV strain - NC99. In addition, it may be a recombinant, with the recombination region around the NS2 gene. To our knowledge, this is the first documented report of ECoV in China, highlighting its risk to horse/donkey breeding. In addition, its potential risk to public health also warrants further investigation.

Identification of novel α-gliadin genes.

Ten novel α-gliadin genes (Gli-ta, Gli-turg1, Gli-turg2, Gli-turg3, Gli-turg4, Gli-turg5, Gli-turg6, Gli-cs1, Gli-cs2, and Gli-cs3) with unique characteristics were isolated from wheat (Triticum aestivumL.), among which Gli-cs1, Gli-cs2, Gli-cs3, and Gli-turg6 were pseudogenes. Gli-cs3 and nine other sequences were much larger and smaller, respectively, than the typical α-gliadins. This variation was caused by insertion or deletion of the unique domain I and a polyglutamine region, possibly the result of illegitimate recombination. Consequently, Gli-cs3 contained 10 cysteine residues, whereas there were 2 cysteine residues only in the other nine sequences. Gli-ta/Gli-ta-like α-gliadin genes are normally expressed during the development of seeds. SDS-PAGE analysis showed that in-vitro-expressed Gli-ta could form intermolecular disulphide bonds and could be chain extenders. A protein band similar in size to Gli-ta has been observed in seed extracts, and mass spectrometry results confirm that the band contains small molecular mass α-gliadins, which is a characteristic of the novel α-gliadins. Mass spectrometry results also indicated that the two cysteine residues of Gli-ta/Gli-ta-like proteins participated in the formation of intermolecular disulphide bonds in vivo.

The gamma-gliadin multigene family in common wheat (Triticum aestivum) and its closely related species.

BACKGROUND: The unique properties of wheat flour primarily depend on gluten, which is the most important source of protein for human being. gamma-Gliadins have been considered to be the most ancient of the wheat gluten family. The complex family structure of gamma-gliadins complicates the determination of their function. Moreover, gamma-gliadins contain several sets of celiac disease epitopes. However, no systematic research has been conducted yet. RESULTS: A total of 170 gamma-gliadin genes were isolated from common wheat and its closely related species, among which 138 sequences are putatively functional. The ORF lengths of these sequences range from 678 to 1089 bp, and the repetitive region is mainly responsible for the size heterogeneity of gamma-gliadins. The repeat motif P(Q/L/S/T/I/V/R/A)F(S/Y/V/Q/I/C/L)P(R/L/S/T/H/C/Y)Q1-2(P(S/L/T/A/F/H)QQ)1-2is repeated from 7 to 22 times. Sequence polymorphism and linkage disequilibrium analyses show that gamma-gliadins are highly diverse. Phylogenic analyses indicate that there is no obvious discrimination between Sitopsis and Ae. tauschii at the Gli-1 loci, compared with diploid wheat. According to the number and placement of cysteine residues, we defined nine cysteine patterns and 17 subgroups. Alternatively, we classified gamma-gliadins into two types based on the length of repetitive domain. Amino acid composition analyses indicate that there is a wide range of essential amino acids in gamma-gliadins, and those gamma-gliadins from subgroup SG-10 and SG-12 and gamma-gliadins with a short repetitive domain are more nutritional. A screening of toxic epitopes shows that gamma-gliadins with a pattern of C9 and gamma-gliadins with a short repetitive domain almost lack any epitopes. CONCLUSION: gamma-Gliadin sequences in wheat and closely related Aegilops species are diverse. Each group/subgroup contributes differently to nutritional quality and epitope content. It is suggested that the genes with a short repetitive domain are more nutritional and valuable. Therefore, it is possible to breed wheat varieties, the gamma-gliadins of which are less, even non-toxic and more nutritional.

Molecular characterization of the pina gene in einkorn wheat.

Fifty-six sequences encoding the pina protein were characterized from three species or subspecies of einkorn wheat. These sequences contained 1,595 nucleotides, including 1,270 conserved sites, 21 single nucleotide polymorphisms (SNPs), and 16 indels. The average frequency of SNPs and indels was one out of 76.1 and 99.9 bases, respectively. Five SNPs and no indels were found in the translated sequences. Fourteen haplotypes were defined, and the accessions in each haplotype ranged from 1 to 18. There were nine haplotypes in Triticum monococcum ssp. aegilopoides, eight in T. monococcum ssp. monococcum, and two in T. urartu. Phylogenetic analysis showed that pina genes from different species or subspecies could be clearly differentiated based on the open reading frame. Genes from T. urartu grouped together, whereas genes from T. monococcum ssp. aegilopoides and T. monococcum ssp. monococcum were shared by three and two clusters, respectively. Both the haplotype and phylogenetic analyses indicated that T. monococcum ssp. aegilopoides was more diverse. These results would contribute to the understanding of functional aspects and efficient utilization of pina genes.

Design, synthesis and biological evaluation of 2-H pyrazole derivatives containing morpholine moieties as highly potent small molecule inhibitors of APC-Asef interaction.

Mutated adenomatous polyposis coli (APC) selectively combining with Asef has been reported to be implicated in promoting colon cancer proliferation, invasion and metastasis in several cancer biotherapy studies. However, there were universally resistance and harsh terms in disrupting APC-Asef interaction in biotherapy. Under the circumstances small-molecule inhibitors as the new APC interface could resolve the problems. In this research, a series of novel dihydropyrazole derivatives containing morpholine as high potent interaction inhibitors between APC and Asef were first synthesized after selection by means of docking simulation and virtual screening. Afterwards they were evaluated interaction inhibition of APC-Asef and pharmacological efficiency both in vitro and in vivo utilizing orthotopic transplantation model with multi-angle of view. Among them, compound 7g exhibited most excellent anti-proliferation activities against HCT116 cells with IC50 of 0.10 ±â€¯0.01 µM than Regorafenib (IC50 = 0.16 ±â€¯0.04 µM). The results favored our rational design intention and provides a new class of small-molecule inhibitors available for the development of colon tumor therapeutics targeting APC-Asef interaction inhibitions.