Risk of advanced neoplasia after removal of colorectal adenomas with high-grade dysplasia.

BACKGROUND: Many studies reported the presence of adenomas with high-grade dysplasia (HGD) at index colonoscopy increased the incidence of advanced neoplasia (AN) and colorectal cancer (CRC) following. However, the conclusion remains obscure due to lack of studies on the specific population of adenomas with HGD. This study aimed to assess the long-term risk of AN and CRC after removal of adenomas with HGD. METHODS: A total of 814 patients who underwent adenomas with HGD removal between 2010 and 2019 were retrospectively analyzed. The outcomes were the incidences of AN and CRC during surveillance colonoscopy. Cox proportional hazards models were utilized to identify risk factors associated with AN and CRC. RESULTS: During more than 2000 person-years of follow-up, we found that AN and CRC incidence densities were 44.3 and 4.4 per 1000 person-years, respectively. The 10-year cumulative incidence of AN and CRC were 39.1% and 5.5%, respectively. In the multivariate model, synchronous low-risk polyps (HR 1.80, 95% CI 1.10-2.93) and synchronous high-risk polyps (HR 3.99, 95% CI 2.37-6.72) were risk factors for AN, whereas participation in surveillance colonoscopy visits (HR 0.56, 95% CI 0.36-0.88 for 1 visit; HR 0.10, 95% CI 0.06-0.19 for ≥ 2 visits) were associated with decreased AN incidence. Additionally, elevated baseline carcinoembryonic antigen (CEA) level (HR 10.19, 95% CI 1.77-58.59) was a risk factor for CRC, while participation in ≥ 2 surveillance colonoscopy visits (HR 0.11, 95% CI 0.02-0.56) were associated with decreased CRC incidence. Interestingly, for 11 patients who developed CRC after removal of adenomas with HGD, immunohistochemistry revealed that 8 cases (73%) were deficient mismatch repair CRCs. CONCLUSIONS: Patients who have undergone adenoma with HGD removal are at higher risk of developing AN and CRC, while surveillance colonoscopy can reduce the risk. Patients with synchronous polyps, or with elevated baseline CEA level are considered high-risk populations and require more frequent surveillance.

Investigation of salt tolerance in cotton germplasm by analyzing agro-physiological traits and ERF genes expression.

The development of genotypes that can tolerate high levels of salt is crucial for the efficient use of salt-affected land and for enhancing crop productivity worldwide. Therefore, incorporating salinity tolerance is a critical trait that crops must possess. Salt resistance is a complex character, controlled by multiple genes both physiologically and genetically. To examine the genetic foundation of salt tolerance, we assessed 16 F1 hybrids and their eight parental lines under normal and salt stress (15 dS/m) conditions. Under salt stress conditions significant reduction was observed for plant height (PH), bolls/plant (NBP), boll weight (BW), seed cotton yield (SCY), lint% (LP), fiber length (FL), fiber strength (FS), potassium to sodium ratio (K+/Na+), potassium contents (K+), total soluble proteins (TSP), carotenoids (Car) and chlorophyll contents. Furthermore, the mean values for hydrogen peroxide (H2O2), sodium contents (Na+), catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), and fiber fineness (FF) were increased under salt stress. Moderate to high heritability and genetic advancement was observed for NBP, BW, LP, SCY, K+/Na+, SOD, CAT, POD, Car, TSP, FL, and FS. Mean performance and multivariate analysis of 24 cotton genotypes based on various agro-physiological and biochemical parameters suggested that the genotypes FBS-Falcon, Barani-333, JSQ-White Hold, Ghauri, along with crosses FBS-FALCON × JSQ-White Hold, FBG-222 × FBG-333, FBG-222 × Barani-222, and Barani-333 × FBG-333 achieved the maximum values for K+/Na+, K+, TSP, POD, Chlb, CAT, Car, LP, FS, FL, PH, NBP, BW, and SCY under salt stress and declared as salt resistant genotypes. The above-mentioned genotypes also showed relatively higher expression levels of Ghi-ERF-2D.6 and Ghi-ERF-7A.6 at 15 dS/m and proved the role of these ERF genes in salt tolerance in cotton. These findings suggest that these genotypes have the potential for the development of salt-tolerant cotton varieties with desirable fiber quality traits.

Gossypium hirsutum calmodulin-like protein (CML 11) interaction with geminivirus encoded protein using bioinformatics and molecular techniques.

Plant viruses are the most abundant destructive agents that exist in every ecosystem, causing severe diseases in multiple crops worldwide. Currently, a major gap is present in computational biology determining plant viruses interaction with its host. We lay out a strategy to extract virus-host protein interactions using various protein binding and interface methods for Geminiviridae, a second largest virus family. Using this approach, transcriptional activator protein (TrAP/C2) encoded by Cotton leaf curl Kokhran virus (CLCuKoV) and Cotton leaf curl Multan virus (CLCuMV) showed strong binding affinity with calmodulin-like (CML) protein of Gossypium hirsutum (Gh-CML11). Higher negative value for the change in Gibbs free energy between TrAP and Gh-CML11 indicated strong binding affinity. Consensus from gene ontology database and in-silico nuclear localization signal (NLS) tools identified subcellular localization of TrAP in the nucleus associated with Gh-CML11 for virus infection. Data based on interaction prediction and docking methods present evidences that full length and truncated C2 strongly binds with Gh-CML11. This computational data was further validated with molecular results collected from yeast two-hybrid, bimolecular fluorescence complementation system and pull down assay. In this work, we also show the outcomes of full length and truncated TrAP on plant machinery. This is a first extensive report to delineate a role of CML protein from cotton with begomoviruses encoded transcription activator protein.

Discovery of Kinetin in inhibiting colorectal cancer progression via enhancing PSMB1-mediated RAB34 degradation.

Colorectal cancer (CRC) is one of the most prevalent malignancies worldwide. Understanding the underlying mechanism driving CRC progression and identifying potential therapeutic drug targets are of utmost urgency. We previously utilized LC-MS-based proteomic profiling to identify proteins associated with postoperative progression in stage II/III CRC. Here, we revealed that proteasome subunit beta type-1 (PSMB1) is an independent predictor for postoperative progression in stage II/III CRC. Mechanistically, PSMB1 binds directly to onco-protein RAB34 and promotes its proteasome-dependent degradation, potentially leading to the inactivation of the MEK/ERK signaling pathway and inhibition of CRC progression. To further identify potential anticancer drugs, we screened a library of 2509 FDA-approved drugs using computer-aided drug design (CADD) and identified Kinetin as a potentiating agent for PSMB1. Functional assays confirmed that Kinetin enhanced the interaction between PSMB1 and RAB34, hence facilitated the degradation of RAB34 protein and decreased the MEK/ERK phosphorylation. Kinetin suppresses CRC progression in patient-derived xenograft (PDX) and liver metastasis models. Conclusively, our study identifies PSMB1 as a potential biomarker and therapeutic target for CRC, and Kinetin as an anticancer drug by enhancing proteasome-dependent onco-protein degradation.

[Preparation of porous organic cage and its use as chiral stationary phase for capillary electrochromatography].

Porous organic cages (POCs) are a unique type of microporous materials composed of discrete molecules with internal cavities that are accessible to various compounds. In this study, a prismatic chiral POC with good thermochemical stability was synthesized by condensing (1R,2R)-diaminocyclohexane and 3,3',5,5'-teturonic-4,4'-biphenediol via the Schiff base reaction and characterized by proton nuclear magnetic resonance spectroscopy, infrared (IR) spectroscopy, thermogravimetric analysis (TGA), and scanning electron microscopy. The IR spectrum of the POC revealed a strong characteristic absorption peak at 1635 cm-1, indicating that it formed imine bonds (C=N). The absorption peak at 3425 cm-1 was attributed to the stretching vibrations of -OH, the absorption peaks at 2925 and 2858 cm-1 were attributed to the stretching vibrations of N=C-H and C-H, and the absorption peaks at 1446 and 1383 cm-1 were attributed to the stretching vibrations of C=C-H and C=C in the benzene ring. High-resolution mass spectral analysis of the POC showed a molecular ion peak at m/z 1363.7228, indicating its successful synthesis. TGA was performed from 25 to 800 ℃ at a rate of 10 ℃/min, and the results of this analysis showed that the POC was stable up to approximately 300 ℃. The POC was dissolved in dichloromethane and uniformly coated on the inner wall of a quartz capillary via the dynamic coating method to prepare a capillary electrochromatographic column. The experimental results revealed that the chiral electrochromatographic column could not only resolve ofloxacin, Troger's base, 2-amino-1-butanol, and 1-phenyl-1-amyl alcohol but also separate the isomers of o-, m-, and p-toluidine and o-, m-, and p-chloroaniline, indicating its good chiral separation ability. Investigation of the optimal separation conditions for ofloxacin, Troger's base, 2-amino-1-butanol, and 1-phenyl-1-amyl alcohol revealed that the voltage, buffer solution concentration, and pH significantly affected their separation degree. In particular, the optimal separation voltage for ofloxacin, Troger's base, and 2-amino-1-butanol was 15 kV, while that for 1-phenyl-1-amyl alcohol was 17 kV. The optimal buffer concentration and pH for ofloxacin, Troger's base, 2-amino-1-butanol, and 1-phenyl-1-amyl alcohol were 0.100 mol/L and 7.5. Under optimal chromatographic conditions, the resolution values for ofloxacin, Troger's base, 2-amino-1-butanol, and 1-phenyl-1-pentanol were 1.80, 3.33, 1.69, and 1.18, respectively. The results collectively demonstrate that the prepared POC may serve as a good chiral stationary phase for capillary electrochromatography with a certain chiral resolution ability and has good application prospects in chromatographic separation.

Aquaporin 9 is involved in CRC metastasis through DVL2-dependent Wnt/ß-catenin signaling activation.

Background: Aquaporin 9 (AQP9) is permeable to water or other small molecules, and plays an important role in various cancers. We previously found that AQP9 was related to the efficacy of chemotherapy in patients with colorectal cancer (CRC). This study aimed to identify the role and regulatory mechanism of AQP9 in CRC metastasis. Methods: The clinical significance of AQP9 was analysed by using bioinformatics and tissue microarray. Transcriptome sequencing, Dual-Luciferase Reporter Assay, Biacore, and co-immunoprecipitation were employed to demonstrate the regulatory mechanism of AQP9 in CRC. The relationship between AQP9 and CRC metastasis was verified in vitro and in vivo by using real-time cell analysis assay, high content screening, and liver metastasis models of nude mice. Results: We found that AQP9 was highly expressed in metastatic CRC. AQP9 overexpression reduced cell roundness and enhanced cell motility in CRC. We further showed that AQP9 interacted with Dishevelled 2 (DVL2) via the C-terminal SVIM motif, resulting in DVL2 stabilization and the Wnt/ß-catenin pathway activation. Additionally, we identified the E3 ligase neural precursor cell expressed developmentally downregulated 4-like (NEDD4L) as a modulator regulating the ubiquitination and degradation of AQP9. Conclusions: Collectively, our study revealed the important role of AQP9 in regulating DVL2 stabilization and Wnt/ß-catenin signaling to promote CRC metastasis. Targeting the NEDD4L-AQP9-DVL2 axis might have therapeutic usefulness in metastatic CRC treatment.

Elucidation of the 1-phenethylisoquinoline pathway from an endemic conifer Cephalotaxus hainanensis.

Cephalotaxines harbor great medical potential, but their natural source, the endemic conifer Cephalotaxus is highly endangered, creating a conflict between biotechnological valorization and preservation of biodiversity. Here, we construct the whole biosynthetic pathway to the 1-phenethylisoquinoline scaffold, as first committed compound for phenylethylisoquinoline alkaloids (PIAs), combining metabolic modeling, and transcriptome mining of Cephalotaxus hainanensis to infer the biosynthesis for PIA precursor. We identify a novel protein, ChPSS, driving the Pictet-Spengler condensation and show that this enzyme represents the branching point where PIA biosynthesis diverges from the concurrent benzylisoquinoline-alkaloids pathway. We also pinpoint ChDBR as crucial step to form 4-hydroxydihydrocinnamaldehyde diverging from lignin biosynthesis. The elucidation of the early PIA pathway represents an important step toward microbe-based production of these pharmaceutically important alkaloids resolving the conflict between biotechnology and preservation of biodiversity.

Microstructural and Thermo-Mechanical Characterization of Furfurylated Douglas Fir.

Fast-growing wood has become a major source of materials for the wood industry in recent years, but defects have limited its use. Therefore, modification is urgently needed for the more efficient application of wood products. In this study, a 30 to 50% solution of furfuryl alcohol (FA) was impregnated into Douglas fir sapwood. The microstructure and thermal properties of the specimens before and after furfurylation were evaluated by different techniques. The weight percentage gain (WPG) of modified wood increased up to 22.97%, with the polymerized FA distributed in cell lumens and cell walls, as well as chemically bound to wood components. The polyfurfuryl alcohol (PFA) was mainly located in the tracheids, ray parenchyma cells, and resin canals. In addition, the furfurylated cell walls were greatly thickened. Raman spectra showed that modified wood had significant background fluorescence that covered other peaks. Differential Scanning Calorimetry analysis revealed that the cross-linking reaction between FA and wood changed the shape of curves, with no endothermic or exothermic peaks within the programmed temperature. Moreover, Thermogravimetry and Dynamic Mechanical Analysis results both confirmed that the furfurylation increased the thermal stability of Douglas fir. The percentage of the final mass loss of untreated specimen was 80.11%, while the highest one of furfurylated specimen was 78.15%, and it gradually decreased with increasing FA concentration. The storage modulus (E') and loss modulus (E″) of the furfurylated wood were both lower, and the damping factor (tan δ) was higher than the untreated one. When the temperature reaches about 75 °C, the untreated specimen began to soften and deform. At 90 °C, it fractured completely while the furfurylatedone remained stable. This study demonstrated that furfurylation can improve wood properties and elongate its service life.

Mapping of a novel clubroot disease resistance locus in Brassica napus and related functional identification.

Clubroot disease, caused by Plasmodiophora brassicae, is a devastating disease that results in substantial yield loss in Brassicaceae crops worldwide. In this study, we identified a clubroot disease resistance (CR) Brassica napus, "Kc84R," which was obtained by mutation breeding. Genetic analysis revealed that the CR trait of "Kc84R" was controlled by a single dominant locus. We used the bulked segregant analysis sequencing (BSA-seq) approach, combined with genetic mapping based on single nucleotide polymorphism (SNP) markers to identify CR loci from the F2 population derived from crossing CR "Kc84R" and clubroot susceptible "855S." The CR locus was mapped to a region between markers BnSNP14198336 and BnSNP14462201 on the A03 chromosome, and this fragment of 267 kb contained 68 annotated candidate genes. Furthermore, we performed the CR relation screening of candidate genes with the model species Arabidopsis. An ERF family transcriptional activator, BnERF034, was identified to be associated with the CR, and the corresponding Arabidopsis homozygous knockout mutants exhibited more pronounced resistance compared with the wild-type Col-0 and the transgenic lines of BnERF034 in response to P. brassicae infection. Additionally, the expression analysis between resistant and susceptible materials indicated that BnERF034 was identified to be the most likely CR candidate for the resistance in Kc84R. To conclude, this study reveals a novel gene responsible for CR. Further analysis of BnERF034 may reveal the molecular mechanisms underlying the CR of plants and provide a theoretical basis for Brassicaceae resistance breeding.

Functional characterization of a Colchicum autumnale L. double-bond reductase (CaDBR1) in colchicine biosynthesis.

MAIN CONCLUSION: An alkenal double-bond reductase enzyme (CaDBR1) was cloned from Colchicum autumnale L. The encoded enzyme catalysed 4-coumaraldehyde to 4-hydroxydihydrocinnamaldehyde (4-HDCA). Its functional characterization increased the understanding of colchicine biosynthesis. As a traditional medical plant, Colchicum autumnale L. is famous for producing colchicine, a widely used drug for alleviating gout attacks. The biosynthetic pathway of colchicine was revealed most recently, and 4-hydroxydihydrocinnamaldehyde (4-HDCA) has been verified as a crucial intermediate derived from L-phenylalanine. However, the functional gene that catalyses the formation of 4-HDCA remains controversial. In this study, the alkenal double-bond reductase (DBR) gene member CaDBR1 was cloned and characterized from C. autumnale. Bioinformatics analysis predicted and characterized the basic physicochemical properties of CaDBR1. Recombinant CaDBR1 protein was heterologously expressed in Escherichia coli and purified by a Ni-NTA column. In vitro enzyme assays indicated that CaDBR1 could catalyse 4-coumaraldehyde to form 4-HDCA but could not generate 4-HDCA by taking cinnamaldehyde as a substrate. Stable transformation into tobacco BY-2 cells revealed that CaDBR1 localized in the cytoplasm, and tissue-specific expression results showed that CaDBR1 had the highest expression in bulbs. All these results verify and confirm the participation and contribution of CaDBR1 in the biosynthesis pathway of 4-HDCA and colchicine alkaloids in C. autumnale.

Forming coumarin C-glycosides via biocatalysis: Characterization of a C-glycosyltransferase from Angelica decursiva.

A glycosyl transferase, isolated from Angelica decursiva a medical herb rich in coumarin, shows C-glycosyl transferase activity by in vitro activity assay using 5,7-dihydroxycoumarin as substrate, producing a C-glycosylated product at position C'8 along with the main product at C'6 position. Catalytic promiscuity assay shows that AdCGT also displays O- or C-glycosylation activity to other coumarins and flavonoids. When phloretin and 2,4,6-trihydroxyacetophenone were fed as substrates, AdCGT catalyzed the formation of di-C-glycosides. Therefore, AdCGT is a multifunctional glycosyltransferase with a broad substrate acceptability. This work highlights the potential of AdCGT as a catalyst for glycosylation of coumarin and reveals a new regio-selective C-glycosyltransferase, providing a basis for exploring the mechanism of coumarin glycosylation.

TSG-6 promotes Cancer Cell aggressiveness in a CD44-Dependent Manner and Reprograms Normal Fibroblasts to create a Pro-metastatic Microenvironment in Colorectal Cancer.

Tumor necrosis factor α stimulated gene 6 (TSG-6), a 30-KD secretory protein, plays an essential role in modulating inflammatory responses and extracellular matrix remodeling. However, little is known regarding the role of TSG-6 in human cancers. Here, we investigated the mechanism of action and the role of TSG-6 in colorectal cancer (CRC) metastasis. We found that TSG-6 was highly expressed in tumor tissues and was associated with poor prognosis and metastasis in CRC. Mechanistically, TSG-6 overexpression in CRC cells resulted in ERK activation and epithelial-mesenchymal transition by means of stabilizing CD44 and facilitating the CD44-EGFR complex formation on the cell membrane. Consequently, this resulted in the promotion of tumor migration and invasion both in vitro and in vivo. Notably, our data showed that CRC cells secreted TSG-6 could trigger a paracrine activation of JAK2-STAT3 signaling and reprogram normal fibroblasts into cancer-associated fibroblasts, which exhibited upregulation of pro-metastatic cytokines (CCL5 and MMP3) and higher movement ability. In animal models, the co-injection of cancer cells and TSG6-reprogrammed fibroblasts led to a significant increase in tumor metastasis. Our findings indicated that TSG-6 overexpression in CRC cells could promote cancer metastasis in both an autocrine and paracrine manner. Therefore, targeting TSG-6 might be a potential therapeutic strategy for the treatment of metastatic CRC.

The Role of RAB GTPases and Its Potential in Predicting Immunotherapy Response and Prognosis in Colorectal Cancer.

Background: Colorectal cancer (CRC) is the third most common cancer worldwide, in which aberrant activation of the RAS signaling pathway appears frequently. RAB proteins (RABs) are the largest Ras small GTPases superfamily that regulates intracellular membrane trafficking pathways. The dysregulation of RABs have been found in various diseases including cancers. Compared with other members of Ras families, the roles of RABs in colorectal cancer are less well understood. Methods: We analyzed the differential expression and clinicopathological association of RABs in CRC using RNA sequencing and genotyping datasets from TCGA samples. Moreover, the biological function of RAB17 and RAB34 were investigated in CRC cell lines and patient samples. Results: Of the 62 RABs we analyzed in CRC, seven (RAB10, RAB11A, RAB15, RAB17, RAB19, RAB20, and RAB25) were significantly upregulated, while six (RAB6B, RAB9B, RAB12, RAB23, RAB31, and RAB34) were significantly downregulated in tumor tissues as compared to normal. We found that the upregulated-RABs, which were highly expressed in metabolic activated CRC subtype (CMS3), are associated with cell cycle related pathways enrichment and positively correlated with the mismatch repair (MMR) genes in CRC, implying their role in regulating cell metabolism and tumor growth. While, high expression of the downregulated-RABs were significantly associated with poor prognostic CRC mesenchymal subtypes (CMS4), immune checkpoint genes, and tumor infiltrating immune cells, indicating their role in predicting prognosis and immunotherapy efficacy. Interestingly, though RAB34 mRNA is downregulated in CRC, its high expression is significantly associated with poor prognosis. In vitro experiments showed that RAB17 overexpression can promote cell proliferation via cell cycle regulation. While, RAB34 overexpression can promote cell migration and invasion and is associated with PD-L1/PD-L2 expression increase in CRC cells. Conclusions: Our study showed that RABs may play important roles in regulating cell cycle and immune-related pathways, therefore might be potential biomarkers in predicting prognosis and immunotherapy response in CRC.

Plant hormone signals regulate trehalose accumulation against osmotic stress in watermelon cells.

Trehalose, one of the most chemically stable sugars, can effectively improve the tolerance of various plants against abiotic stress by protecting and stabilizing protein and cell membranes. However, the signaling pathway in trehalose biosynthesis triggered by abiotic stresses is still unclear. In the study, it can be shown that exogenous trehalose can alleviate the inhibitory effect of osmotic stress on cell growth, suppress extracellular alkalization, ROS burst, and maintain the integrity of the microtubular cytoskeleton. Trehalose-6-phosphate synthase (TPS) is the key limiting enzyme for trehalose synthesis and is encoded by 7 ClTPS genes, located in 7 different chromosomes of the watermelon genome. Expression analysis by qRT-PCR indicated that osmotic stress could upregulate the expression of all the family members of ClTPS and promote the accumulation of trehalose in watermelon cells accordingly. Exogenous methyl jasmonate (MeJA), ethephon (ETH), abscisic acid (ABA), or salicylic acid (SA) induced trehalose accumulation, with MeJA being the most effective treatment. When fluridone (FL), an ABA biosynthesis inhibitor, was pre-perfused into the cells before osmotic stress, trehalose accumulation and packed cell volume were suppressed significantly, whereas inhibition of ethylene biosynthesis could even restore cell growth. Moreover, inhibition of trehalose hydrolysis could also increase the tolerance against osmotic stress. This study shows that trehalose biosynthesis is phytohormone-dependent and the hydrolysis of trehalose is involved in osmotic tolerance regulation.

Integrated Transcriptome Analysis and Single-Base Resolution Methylomes of Watermelon (Citrullus lanatus) Reveal Epigenome Modifications in Response to Osmotic Stress.

DNA methylation plays an important role against adverse environment by reshaping transcriptional profile in plants. To better understand the molecular mechanisms of watermelon response to osmotic stress, the suspension cultured watermelon cells were treated with 100mM mannitol, and then a methylated cytosines map was generated by whole genome bisulfite sequencing (WGBS). Combined with transcriptome sequencing, the effects of osmotic stress on differentially methylated expressed genes (DMEGs) were assessed. It was found that genes related to plant hormone synthesis, signal transduction, osmoregulatory substance-related and reactive oxygen species scavenging-related enzyme could rapidly respond to osmotic stress. The overall methylation level of watermelon decreased after osmotic stress treatment, and demethylation occurred in CG, CHG, and CHH contexts. Moreover, differentially methylated expressed genes (DMEGs) were significantly enriched in RNA transport, starch and sucrose metabolism, plant hormone signal transduction and biosynthesis of secondary metabolites, especially in biosynthesis of osmolytes synthase genes. Interestingly, demethylation of a key enzyme gene Cla014489 in biosynthesis of inositol upregulated its expression and promoted accumulation of inositol, which could alleviate the inhibition of cell growth caused by osmotic stress. Meanwhile, a recombinant plasmid pET28a-Cla014489 was constructed and transferred into Escherichia coli BL21 for prokaryotic expression and the expression of ClMIPS protein could improve the tolerance of E. coli to osmotic stress. The effect of methylation level on the expression properties of inositol and its related genes was further confirmed by application of DNA methylation inhibitor 5-azacytidine. These results provide a preliminary insight into the altered methylation levels of watermelon cells in response to osmotic stress and suggest a new mechanism that how watermelon cells adapt to osmotic stress.

A Gain-of-Function Mutant of IAA7 Inhibits Stem Elongation by Transcriptional Repression of EXPA5 Genes in Brassica napus.

Plant height is one of the most important agronomic traits of rapeseeds. In this study, we characterized a dwarf Brassica napus mutant, named ndf-2, obtained from fast neutrons and DES mutagenesis. Based on BSA-Seq and genetic properties, we identified causal mutations with a time-saving approach. The ndf-2 mutation was identified on chromosome A03 and can result in an amino acid substitution in the conserved degron motif (GWPPV to EWPPV) of the Auxin/indole-3-acetic acid protein 7 (BnaA03.IAA7) encoded by the causative gene. Aux/IAA protein is one of the core components of the auxin signaling pathway, which regulates many growth and development processes. However, the molecular mechanism of auxin signal regulating plant height is still not well understood. In the following work, we identified that BnaARF6 and BnaARF8 as interactors of BnaA03.IAA7 and BnaEXPA5 as a target of BnaARF6 and BnaARF8. The three genes BnaA03.IAA7, BnaARF6/8 and BnaEXPA5 were highly expressed in stem, suggesting that these genes were involved in stem development. The overexpression of BnaEXPA5 results in larger rosettes leaves and longer inflorescence stems in Arabidopsis thaliana. Our results indicate that BnaA03.IAA7- and BnaARF6/8-dependent auxin signal control stem elongation and plant height by regulating the transcription of BnaEXPA5 gene, which is one of the targets of this signal.

Vesicle transporter GOLT1B mediates the cell membrane localization of DVL2 and PD-L2 and promotes colorectal cancer metastasis.

BACKGROUND: Colorectal cancer (CRC) is the third most diagnosed and second leading cause of cancer death worldwide. Hallmark proteins processing is usually dysregulated in cancers. Finding key regulatory molecules is of great importance for CRC metastasis intervention. GOLT1B is a vesicle transport protein which is involved in cytosolic proteins trafficking. However, its role in cancer has never been addressed. METHODS: CRC cell lines and subcutaneous xenograft animal model were utilized to investigate the biological function of GOLT1B. Patients samples were used to validate the correlation between GOLT1B and clinical outcome. In vivo targeted delivery of GOLT1B-siRNA was investigated in PDX (Patient derived tumor xenograft) model. RESULTS: We found that GOLT1B was highly expressed in CRC, and was an independent prognostic marker of overall survival (OS) and progression free survival (PFS). GOLT1B could promote CRC metastasis in vitro and in vivo. GOLT1B overexpression could increase DVL2 level and enhance its plasma membrane translocation, which subsequently activated downstream Wnt/ß-catenin pathway and increase the nuclear ß-catenin level, hence induce epithelial-mesenchymal transition (EMT). In addition, GOLT1B could also interact with PD-L2 and increase its membrane level. Co-culture of GOLT1B-overexpresed CRC cells with Jurkat cells significantly induced T cells apoptosis, which might further promote cancer cell the migration and invasion. Further, targeted delivery of GOLT1B siRNA could significantly inhibit tumor progression in GOLT1B highly expressed PDX model. CONCLUSION: Taken together, our findings suggest that the vesicle transporter GOLT1B could promote CRC metastasis not only by assisting DVL2 translocation and activating Wnt/ß-catenin pathway, but also facilitating PD-L2 membrane localization to induce immune suppression. Targeted inhibition of GOLT1B could be a potential therapeutic strategy for CRC treatment.

Dynamic analysis of pulmonary computed tomography (CT) characteristics in cured coronavirus disease 2019 (COVID-19) patients.

BACKGROUND: To retrospectively analyze the pulmonary computed tomography (CT) characteristics and dynamic changes in the lungs of cured coronavirus disease 2019 (COVID-19) patients at discharge and reexamination. METHODS: A total of 155 cured COVID-19 patients admitted to designated hospitals in Yunnan Province, China, from February 1, 2020, to March 20, 2020, were included. All patients underwent pulmonary CT at discharge and at 2 weeks after discharge (during reexamination at hospital). A retrospective analysis was performed using these two pulmonary CT scans of the cured patients to observe changes in the number, distribution, morphology, and density of lesions. RESULTS: At discharge, the lung CT images of 15 cured patients showed no obvious lesions, while those of the remaining 140 patients showed different degrees of residual lesions. Patients with moderate disease mostly had multiple pulmonary lesions, mainly in the lower lobes of both lungs. At reexamination, the lung lesions in the patients with moderate disease had significantly improved (P<0.05), and the lung lesions in the patients with severe disease had partially improved, especially in patients with multi-lobe involvement (χ 2 =3.956, P<0.05). At reexamination, the lung lesions of patients with severe disease did not show significant changes (P>0.05). CONCLUSIONS: The pulmonary CT manifestations of cured COVID-19 patients had certain characteristics and variation patterns, providing a reference for the clinical evaluation of treatment efficacy and prognosis of patients.

Characterisation, expression and functional analysis of PAL gene family in Cephalotaxus hainanensis.

Phenylalanine ammonia lyase (PAL) is the first committed step in the formation of phenylpropanoids, and catalyses the deamination of L-phenylalanine (L-Phe) to yield cinnamic acid. While PALs are common in plants, PAL genes involved in alkaloid biosynthesis in Cephalotaxus hainanensis have never been described. To obtain better knowledge of PAL genes and their number and function involved in Cephalotaxus alkaloid biosynthesis four PAL genes were screened and cloned. In vitro enzymatic analysis showed that all four PAL recombinant proteins could convert L-Phe to product trans-cinnamic acid, and showed strict substrate specificity. Moreover, the expression profiles of four ChPALs were analysed using qRT-PCR, which showed that they had higher transcript levels in roots and stems, and that different ChPALs displayed different response sensitivities and change patterns in response to stimuli. Several metabolic compounds were measured in stimulated leaves using UPLC-MS, and indicating the concentration of Cephalotaxus alkaloids and cinnamic acid in leaves subjected to different conditions. These concentrations increased significantly after treatment with 100 mM NaCl, 100 mM mannitol, 100 µM SA and 10 µM ABA. The expression levels of four PAL genes showed indications of upregulation after treatment. These results supply an important foundation for further research on candidate genes involved in the biosynthesis of Cephalotaxus alkaloids.