ILKAP Promotes the Metastasis of Hepatocellular Carcinoma Cells by Inhibiting ß-Catenin Degradation and Enhancing the WNT Signaling Pathway.

The incidence of Hepatocellular carcinoma (HCC) and HCC-related deaths have remarkably increased over the recent decades. It has been reported that ß-catenin activation can be frequently observed in HCC cases. This study identified the integrin-linked kinase-associated phosphatase (ILKAP) as a novel ß-catenin-interacting protein. ILKAP is localized both in the nucleus and cytoplasm and regulates the WNT pathway in different ways. First, it is demonstrated that ILKAP activates the WNT pathway in HCC cells by increasing the protein level of ß-catenin and other proteins associated with the WNT signaling, such as c-Myc and CyclinD1. Next, it is shown that ILKAP promotes the metastasis of HCC both in vitro and in vivo in a zebrafish xenograft model. It is also found that ILKAP dephosphorylates the GSK3ß and CK1, contributing to the reduced ubiquitination of ß-catenin. Furthermore, it is identified that ILKAP functions by mediating binding between TCF4 and ß-catenin to enhance expression of WNT target genes. Taken together, the study demonstrates a critical function of ILKAP in metastasis of HCC, since ILKAP is crucial for the activation of the WNT pathway via stabilization of ß-catenin and increased binding between TCF4 and ß-catenin.

Elevating plant immunity by translational regulation of a rice WRKY transcription factor.

Plants have intricate mechanisms that tailor their defence responses to pathogens. WRKY transcription factors play a pivotal role in plant immunity by regulating various defence signalling pathways. Many WRKY genes are transcriptionally activated upon pathogen attack, but how their functions are regulated after transcription remains elusive. Here, we show that OsWRKY7 functions as a crucial positive regulator of rice basal immunity against Xanthomonas oryzae pv. oryzae (Xoo). The activity of OsWRKY7 was regulated at both translational and post-translational levels. Two translational products of OsWRKY7 were generated by alternative initiation. The full-length OsWRKY7 protein is normally degraded by the ubiquitin-proteasome system but was accumulated following elicitor or pathogen treatment, whereas the alternate product initiated from the downstream in-frame start codon was stable. Both the full and alternate OsWRKY7 proteins have transcriptional activities in yeast and rice cells, and overexpression of each form enhanced resistance to Xoo infection. Furthermore, disruption of the main AUG in rice increased the endogenous translation of the alternate stabilized form of OsWRKY7 and enhanced bacterial blight resistance. This study provides insights into the coordination of alternative translation and protein stability in the regulation of plant growth and basal defence mediated by the OsWRKY7 transcription factor, and also suggests a promising strategy to breed disease-resistant rice by translation initiation control.

Fatigue at sea during and after the COVID-19 pandemic: A comparative study of two matched samples of seafarers.

This paper examines seafarers' experience of fatigue during and after the pandemic. A multi-phase mixed methods research design was used, including two quantitative surveys (Nduring-pandemic=501 and Nafter-pandemic=412) and 36 in-depth interviews. Applying propensity score matching the two samples to approximate the conditions of a randomized controlled experiment, the study shows that surprisingly seafarers reported higher levels of fatigue after the pandemic. Qualitative interviews with seafarers and ship managers reveal the underlying reason - the intensified ship inspection regime together with policy and regulatory updates/revisions in the immediate aftermath of the pandemic increased seafarers' workload and made seafarers more fatigued. The results of the two surveys also show that while fatigue risk factors differed between the two periods, fatigue risk can be managed and mitigated in both periods by implementing fatigue risk management policies and practices. Policy and management implications for improving seafarers' occupational health and safety are discussed at the end of the paper.

Fatigue during the COVID-19 pandemic: The experiences of Chinese seafarers.

Though the COVID-19 pandemic is drawing to a close, very little is known about the impact of China's zero-COVID policy on Chinese seafarers. This paper examines Chinese seafarers' experiences of fatigue during the pandemic. It adopted a mix-method research design involving two quantitative surveys conducted with seafarers before and during the pandemic and 35 in-depth interviews with both seafarers and managers. All the participants were from two Chinese shipping companies. The comparisons between the two surveys show that Chinese seafarers experienced significantly higher levels of fatigue during the pandemic. The interview data suggest a range of factors underpinning the higher levels of fatigue including fear of being infected, increased workload, wearing of four-piece personal protection equipment (PPE), the deprivation of shore leaves, and the prolonged service time. More importantly, the data indicate that the draconian zero-COVID policy in China and the related policy guidelines for Chinese shipping companies and seafarers reinforce these factors. This research extends previous research by providing an exclusive and comprehensive examination of seafarer fatigue during the pandemic and revealing that the policies adopted by seafarers' home countries can have profound implications for seafarers' experiences of fatigue. Suggestions are provided at the end of the paper.

[Cloning and functional analysis of flavanone 3-hydroxylase gene in Rhododendron hybridum Hort.]

Flavanone 3-hydroxylase (F3H) is a key enzyme in the synthesis of phycocyanidins. In this experiment, the petals of red Rhododendron hybridum Hort. at different developmental stages were used as experimental materials. The R. hybridum flavanone 3-hydroxylase (RhF3H) gene was cloned using reverse transcription PCR (RT-PCR) and rapid-amplification of cDNA ends (RACE) techniques, and bioinformatics analyses were performed. Petal RhF3H gene expression at different developmental stages were analyzed by using quantitative real-time polymerase chain reaction (qRT-PCR). A pET-28a-RhF3H prokaryotic expression vector was constructed for the preparation and purification of RhF3H protein. A pCAMBIA1302-RhF3H overexpression vector was constructed for genetic transformation in Arabidopsis thaliana by Agrobacterium-mediated method. The results showed that the R. hybridum Hort. RhF3H gene is 1 245 bp long, with an open reading frame of 1 092 bp, encoding 363 amino acids. It contains a Fe2+ binding motif and a 2-ketoglutarate binding motif of the dioxygenase superfamily. Phylogenetic analysis showed that the R. hybridum RhF3H protein is most closely related to the Vaccinium corymbosum F3H protein. qRT-PCR analysis showed that the expression level of the red R. hybridum RhF3H gene tended to increase and then decrease in the petals at different developmental stages, with the highest expression at middle opening stage. The results of the prokaryotic expression showed that the size of the induced protein of the constructed prokaryotic expression vector pET-28a-RhF3H was about 40 kDa, which was similar to the theoretical value. Transgenic RhF3H Arabidopsis thaliana plants were successfully obtained, and PCR identification and ß-glucuronidase (GUS) staining demonstrated that the RhF3H gene was integrated into the genome of A. thaliana plants. qRT-PCR, total flavonoid and anthocyanin contentanalysis showed that RhF3H was significantly higher expressed in the transgenic A. thaliana relative to that of the wild type, and its total flavonoid and anthocyanin content were significantly increased. This study provides a theoretical basis for investigating the function of RhF3H gene, as well as for studying the molecular mechanism of flower color in R. simsiib Planch.

Molecular characterization of a chalcone synthase gene RhCHS from Rhododendron × hybridum Hort.

Chalcone synthase (CHS) plays a vital role in anthocyanin biosynthesis pathway, which is associated with petal color of flower. To date, lots of CHS genes have been obtained from plants, while few were from Rhododendron genus. In this study we got a new CHS gene named RhCHS (MW358095) from Rhododendron × hybridum Hort. It had a 2040 bp coding region consisting of two exons and one intron. By using the deduced RhCHS protein as a query sequence, 15 CHS homologous family genes with sequence similarity from 60% to 98%, designated as RgCHS-D(x), were retrieved from the genome assembly of Rhododendron griersonianum (RGv1.1) by TBlastN. 12 CHS family genes were found locating in No.9 chromosome arranged in clusters, while only 3 of them exhibited in No.1, 2, and 8 chromosomes, respectively. The results revealed gene duplication of CHS in evolutionary process. Multiple alignment of the deduced amino acid sequence of RhCHS showed high similarity of the active site, the catalytic residue, and the signature motif, the conserved characteristics of which were also exhibited in the tertiary structure prediction of the RhCHS, as well as the phylogenetic tree, all these demonstrated the RhCHS belonging to the type III PKS superfamily. HPLC-MS/MS of flower petals detected the total concentration of CC, DC, and PelC. These anthocyanidins showed an overall increasing trend during the flowering period and reached the peak in the full-blooming stage, which was consistence with the changeable rule of RhCHS expression level. The promoter, which was 1507 bp exhibiting high ß-glucuronidase (GUS) staining activity, was predicted containing many cis-acting elements, especially light and transcription factor such as bHLH, MYB, WRKY, Dof, and ERF. In short, this study may provide the help to Rhododendron × hybridum Hort. not only in the mechanism research of petals color exhibition, but also in molecular breeding of CHS practice value.

Transcriptomic and metabolomic data reveal key genes that are involved in the phenylpropanoid pathway and regulate the floral fragrance of Rhododendron fortunei.

BACKGROUND: To reveal the key genes involved in the phenylpropanoid pathway, which ultimately governs the fragrance of Rhododendron fortunei, we performed a comprehensive transcriptome and metabolomic analysis of the petals of two different varieties of two alpine rhododendrons: the scented R. fortunei and the unscented Rhododendron 'Nova Zembla'. RESULTS: Our transcriptomic and qRT-PCR data showed that nine candidate genes were highly expressed in R. fortunei but were downregulated in Rhododendron 'Nova Zembla'. Among these genes, EGS expression was significantly positively correlated with various volatile benzene/phenylpropanoid compounds and significantly negatively correlated with the contents of various nonvolatile compounds, whereas CCoAOMT, PAL, C4H, and BALDH expression was significantly negatively correlated with the contents of various volatile benzene/phenylpropanoid compounds and significantly positively correlated with the contents of various nonvolatile compounds. CCR, CAD, 4CL, and SAMT expression was significantly negatively correlated with the contents of various benzene/phenylpropanoid compounds. The validation of RfSAMT showed that the RfSAMT gene regulates the synthesis of aromatic metabolites in R. fortunei. CONCLUSION: The findings of this study indicated that key candidate genes and metabolites involved in the phenylpropanoid biosynthesis pathway may govern the fragrance of R. fortunei. This lays a foundation for further research on the molecular mechanism underlying fragrance in the genus Rhododendron.

[Identification of terpene synthase gene family members in Rhododendron and its relationship with terpenoid metabolism].

Terpene synthase (TPS) plays important roles in the synthesis of terpenoids which are the main fragrances in Rhododendron flowers. To understand the function of TPS genes in terpenoid metabolism in relation to flower aroma formation, we identified all TPS gene family members in Rhododendron by analyzing its genome database. We then used a transcriptomic approach to analyze the differential gene expression patterns of TPS gene family members in the scented flower Rhododendron fortunei compared to the non-scented flower Rhododendron 'Nova Zembla'. The contents of terpenoid compounds in petals of the above two Rhododendron species at different developmental stages were also measured by using qRT-PCR and head space-solid phase micro-extraction combined with gas chromatography-mass spectrometry. Our results showed that a total of 47 RsTPS members, with individual lengths ranged from 591 to 2 634 bp, were identified in the Rhododendron genome. The number of exons in RsTPS gene ranged from 3 to 12, while the length of each protein encoded ranged from 196 to 877 amino acids. Members of the RsTPS family are mainly distributed in the chloroplast and cytoplasm. Phylogenetic analysis showed that RsTPS genes can be clustered into 5 subgroups. Seven gene family members can be functionally annotated as TPS gene family since they were temporally and spatially expressed as shown in the transcriptome data. Notably, TPS1, TPS10, TPS12 and TPS13 in Rhododendron fortunei were expressed highly in flower buds reached the peak in the full blossoming. Correlation analysis between gene expression levels and terpenoid content indicates that the expression levels of TPS1, TPS4, TPS9, TPS10, TPS12 and TPS13 were positively correlated with the content of terpenoids in the petals of R. fortunei at all flower developmental stages, suggesting that these six genes might be involved in the aroma formation in R. fortunei.

[Transcriptional analysis of grape in response to weak light stress].

Grape (Vitis vinifera L.) in production is frequently exposed to inadequate light, which significantly affects its agronomic traits via inhibiting their physiological, metabolic and developmental processes. To explore the mechanism how the grape plants respond to the weak light stress, we used 'Yinhong' grape and examined their physiology-biochemistry characteristics and transcriptional profile under different levels of weak light stress. The results showed that grape seedlings upon low intensity shading treatments were not significantly affected. As the shading stress intensity was strengthened, the epidermis cells, palisade tissue, and spongy tissue in the leaves were thinner, the intercellular space between the palisade tissue and spongy tissue was larger compared with that of the control, and the activities of superoxide dismutase, catalase and peroxidase were decreased gradually. Additionally, the soluble protein content increased and the free proline content decreased gradually. Compared with the control, significant changes in plant photosynthetic characteristics and physiology-biochemistry characteristics were observed under high intensity of shading (80%). RNA-seq data showed that the differentially expressed genes between CK and T2, CK and T4, T2 and T4 were 13 913, 13 293 and 14 943, respectively. Most of the enrichment pathways were closely related with the plant's response to stress. Several signaling pathways in response to stress-resistance, e.g. JA/MYC2 pathway and MAPK signal pathway, were activated under weak light stress. The expression level of a variety of genes related to antioxidation (such as polyphenol oxidase and thioredoxin), photosynthesis (such as phytochrome) was altered under weak light stress, indicating that 'Yinhong' grape may activate the antioxidation related pathways to cope with reactive oxygen species (ROS). In addition, it may activate the expression of photosynthetic pigment and light reaction structural protein to maintain the photosynthesis activity. This research may help better understand the relevant physiological response mechanism and facilitate cultivation of grape seedlings under weak light.

[Cloning and functional analysis of the phenylalaninammo-nialyase gene from Rhododendron fortunei].

Phenylalaninammo-nialyase (PAL) is a key enzyme in the synthesis of methyl benzoate - a plant aroma compound. In order to understand the function of this enzyme in the formation of fragrance in the scented Rhododendron species-Rhododendron fortunei, we cloned a gene encoding this enzyme and subsequently examined the gene expression patterns and the profile of enzyme activity during development in various tissues. The full length of RhPAL gene was cloned by reverse transcription-PCR (RT-PCR) and rapid amplification of cDNA ends (RACE) techniques. The expression levels of RhPAL gene were measured by real-time quantitative reverse transcription PCR (qRT-PCR) and the amount of phenylalanine and cinnamic acid were assayed with LC-MS. The results showed that the ORF sequence of RhPAL gene amplified from the cDNA templates of flower buds had 2 145 bp, encoding 715 amino acids, and shared 90% homology to the PAL amino acid sequences from other species. qRT-PCR analysis showed that the expression of RhPAL in petals during flowering kept in rising even until the flowers wilted. The expression of RhPAL in pistil was much higher than that in stamen, while the expression in the younger leaves was higher than in old leaves. However, the expression level was relatively lower in petal and stamen compared to that in leaves. We also measured the PAL activity by Enzyme-linked immuno sorbent assay in the petals of flowers at different flowering stages. The results showed that PAL activity reached the highest at the bud stage and then decreased gradually to the lowest when the flowers wilted, which followed a similar trend in the emission of the flower fragrance. The phenylalanine and cinnamic acid contents measured by LC-MS were highly correlated to the expression level of RhPAL in various tissues and at different flowering stages, implying that RhPAL plays an important role in the formation of the flower fragrance. This work may facilitate the breeding and improvement of new fragrant Rhododendron cultivars.

Analysis on genetic diversity of 51 Grape germplasm resources / Análise da diversidade genética da videira 51

ABSTRACT: The objective of this study is to research the genetic diversity of the ' Zuijinxiang ' grape and its mutant breeding F1 plants, we screened the excellent mutant plants with potential breeding value. 50 mutated single plants obtained from 137Cs-γ irradiated 'Zuijinxiang' grape seeds were used as research objects, and SCoT molecular marker technology was used for genetic diversity and variation analysis, and clustering research was carried out. The results showed that: (1) 36 SCoT primers produced abundant polymorphisms, and the amplification results showed obvious bright bands, and the amplification efficiency and polymorphism rate were 100%. (2) A total of 221 bands were amplified by 36 primers, of which 175 were rich in polymorphism, the average polymorphic percentage was 80.3%, and the average genetic similarity coefficient was 0.916. (3) The number of observed alleles (Na) ranged from 4 to 8, with an average of 6.1389; the number of effective alleles (Ne) ranged from 1.2772 to 5.6322 with an average of 3.5968; the desired heterozygosity (He) The range is from 0.2192 to 0.8344, the average is 0.6965; the observed heterozygosity (Ho) ranges from 0.1656 to 0.7808 with an average of 0.3035; the Nei's gene diversity index (H) ranges from 0.2170 to 0.8224 with an average of 0.6863; Shannon-Wiener The index (I) ranges from 0.5186 to 1.8597 with an average of 1.4517. (4) UPGMA clustering of 51 materials showed that the test materials could be divided into three groups when the genetic distance was 0.856. The experiment shows that the genetic diversity of the 'Zuijinxiang' radiation variation germplasm resources is rich. In addition, SCoT molecular marker technology can distinguish the materials with close genetic distance, and can be used for early identification techniques of grape mutant materials. This study provides a theoretical basis for the development of excellent mutant germplasm of 'Zuijinxiang' grapes.

RESUMO: O objetivo deste estudo é investigar a diversidade genética da uva 'Zuijinxiang' e de suas plantas F1 reprodutoras mutantes. Foram selecionadas as melhores plantas mutantes com potencial e valor genético. Utilizaram-se como objeto de pesquisa 50 plantas individuais mutantes obtidas de sementes de uva irradiadas com 137Cs-γ 'Zuijinxiang', e a tecnologia de marcadores moleculares SCoT para análise de diversidade genética e variação, e foi realizada uma pesquisa de agrupamento. Os resultados mostraram que: (1) 36 iniciadores de SCoT produziram polimorfismos abundantes, e os resultados de amplificação mostraram bandas claras óbvias, e a eficiência de amplificação e taxa de polimorfismo foram de 100%. (2) Um total de 221 bandas foi amplificado por 36 iniciadores, dos quais 175 eram ricos em polimorfismo, a porcentagem polimórfica média foi de 80,3% e o coeficiente médio de similaridade genética foi de 0,916. (3) O número de alelos observados (Na) variou de 4 a 8, com uma média de 6,1389; o número de alelos efetivos (Ne) variou de 1,2772 a 5,6322 com uma média de 3,5968; a heterozigosidade desejada (He), o intervalo é de 0,2192 a 0,8344, a média é de 0,6965; a heterozigosidade observada (Ho) varia de 0,1656 a 0,7808 com uma média de 0,3035; o índice de diversidade genética (H) de Nei varia de 0,2170 a 0,8224 com uma média de 0,6863; Shannon-Wiener o índice (I) varia de 0,5186 a 1,8597 com uma média de 1,4517. (4) O agrupamento de 51 materiais da UPGMA mostrou que os materiais de teste poderiam ser divididos em três grupos quando a distância genética era de 0,856. O experimento mostra que a diversidade genética dos recursos de germoplasma de variação de radiação "Zuijinxiang" é rica. Além disso, a tecnologia de marcadores moleculares da SCoT pode distinguir os materiais com uma distância genética próxima, e pode ser usada para técnicas de identificação precoce de materiais mutantes da uva. Este estudo fornece uma base teórica para o desenvolvimento de germoplasma mutante excelente de uvas "Zuijinxiang".

[Effects of light intensity on associated enzyme activity and gene expression during callus formation of Vitis vinifera].

We analyzed the best light intensity for callus induction and maintenance in Vitis vinifera and explored the mechanism of grape callus browning. Tender stem segments of grape cultivar "gold finger" were used to study the effects of different light intensities (0, 500, 1 000, 1 500, 2 000, 2 500, 3 000 and 4 000 Lx) on the induction rate, browning rate and associated enzyme activity and gene expression during Vitis vinifera callus formation. The callus induction rate under 0, 500, 1 000 and 1 500 Lx was more than 92%, significantly higher than in other treatments (P < 0.05). A lower browning rate and better callus growth were also observed during subculture under 1 000 and 1 500 Lx treatments. We found that chlorogenic acid, caffeic acid, p-hydroxybenzoic acid and coumaric acid contents were correlated with the browning rate of callus, among which chlorogenic acid content was positively correlated with the browning rate (P < 0.05). Peroxidase (POD) and polyphenol oxidase (PPO) activities were negatively correlated with the browning rate of callus (P < 0.01). The POD, PPO and phenylalanine ammonialyase (PAL) expression levels were positively correlated with the browning rate at P < 0.05 or P < 0.01. An appropriate light intensity for the tissue culture of Vitis vinifera was 1 000-1 500 Lx, higher or lower light intensities significantly impaired normal callus growth.