R2R3 MYB-dependent auxin signalling regulates trichome formation, and increased trichome density confers spider mite tolerance on tomato.

Unicellular and multicellular tomato trichomes function as mechanical and chemical barriers against herbivores. Auxin treatment increased the formation of II, V and VI type trichomes in tomato leaves. The auxin response factor gene SlARF4, which was highly expressed in II, V and VI type trichomes, positively regulated the auxin-induced formation of II, V and VI type trichomes in the tomato leaves. SlARF4 overexpression plants with high densities of these trichomes exhibited tolerance to spider mites. Two R2R3 MYB genes, SlTHM1 and SlMYB52, were directly targeted and inhibited by SlARF4. SlTHM1 was specifically expressed in II and VI type trichomes and negatively regulated the auxin-induced formation of II and VI type trichomes in the tomato leaves. SlTHM1 down-regulation plants with high densities of II and VI type trichomes also showed tolerance to spider mites. SlMYB52 was specifically expressed in V type trichomes and negatively regulated the auxin-induced formation of V type trichome in the tomato leaves. The regulation of SlARF4 on the formation of II, V and VI type trichomes depended on SlTHM1 and SlMYB52, which directly targeted cyclin gene SlCycB2 and increased its expression. In conclusion, our data indicates that the R2R3 MYB-dependent auxin signalling pathway regulates the formation of II, V and VI type trichomes in tomato leaves. Our study provides an effective method for improving the tolerance of tomato to spider mites.

A SlMYB75-centred transcriptional cascade regulates trichome formation and sesquiterpene accumulation in tomato.

Tomato trichomes act as a mechanical and chemical barrier against pests. An R2R3 MYB transcription factor gene, SlMYB75, is highly expressed in type II, V, and VI trichomes. SlMYB75 protein is located in the nucleus and possesses transcriptional activation activity. Down-regulation of SlMYB75 increased the formation of type II, V, and VI trichomes, accumulation of δ-elemene, ß-caryophyllene, and α-humulene in glandular trichomes, and tolerance to spider mites in tomato. In contrast, overexpression of SlMYB75 inhibited trichome formation and sesquiterpene accumulation, and increased plant sensitivity to spider mites. RNA-Seq analyses of the SlMYB75 RNAi line indicated massive perturbation of the transcriptome, with a significant impact on several classes of transcription factors. Expression of the MYB genes SlMYB52 and SlTHM1 was strongly reduced in the RNAi line and increased in the SlMYB75-overexpressing line. SlMYB75 protein interacted with SlMYB52 and SlTHM1 and activated their expression. SlMYB75 directly targeted the promoter of the cyclin gene SlCycB2, increasing its activity. The auxin response factor SlARF4 directly targeted the promoter of SlMYB75 and inhibited its expression. SlMYB75 also bound to the promoters of the terpene synthase genes SlTPS12, SlTPS31, and SlTPS35, inhibiting their transcription. Our findings indicate that SlMYB75 perturbation affects several transcriptional circuits, resulting in altered trichome density and metabolic content.

SlARF10, an auxin response factor, is involved in chlorophyll and sugar accumulation during tomato fruit development.

The photosynthesis of green tomatoes contributes to fruit growth and carbon economy. The tomato auxin response factor 10 (SlARF10) belongs to the ARF family and is located in nucleus. In this study, we found that SlARF10 was highly expressed in green fruit. Overexpression of SlARF10 in fruit produced a dark-green phenotype whilst knock-down by RNAi produced a light-green phenotype. Autofluorescence and chlorophyll content analyses confirmed the phenotypes, which indicated that SlARF10 plays an important role in chlorophyll accumulation. Overexpression of SlARF10 positively affected photosynthesis in both leaves and fruit. Furthermore, SlARF10-overexpression lines displayed improved accumulation of starch, fructose, and sucrose in fruit, whilst SlARF10-RNAi lines showed decreased accumulation of starch and sucrose. Regulation of SlARF10 expression altered the expression of AGPase starch biosynthesis genes. SlARF10 positively regulated the expression of SlGLK1, POR, CBP1, and CBP2, which are related to chlorophyll metabolism and regulation. Electrophoretic mobility shift assays confirmed that SlARF10 directly targets to the SlGLK1 promoter. Our results thus indicate that SlARF10 is involved in chlorophyll accumulation by transcriptional activation of SlGLK1 expression in tomato fruit, and provide insights into the link between auxin signaling, chloroplast activity, and sugar metabolism during tomato fruit development.

KLHL21, a novel gene that contributes to the progression of hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) has very high prevalence and associated-mortality. However, targeted therapies that are currently used in clinical practice for HCC have certain limitations, in part because of the lack of reliable and clinically applicable biomarkers that can be used for diagnosis and prognosis assessments and for the surveillance of treatment effectiveness. METHODS: Meta-analysis was used to analyze the integrated microarray data for global identification of a set of robust biomarkers for HCC. Quantitative RT-PCR (qRT-PCR) was performed to validate the expression levels of selected genes. Gene expression was inhibited by siRNA. CellTiter 96® AQueous One Solution Cell Proliferation assays were used to determine cell proliferation, and Transwell assays were used to determine cell migration and invasion potential. RESULTS: Meta-analysis of the expression data provided a gene expression signature from a total of 1525 patients with HCC, showing 1529 up-regulated genes and 478 down-regulated genes in cancer samples. The expression levels of genes having strong clinical significance were validated by qRT-PCR using primary HCC tissues and the paired adjacent noncancerous liver tissues. Up-regulation of VPS45, WIPI1, TTC1, IGBP1 and KLHL21 genes and down-regulation of FCGRT gene were confirmed in clinical HCC samples. KLHL21 was the most promising gene for potential use as a bioclinical marker in this analysis. Abrogating expression of it significantly inhibited cell proliferation, migration and invasion. CONCLUSIONS: Our study suggests that KLHL21 is a potential target for therapeutic intervention. Our findings also provide novel candidate genes on a genome-wide scale, which may have significant impact on the design and execution of effective therapy of HCC patients.

Search of Novel Small Molecule Inhibitors for the Main Protease of SARS-CoV-2.

The current outbreak of coronavirus disease 2019 (COVID-19) has prompted the necessity of efficient treatment strategies. The COVID-19 pandemic was caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Main protease (Mpro), also called 3-chymotrypsin-like protease (3CL protease), plays an essential role in cleaving virus polyproteins for the functional replication complex. Therefore, Mpro is a promising drug target for COVID-19 therapy. Through molecular modelling, docking and a protease activity assay, we found four novel inhibitors targeting Mpro with the half maximal inhibitory concentration (IC50) and their binding affinities shown by the dissociation constants (KDs). Our new inhibitors CB-21, CB-25, CP-1 and LC24-20 have IC50s at 14.88 µM (95% Confidence Interval (95% CI): 10.35 µM to 20.48 µM), 22.74 µM (95% CI: 13.01 µM to 38.16 µM), 18.54µM (95% CI: 6.54 µM to 36.30 µM) and 32.87µM (95% CI: 18.37 µM to 54.80 µM)), respectively. The evaluation of interactions suggested that each inhibitor has a hydrogen bond or hydrophobic interactions with important residues, including the most essential catalytic residues: His41 and Cys145. All the four inhibitors have a much higher 50% lethal dose (LD50) compared with the well-known Mpro inhibitor GC376, demonstrating its low toxicity. These four inhibitors can be potential drug candidates for further in vitro and in vivo studies against COVID-19.

Human endeavor for anti-SARS-CoV-2 pharmacotherapy: A major strategy to fight the pandemic.

The global spread of COVID-19 constitutes the most dangerous pandemic to emerge during the last one hundred years. About seventy-nine million infections and more than 1.7 million death have been reported to date, along with destruction of the global economy. With the uncertainty evolved by alarming level of genome mutations, coupled with likelihood of generating only a short lived immune response by the vaccine injections, the identification of antiviral drugs for direct therapy is the need of the hour. Strategies to inhibit virus infection and replication focus on targets such as the spike protein and non-structural proteins including the highly conserved RNA-dependent-RNA-polymerase, nucleotidyl-transferases, main protease and papain-like proteases. There is also an indirect option to target the host cell recognition systems such as angiotensin-converting enzyme 2 (ACE2), transmembrane protease, serine 2, host cell expressed CD147, and the host furin. A drug search strategy consensus in tandem with analysis of currently available information is extremely important for the rapid identification of anti-viral. An unprecedented display of cooperation among the scientific community regarding SARS-CoV-2 research has resulted in the accumulation of an enormous amount of literature that requires curation. Drug repurposing and drug combinations have drawn tremendous attention for rapid therapeutic application, while high throughput screening and virtual searches support de novo drug identification. Here, we examine how certain approved drugs targeting different viruses can play a role in combating this new virus and analyze how they demonstrate efficacy under clinical assessment. Suggestions on repurposing and de novo strategies are proposed to facilitate the fight against the COVID-19 pandemic.

Steroid enzyme and receptor expression and regulations in breast tumor samples - A statistical evaluation of public data.

In spite of the significant progress of estrogen-dependent breast cancer (BC) treatment, aromatase inhibitor resistance is a major problem limiting the clinical benefit of this frontier endocrine-therapy. The aim of this study was to determine the differential expression of steroid-converting enzymes between tumor and adjacent normal tissues, as well as their correlation in modulating intratumoral steroid-hormone levels in post-menopausal estrogen-dependent BC. RNA sequencing dataset (n = 1097) of The-Cancer-Genome-Atlas (Breast Invasive Carcinoma) retrieved through the data portal of Genomic Data Commons was used for differential expressions and expression correlation analyses by Mann-Whitney U and Spearman's rank test, respectively. The results showed significant up-regulation of 17ß-HSD7 (2.50-fold, p < 0.0001) in BC, supporting its effect in sex-hormone control. Besides, suppression of 11ß-HSD1 expression (-8.29-fold, p < 0.0001) and elevation of 11ß-HSD2 expression (2.04-fold, p < 0.0001) provide a low glucocorticoid environment diminishing BC anti-proliferation. Furthermore, 3α-HSDs were down-regulated (-1.59-fold, p < 0.01; -8.18-fold, p < 0.0001; -33.96-fold, p < 0.0001; -31.85-fold, p < 0.0001 for type 1-4, respectively), while 5α-reductases were up-regulated (1.41-fold, p < 0.0001; 2.85-fold, p < 0.0001; 1.70-fold, p < 0.0001 for type 1-3, respectively) in BC, reducing cell proliferation suppressers 4-pregnenes, increasing cell proliferation stimulators 5α-pregnanes. Expression analysis indicates significant correlations between 11ß-HSD1 with 3α-HSD4 (r = 0.605, p < 0.0001) and 3α-HSD3 (r = 0.537, p < 0.0001). Significant expression correlations between 3α-HSDs were also observed. Our results systematically present the regulation of steroid-converting enzymes and their roles in modulating the intratumoral steroid-hormone levels in BC with a vivid 3D-schema, supporting novel therapy targeting the reductive 17ß-HSD7 and proposing a new combined therapy targeting 11ß-HSD2 and 17ß-HSD7.

Meta-Analysis of steroid-converting enzymes and related receptors in prostate cancer suggesting novel combined therapies.

Androgen receptor (AR) signaling is essential for prostate cancer (PC) progression and treatment. Experiments have demonstrated that the intratumoral androgen levels are not affected by circulating androgen levels, but rather modulated by local steroid-converting enzyme activities. The expression modulation status of human steroid-converting enzymes and nuclear receptors are of great promise to identify novel therapeutic targets. Meta-analysis was performed with 9 cohorts (1093 specimens) from Gene Expression Omnibus, 16 cohorts (933 specimens) from Oncomine and the TCGA cohort (550 specimens). We found significant up regulation of 5α-reductase type 1 and type 3 in both primary and metastatic PC, together with the down regulation of AKR1C2 in primary PC, contributing to the high intratumoral DHT levels. The expression of AR in metastatic PC was up regulated, indicating the importance of AR signaling in the progression of this cancer. The down regulations of HSD11B1 and NR3C1 in primary and metastatic PC may diminish the anti-inflammation and anti-proliferation effects of glucocorticoids signaling. Furthermore, the decrease of progesterone receptor (PGR) expression in primary and metastatic PC was also observed, relieving the suppression effect of PGR on PC proliferation. The clinical evidences of the remarkable expression modulation of steroid-converting enzymes and receptors in PC may indicate novel combined treatment against this highly incident cancer.

Novel Coronavirus Polymerase and Nucleotidyl-Transferase Structures: Potential to Target New Outbreaks.

The pandemic outbreak of a new coronavirus (CoV), SARS-CoV-2, has captured the world's attention, demonstrating that CoVs represent a continuous global threat. As this is a highly contagious virus, it is imperative to understand RNA-dependent-RNA-polymerase (RdRp), the key component in virus replication. Although the SARS-CoV-2 genome shares 80% sequence identity with severe acute respiratory syndrome SARS-CoV, their RdRps and nucleotidyl-transferases (NiRAN) share 98.1% and 93.2% identity, respectively. Sequence alignment of six coronaviruses demonstrated higher identity among their RdRps (60.9%-98.1%) and lower identity among their Spike proteins (27%-77%). Thus, a 3D structural model of RdRp, NiRAN, non-structural protein 7 (nsp7), and nsp8 of SARS-CoV-2 was generated by modeling starting from the SARS counterpart structures. Furthermore, we demonstrate the binding poses of three viral RdRp inhibitors (Galidesivir, Favipiravir, and Penciclovir), which were recently reported to have clinical significance for SARS-CoV-2. The network of interactions established by these drug molecules affirms their efficacy to inhibit viral RNA replication and provides an insight into their structure-based rational optimization for SARS-CoV-2 inhibition.

Cold-active extracellular lipase: Expression in Sf9 insect cells, purification, and catalysis.

Cold-active lipases are gaining special attention nowadays as they are increasingly used in various industries such as fine chemical synthesis, food processing, and washer detergent. In the present study, an extracellular lipase gene from Yarrowia lipolytica (LIPY8) was cloned and expressed by baculovirus expression system. The recombinant lipase (LipY8p) was purified using chromatographic techniques, resulting in a purification factor of 25.7-fold with a specific activity of 1102.9U/mg toward olive oil. The apparent molecular mass of purified LipY8p was 40 kDa. The enzyme was most active at pH 7.5 and 17 °C. It exhibited maximum activity toward medium chain (C10) esters. The presence of transition metals such as Zn2+, Cu2+, and Ni2+ strongly inhibited the enzyme activity, which was enhanced by EDTA. The lipase activity was affected by detergents and was elevated by various organic solvents at 10% (v/v). These enzymatic properties make this lipase of considerable potential for biotechnological applications.

Integrative comparative analyses of metabolite and transcript profiles uncovers complex regulatory network in tomato (Solanum lycopersicum L.) fruit undergoing chilling injury.

Tomato fruit are especially susceptible to chilling injury (CI) when continuously exposed to temperatures below 12 °C. In this study, integrative comparative analyses of transcriptomics and metabolomics data were performed to uncover the regulatory network in CI tomato fruit. Metabolite profiling analysis found that 7 amino acids, 27 organic acids, 16 of sugars and 22 other compounds had a significantly different content while transcriptomics data showed 1735 differentially expressed genes (DEGs) were down-regulated and 1369 were up-regulated in cold-stored fruit. We found that the contents of citrate, cis-aconitate and succinate were increased, which were consistent with the expression of ATP-citrate synthase (ACS) and isocitrate dehydrogenase (IDH) genes in cold-treated tomato fruit. Cold stress promotes the expression of ACS and IDH which may increase the synthesis of citrate, cis-aconitate and succinate. Alanine and leucine had increased contents, which may result from alanine aminotransferase (ALT) and branched-chain amino acid aminotransferase (BcAT)'s high expression levels, respectively. Overall the transcriptomics and metabolomics data in our study explain the molecular mechanisms of the chilling injury and expands our understanding of the complex regulatory mechanisms of a metabolic network in response to chilling injury in tomato fruit.

Auxin response factor 6A regulates photosynthesis, sugar accumulation, and fruit development in tomato.

Auxin response factors (ARFs) are involved in auxin-mediated transcriptional regulation in plants. In this study, we performed functional characterization of SlARF6A in tomato. SlARF6A is located in the nucleus and exhibits transcriptional activator activity. Overexpression of SlARF6A increased chlorophyll contents in the fruits and leaves of tomato plants, whereas downregulation of SlARF6A decreased chlorophyll contents compared with those of wild-type (WT) plants. Analysis of chloroplasts using transmission electron microscopy indicated increased sizes of chloroplasts in SlARF6A-overexpressing plants and decreased numbers of chloroplasts in SlARF6A-downregulated plants. Overexpression of SlARF6A increased the photosynthesis rate and accumulation of starch and soluble sugars, whereas knockdown of SlARF6A resulted in opposite phenotypes in tomato leaves and fruits. RNA-sequence analysis showed that regulation of SlARF6A expression altered the expression of genes involved in chlorophyll metabolism, photosynthesis and sugar metabolism. SlARF6A directly bound to the promoters of SlGLK1, CAB, and RbcS genes and positively regulated the expression of these genes. Overexpression of SlARF6A also inhibited fruit ripening and ethylene production, whereas downregulation of SlARF6A increased fruit ripening and ethylene production. SlARF6A directly bound to the SAMS1 promoter and negatively regulated SAMS1 expression. Taken together, these results expand our understanding of ARFs with regard to photosynthesis, sugar accumulation and fruit development and provide a potential target for genetic engineering to improve fruit nutrition in horticulture crops.

FGF19 Protects Hepatocellular Carcinoma Cells against Endoplasmic Reticulum Stress via Activation of FGFR4-GSK3ß-Nrf2 Signaling.

The tumor microenvironment induces endoplasmic reticulum (ER) stress in tumor cells, an event that can promote progression, but it is unknown how tumor cells adapt to this stress. In this study, we show that the fibroblast growth factor FGF19, a gene frequently amplified in hepatocellular carcinoma (HCC), facilitates a survival response to ER stress. Levels of FGF19 expression were increased in stressed HCC cells in culture and in a mouse xenograft model. Induction of ER stress required the transcription factor ATF4, which directly bound the FGF19 promoter. In cells where ER stress was induced, FGF19 overexpression promoted HCC cell survival and increased resistance to apoptosis, whereas FGF19 silencing counteracted these effects. Mechanistic investigations implicated glycogen synthase kinase-3ß (GSK3ß) in regulating nuclear accumulation of the stress-regulated transcription factor Nrf2 activated by FGF19. Our findings show how FGF19 provides a cytoprotective role against ER stress by activating a FGFR4-GSK3ß-Nrf2 signaling cascade, with implications for targeting this signaling node as a candidate therapeutic regimen for HCC management. Cancer Res; 77(22); 6215-25. ©2017 AACR.

Critical role of DEK and its regulation in tumorigenesis and metastasis of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related mortality globally. Therefore, it is quite essential to identify novel HCC-related molecules for the discovery of new prognostic markers and therapeutic targets. As an oncogene, DEK plays an important role in cell processes and participates in a variety of cellular metabolic functions, and its altered expression is associated with several human malignancies. However, the functional significance of DEK and the involved complex biological events in HCC development and progression are poorly understood. Here, combing the results from clinical specimens and cultured cell lines, we uncover a critical oncogenic role of DEK, which is highly expressed in HCC cells. DEK protein encompasses two isoforms (isoforms 1 and 2) and isoform 1 is the most frequently expressed DEK isoform in HCC cells. DEK depletion by using shRNA inhibited the cell proliferation and migration in vitro and suppressed tumorigenesis and metastasis in mouse models. Consistently, DEK overexpression regardless of which isoform produced the opposite effects. Further studies showed that DEK induced cell proliferation through upregulating cell cycle related CDK signaling, and promoted cell migration and EMT, at least in part, through the repression of ß-catenin/E-cadherin axis. Interestingly, isoform 1 induced cell proliferation more efficiently than isoform 2, however, no functional differences existed between these two isoforms in cell migration. Together, our study indicates that DEK expression is required for tumorigenesis and metastasis of HCC, providing molecular insights for DEK-related pathogenesis and a basis for developing new strategies against HCC.

FGF19 promotes epithelial-mesenchymal transition in hepatocellular carcinoma cells by modulating the GSK3ß/ß- catenin signaling cascade via FGFR4 activation.

Compelling evidence suggests that the epithelial-mesenchymal transition (EMT) correlates with aggressiveness of tumors and poor survival. FGF19 has been shown to be involved in EMT in cholangiocarcinoma and colorectal cancer, however, molecular mechanisms underlying FGF19-induced EMT process in hepatocellular carcinoma (HCC) remain largely unknown. Here, we show the expression of FGF19 is significantly elevated and negatively associated with the expression of E-cadherin in HCC tissues and cell lines. Ectopic FGF19 expression promotes EMT and invasion in epithelial-like HCC cells through repression of E-cadherin expression, whereas FGF19 knockdown enhances E-cadherin expression and hence diminishes EMT traits in mesenchymal-like HCC cells, suggesting FGF19 exerts its tumor progressing functions as an EMT inducer. Interestingly, depletion of FGF19 cannot abrogate EMT traits in the presence of GSK3ß inhibitors. Furthermore, FGF19-induced EMT can be markedly attenuated when FGFR4 is knocked out. These observations clearly indicate that FGFR4/GSK3ß/ß-catenin axis may play a pivotal role in FGF19-induced EMT in HCC cells. As FGF19 and its specific receptor FGFR4 are frequently amplified in HCC cells, selective targeting this signaling node may lend insights into a potential effective therapeutic approach for blocking metastasis of HCC.