Genome-wide identification of cannabinoid biosynthesis genes in non-drug type Cannabis (Cannabis sativa L.) cultivar.

BACKGROUND: Cannabis sativa cultivars can be classified as marijuana or hemp, depending on its amount of the psychoactive cannabinoid Δ9-tetrahydrocannabinolic acid (THCA). Hemp Cheungsam is a non-drug type Cannabis sativa that is characterized by low THCA content. However, the transcripts and expression profile of cannabinoid biosynthesis pathway genes of hemp Cheungsam have not been investigated. METHODS: RNA-sequencing (RNA-seq) was performed on three different tissue types (flower, leaf, and stem) of hemp Cheungsam to understand their transcriptomes. The expression of cannabinoid biosynthesis pathway genes was further analyzed in each tissue type. Multiple sequence alignment and conserved domain analyses were used to investigate the homologs of cannbinoid biosynthesis genes. RESULTS: We found that the cannabinoid biosynthesis pathway was mainly expressed in the flowers of hemp Cheungsam, similar to other Cannabis cultivars. However, expression of cannabidiolic acid (CBDA) synthase was much higher than THCA synthase and cannabichromenic acid (CBCA) synthase, suggesting that the transcription profile favors CBDA biosynthesis. Sequence analysis of cannabinoid biosynthesis pathway genes suggested the identity of orthologs in hemp Cheungsam. CONCLUSIONS: Cannabinoid biosynthesis in hemp Cheungsam mostly occurs in the flowers, compared to other plant organs. While CBDA synthase expression is high, THCA and CBCA synthase expression is considerably low, indicating lesser THCA biosynthesis and thus low THCA content. Sequence analysis of key genes (CBDA, THCA, and CBCA synthases) of the cannabinoid biosynthetic pathway indicates that orthologs are present in hemp Cheungsam.

TIPRL Regulates Stemness and Survival in Lung Cancer Stem Cells through CaMKK2-CaMK4-CREB Feedback Loop Activation.

Frequent recurrence and metastasis caused by cancer stem cells (CSCs) are major challenges in lung cancer treatment. Therefore, identifying and characterizing specific CSC targets are crucial for the success of prospective targeted therapies. In this study, it is found that upregulated TOR Signaling Pathway Regulator-Like (TIPRL) in lung CSCs causes sustained activation of the calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) signaling pathway by binding to CaMKK2, thereby maintaining stemness and survival. CaMKK2-mediated activation of CaM kinase 4 (CaMK4) leads to phosphorylation of cAMP response element-binding protein (CREB) at Ser129 and Ser133, which is necessary for its maximum activation and the downstream constitutive expression of its target genes (Bcl2 and HMG20A). TIPRL depletion sensitizes lung CSCs to afatinib-induced cell death and reduces distal metastasis of lung cancer in vivo. It is determined that CREB activates the transcription of TIPRL in lung CSCs. The positive feedback loop consisting of CREB and TIPRL induces the sustained activation of the CaMKK2-CaMK4-CREB axis as a driving force and upregulates the expression of stemness- and survival-related genes, promoting tumorigenesis in patients with lung cancer. Thus, TIPRL and the CaMKK2 signaling axis may be promising targets for overcoming drug resistance and reducing metastasis in lung cancer.

1-Aminocyclopropane-1-carboxylic Acid Enhances Phytoestrogen Accumulation in Soy Plants (Glycine max L.) by Its Acceleration of the Isoflavone Biosynthetic Pathway.

The low levels of bioactive metabolites in target plants present a bottleneck for the functional food industry. The major disadvantage of soy leaves is their low phytoestrogen content despite the fact that these leaves are an enriched source of flavonols. Our study demonstrated that simple foliar spraying with 1-aminocyclopropane-1-carboxylic acid (ACC) significantly enhanced the phytoestrogen contents of the whole soy plant, including its leaves (27-fold), stalks (3-fold), and roots (4-fold). In particular, ACC continued to accelerate the biosynthesis pathway of isoflavones in the leaves for up to 3 days after treatment, from 580 to 15,439 µg/g. The detailed changes in the levels of this metabolite in soy leaves are disclosed by quantitative and metabolomic analyses based on HPLC and UPLC-ESI-TOF/MS. The PLS-DA score plot, S-plot, and heatmap provide comprehensive evidence to clearly distinguish the effect of ACC treatment. ACC was also proved to activate a series of structural genes (CHS, CHR, CHI, IFS, HID, IF7GT, and IF7MaT) along the isoflavone biosynthesis pathway time-dependently. In particular, ACC oxidase genes were turned on 12 h after ACC treatment, which was rationalized to start activating the synthetic pathway of isoflavones.

Reduced Expression of PRX2/ATPRX1, PRX8, PRX35, and PRX73 Affects Cell Elongation, Vegetative Growth, and Vasculature Structures in Arabidopsis thaliana.

Class III peroxidases (PRXs) are involved in a broad spectrum of physiological and developmental processes throughout the life cycle of plants. However, the specific function of each PRX member in the family remains largely unknown. In this study, we selected four class III peroxidase genes (PRX2/ATPRX1, PRX8, PRX35, and PRX73) from a previous genome-wide transcriptome analysis, and performed phenotypic and morphological analyses, including histochemical staining, in PRX2RNAi, PRX8RNAi, PRX35RNAi, and PRX73RNAi plants. The reduced mRNA levels of corresponding PRX genes in PRX2RNAi, PRX8RNAi, PRX35RNAi, and PRX73RNAi seedlings resulted in elongated hypocotyls and roots, and slightly faster vegetative growth. To investigate internal structural changes in the vasculature, we performed histochemical staining, which revealed alterations in cell wall structures in the main vasculature of hypocotyls, stems, and roots of each PRXRNAi plant compared to wild-type (Col-0) plants. Furthermore, we found that PRX35RNAi plants displayed the decrease in the cell wall in vascular regions, which are involved in downregulation of lignin biosynthesis and biosynthesis-regulated genes' expression. Taken together, these results indicated that the reduced expression levels of PRX2/ATPRX1, PRX8, PRX35, and PRX73 affected hypocotyl and root elongation, vegetative growth, and the vasculature structures in hypocotyl, stem, and root tissues, suggesting that the four class III PRX genes play roles in plant developmental processes.

Immunological Effects of Aster yomena Callus-Derived Extracellular Vesicles as Potential Therapeutic Agents against Allergic Asthma.

Plant-derived extracellular vesicles, (EVs), have recently gained attention as potential therapeutic candidates. However, the varying properties of plants that are dependent on their growth conditions, and the unsustainable production of plant-derived EVs hinder drug development. Herein, we analyzed the secondary metabolites of Aster yomena callus-derived EVs (AYC-EVs) obtained via plant tissue cultures and performed an immune functional assay to assess the potential therapeutic effects of AYC-EVs against inflammatory diseases. AYC-EVs, approximately 225 nm in size, were isolated using tangential flow filtration (TFF) and cushioned ultracentrifugation. Metabolomic analysis, using ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS), revealed that AYC-EVs contained 17 major metabolites. AYC-EVs inhibited the phenotypic and functional maturation of LPS-treated dendritic cells (DCs). Furthermore, LPS-treated DCs exposed to AYC-EVs showed decreased immunostimulatory capacity during induction of CD4+ and CD8+ T-cell proliferation and activation. AYC-EVs inhibited T-cell reactions associated with the etiology of asthma in asthmatic mouse models and improved various symptoms of asthma. This regulatory effect of AYC-EVs resembled that of dexamethasone, which is currently used to treat inflammatory diseases. These results provide a foundation for the development of plant-derived therapeutic agents for the treatment of various inflammatory diseases, as well as providing an insight into the possible mechanisms of action of AYC-EVs.

Highly Efficient Bioconversion of trans-Resveratrol to δ-Viniferin Using Conditioned Medium of Grapevine Callus Suspension Cultures.

δ-Viniferin is a resveratrol dimer that possesses potent antioxidant properties and has attracted attention as an ingredient for cosmetic and nutraceutical products. Enzymatic bioconversion and plant callus and cell suspension cultures can be used to produce stilbenes such as resveratrol and viniferin. Here, δ-viniferin was produced by bioconversion from trans-resveratrol using conditioned medium (CM) of grapevine (Vitis labruscana) callus suspension cultures. The CM converted trans-resveratrol to δ-viniferin immediately after addition of hydrogen peroxide (H2O2). Peroxidase activity and bioconversion efficiency in CM increased with increasing culture time. Optimized δ-viniferin production conditions were determined regarding H2O2 concentration, incubation time, temperature, and pH. Maximum bioconversion efficiency reached 64% under the optimized conditions (pH 6.0, 60 °C, 30 min incubation time, 6.8 mM H2O2). In addition, in vitro bioconversion of trans-resveratrol was investigated using CM of different callus suspension cultures, showing that addition of trans-resveratrol and H2O2 to the CM led to production of δ-viniferin via extracellular peroxidase-mediated oxidative coupling of two molecules of trans-resveratrol. We thus propose a simple and low-cost method of δ-viniferin production from trans-resveratrol using CM of plant callus suspension cultures, which may constitute an alternative approach for in vitro bioconversion of valuable molecules.

PBK/TOPK Is a Favorable Prognostic Biomarker Correlated with Antitumor Immunity in Colon Cancers.

Immune checkpoint inhibitor therapy has proven efficacy in a subset of colon cancer patients featuring a deficient DNA mismatch repair system or a high microsatellite instability profile. However, there is high demand for more effective biomarkers to expand the colon cancer population responding to ICI therapy. PBK/TOPK, a serine/threonine kinase, plays a role in cell cycle regulation and mitotic progression. Here, we investigated the correlation between PBK/TOPK expression and tumor immunity and its prognostic value in colon cancer. Based on large-scale bioinformatics analysis, we discovered that elevated PBK/TOPK expression predicted a favorable outcome in patients with colon cancer and was positively associated with immune infiltration levels of CD8+ T cells, CD4+ T cells, natural killer cells, and M1 macrophages. In contrast, a negative correlation was found between PBK/TOPK expression and immune suppressor cells, including regulatory T cells and M2 macrophages. Furthermore, the expression of PBK/TOPK was correlated with the expression of T-cell cytotoxicity genes in colon cancer. Additionally, high PBK/TOPK expression was associated with mutations in DNA damage repair genes, and thus with increased tumor mutation and neoantigen burden. These findings suggest that PBK/TOPK may serve as a prognostic and predictive biomarker for immunotherapy in colon cancer.

The sulfiredoxin-peroxiredoxin redox system regulates the stemness and survival of colon cancer stem cells.

Cancer stem cells (CSCs) initiate tumor formation and are known to be resistant to chemotherapy. A metabolic alteration in CSCs plays a critical role in stemness and survival. However, the association between mitochondrial energy metabolism and the redox system remains undefined in colon CSCs. In this study, we assessed the role of the Sulfiredoxin-Peroxiredoxin (Srx-Prx) redox system and mitochondrial oxidative phosphorylation (OXPHOS) in maintaining the stemness and survival of colon CSCs. Notably, Srx contributed to the stability of PrxI, PrxII, and PrxIII proteins in colon CSCs. Increased Srx expression promoted the stemness and survival of CSCs and was important for the maintenance of the mitochondrial OXPHOS system. Furthermore, Nrf2 and FoxM1 led to OXPHOS activation and upregulated expression of Srx-Prx redox system-related genes. Therefore, the Nrf2/FoxM1-induced Srx-Prx redox system is a potential therapeutic target for eliminating CSCs in colon cancer.

Inhibition of melanogenesis by Aster yomena callus pellet extract in melanoma cells and patients with skin pigmentation.

Plant tissue culture holds immense potential for the production of secondary metabolites with various physiological functions. We recently established a plant tissue culture system capable of producing secondary metabolites from Aster yomena. This study aimed to uncover the mechanisms underlying the potential therapeutic effects of Aster yomena callus pellet extract (AYC-P-E) on photoaging-induced skin pigmentation. Excessive melanogenesis was induced in B16F10 melanoma cells using α-melanocyte stimulating hormone (α-MSH). The effects of AYC-P-E treatment on melanin biosynthesis inducers and melanin synthesis inhibition were assessed. Based on the results, a clinical study was conducted in subjects with skin pigmentation. AYC-P-E inhibited melanogenesis in α-MSH-treated B16F10 cells, accompanied by decreased mRNA and protein expression of melanin biosynthesis inducers, including cyclic AMP response element-binding protein (CREB), tyrosinase, microphthalmia-associated transcription factor (MITF), tyrosinase related protein-1 (TRP-1), and TRP-2. This anti-melanogenic effect was mediated by mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) and protein kinase B (AKT) phosphorylation. Treatment of subjects with skin pigmentation with AYC-P-E-containing cream formulations resulted in 3.33%, 7.06%, and 8.68% improvement in the melanin levels at 2, 4, and 8 weeks, respectively. Our findings suggest that AYC-P-E inhibits excessive melanogenesis by activating MEK/ERK and AKT signaling, potentiating its cosmetic applications in hyperpigmentation treatment.

Synergistic Effect of Methyl Jasmonate and Abscisic Acid Co-Treatment on Avenanthramide Production in Germinating Oats.

The oat (Avena sativa L.) is a grain of the Poaceae grass family and contains many powerful anti-oxidants, including avenanthramides as phenolic alkaloids with anti-inflammatory, anti-oxidant, anti-itch, anti-irritant, and anti-atherogenic activities. Here, the treatment of germinating oats with methyl jasmonate (MeJA) or abscisic acid (ABA) resulted in 2.5-fold (582.9 mg/kg FW) and 2.8-fold (642.9 mg/kg FW) increase in avenanthramide content, respectively, relative to untreated controls (232.6 mg/kg FW). Moreover, MeJA and ABA co-treatment synergistically increased avenanthramide production in germinating oats to 1505 mg/kg FW. Individual or combined MeJA and ABA treatment increased the expression of genes encoding key catalytic enzymes in the avenanthramide-biosynthesis pathway, including hydroxycinnamoyl-CoA:hydrocyanthranilate N-hydroxycinnamoyl transferase (HHT). Further analyses showed that six AsHHT genes were effectively upregulated by MeJA or ABA treatment, especially AsHHT4 for MeJA and AsHHT5 for ABA, thereby enhancing the production of all three avenanthramides in germinating oats. Specifically, AsHHT5 exhibited the highest expression following MeJA and ABA co-treatment, indicating that AsHHT5 played a more crucial role in avenanthramide biosynthesis in response to MeJA and ABA co-treatment of germinating oats. These findings suggest that elicitor-mediated metabolite farming using MeJA and ABA could be a valuable method for avenanthramide production in germinating oats.