FBXO31 is upregulated by METTL3 to promote pancreatic cancer progression via regulating SIRT2 ubiquitination and degradation.

FBXO31, a member of F-box family to comprise of SCF complex, contributes to a pivotal role in cancer progression. However, the possible involvements of FBXO31 in PC are unelucidated. Here, we reported that FBXO31 was overexpressed in PC patients, which was negatively associated with survival in PC patients. Furthermore, FBXO31 significantly enhanced growth, migration and invasion of PC cells in vitro. Consistently, FBXO31 overexpression promoted tumor growth in nude mice. Mechanistically, SIRT2 was a target of FBXO31 and interacted with FBXO31. Protein half-life and ubiquitination analysis demonstrated that FBXO31 promoted proteasome-dependent degradation of SIRT2. In addition, FBXO31 binds to sirtuin-type domain of SIRT2. Moreover, SIRT2 is required for the oncogenic role of FBXO31 in PC progression. Impressively, METTL3 induced m6A modification of FBXO31 and up-regulated FBXO31 expression, subsequently leading to SIRT2 down-regulation in PC cells. The results showed that METTL3 enhanced FBXO31 mRNA translation in YTHDF1-dependent manner. Taken together, we suggest that METTL3-FBXO31-SIRT2 axis was involved in PC tumorigenesis, which could identify new targets for PC treatment.

Nosip is a potential therapeutic target in hepatocellular carcinoma cells.

Nitric oxide synthase-interacting protein (Nosip) interacts with nitric oxide synthase (NOS) and regulates NO synthesis and release, which participates in various critical physiological and pathological processes. However, the role of Nosip in hepatocellular carcinoma (HCC) is unclear. In this study, Nosip expression was found to be elevated in HCC tissues and cells. Nosip siRNA transfection inhibited the proliferation and motility of HCC cells and promoted apoptosis. In contrast, overexpression of Nosip promoted proliferation and migration and invasion, and inhibited apoptosis of HCC cells. As a natural compound, quercetin exerted the effect of inhibiting the proliferation and motility of HCC cells, and this anticancer activity probably via repressing the expression of Nosip. Our results suggest that Nosip could act as an oncogene in the progression of HCC and that quercetin may be a potential natural compound for treating HCC by inhibiting the expression of Nosip.

Metabolic mechanism of astaxanthin biosynthesis in Xanthophyllomyces dendrorhous in response to sodium citrate treatment.

Astaxanthin is an important ketocarotenoid widely used in industries. However, its application is limited because of its low yield. Sodium citrate (Na-citrate), one of the major carbon sources for microorganisms, can promote cell growth and product accumulation. The basidiomycetous red yeast Xanthophyllomyces dendrorhous was thus used to study the effect of Na-citrate on cell growth and astaxanthin synthesis. The highest biomass and astaxanthin yield (6.0 g/L and 22.5 mg/L) were obtained in shake-flask when 3 g/L Na-citrate was added at 24 h and were 1.8 and 2.0 times higher than those of the control group, respectively. Furthermore, metabolomics and real-time reverse transcription PCR (qRT-PCR) analysis were conducted to study the metabolic pathways of X. dendrorhous in response to Na-citrate. The qRT-PCR assay revealed that Na-citrate facilitated glucose consumption, promoted the metabolic flux from glycolysis, and regulated the tricarboxylic acid (TCA) cycle, providing more energy and substrates for the synthesis of astaxanthin. The gene analysis revealed that adding Na-citrate significantly upregulated the expression of six key genes (ICL, HMGS, crtE, crtYB, crtI, and crtS) involved in pathways related to astaxanthin biosynthesis. These results suggest that exogenous Na-citrate treatment is a potentially valuable strategy to stimulate astaxanthin production in X. dendrorhous.

A Novel Bioflocculant Produced by Cobetia marina MCCC1113: Optimization of Fermentation Conditions by Response Surface Methodology and Evaluation of Flocculation Performance when Harvesting Microalgae.

A preliminary study was carried out to optimize the culture medium conditions for producing a novel microbial flocculant from the marine bacterial species Cobetia marina. The optimal glucose, yeast extract, and glutamate contents were 30, 10, and 2 g/l, respectively, while the optimal initial pH of the culture medium was determined to be 8. Following response surface optimization, the maximum bioflocculant production level of 1.36 g/l was achieved, which was 43.40% higher than the original culture medium. Within 5 min, a 20.0% (v/v) dosage of the yielded bioflocculant applied to algal cultures resulted in the highest flocculating efficiency of 93.9% with Spirulina platensis. The bioflocculant from C. marina MCCC1113 may have promising application potential for highly productive microalgae collection, according to the findings of this study.

Photoprotective carbon redistribution in mixotrophic Haematococcus pluvialis under high light stress.

Mixotrophy of Haematococcus pluvialis is a potential strategy for producing astaxanthin. However, this strategy has not been extensively commercialized because the mixotrophic mechanisms by which H. pluvialis overcomes high light stress are unclear. This study analyzed the biochemical compositions and differential proteomics of mixotrophic H. pluvialis under different light conditions. High light exposure substantially increased astaxanthin, carbohydrate, and fatty acid contents. A total of 119 and 81 proteins were significantly up- and down-regulated after two days of high light exposure. These proteins mainly enriched pathways for photosynthetic metabolism, glyoxylate cycle, and biosynthesis of secondary metabolites. This study proposed a regulatory model through which mixotrophic H. pluvialis copes with high light stress. The model includes pathways for modulating photosynthetic apparatus, increasing astaxanthin accumulation by enhancing photorespiration, pentose phosphate and Embden-Meyerhof-Parna pathways, while thickening the cell wall by malate-oxaloacetate shuttle.

The role of Fbxo5 in the development of human malignant tumors.

Fbxo5 (F-Box only protein 5), as a substrate recognition subunit of SCF (SKP1-Cullin1-Fbox) protein, plays a crucial role in various cellular processes through ubiquitination and degradation of multiple proteins. In recent years, many studies have pointed out that Fbxo5 is critically involved in carcinogenesis. Moreover, targeting Fbxo5 could have a therapeutic potential for cancer therapy. This review focuses on the functions of Fbxo5 in various types of human malignancies and its underlying molecular mechanisms. This review might lay the foundation for enhancing future investigation on Fbxo5 functions in cancer development and progression.

Fbxo45 facilitates pancreatic carcinoma progression by targeting USP49 for ubiquitination and degradation.

Fbxo45, a conserved F-box protein, comprises of an atypical SKP1, CUL1, F-box protein (SCF) ubiquitin ligase complex that promotes tumorigenesis and development. However, the biological function and molecular mechanisms of Fbxo45 involved in pancreatic carcinogenesis are ambiguous. We conducted several approaches, including transfection, coIP, real-time polymerase chain reaction (RT-PCR), Western blotting, ubiquitin assays, and animal studies, to explore the role of Fbxo45 in pancreatic cancer. Here, we report that USP49 stability is governed by Fbxo45-mediated ubiquitination and is enhanced by the absence of Fbxo45. Moreover, Fbxo45 binds to a short consensus sequence of USP49 through its SPRY domain. Furthermore, Fbxo45-mediated USP49 ubiquitination and degradation are enhanced by NEK6 kinase. Functionally, Fbxo45 increases cell viability and motility capacity by targeting USP49 in pancreatic cancer cells. Xenograft mouse experiments demonstrated that ectopic expression of Fbxo45 enhanced tumor growth in mice and that USP49 overexpression inhibited tumor growth in vivo. Notably, Fbxo45 expression was negatively associated with USP49 expression in pancreatic cancer tissues. Fbxo45 serves as an oncoprotein to facilitate pancreatic oncogenesis by regulating the stability of the tumor suppressor USP49 in pancreatic cancer.

ADAM metallopeptidase domain 12 overexpression correlates with prognosis and immune cell infiltration in clear cell renal cell carcinoma.

This study investigated the role of ADAM metallopeptidase domain 12 (ADAM12) in clear cell renal cell carcinoma (ccRCC). The mRNA expression of ADAM12 was analyzed using The Cancer Genome Atlas (TCGA) database and the Gene Expression Omnibus (GEO) database, and the protein expression level of ADAM12 in renal clear cell carcinoma cell lines was detected by Western blot analysis. The Wilcoxon rank-sum test, logistic regression analysis, Cox regression analysis, and Kaplan-Meier analysis were used to assess the relationship between the clinicopathological characteristics and the prognosis of ccRCC patients and ADAM12 expression. The miRNAs and lncRNAs associated with ADAM12 were predicted, and a ceRNA network was constructed using the Starbase database. Gene Set Enrichment Analysis (GSEA) and Gene Ontology (GO) analysis were used to identify relevant pathways. The relationship between ADAM12 and immune infiltration and checkpoints was analyzed using the TIMER and Gene Expression Profiling Interactive Analysis (GEPIA) databases. The results showed that ADAM12 expression was increased in ccRCC tissues and cells and significantly correlated with patient gender, Tumor stage, Metastasis stage, Node stage, and clinical grade. Survival analysis showed that ccRCC patients with high ADAM12 expression had a low overall survival rate. Univariate and multivariate Cox regression analyses showed that ADAM12 was an independent prognostic factor. Enrichment analysis showed that ADAM12 expression was associated with immune-related pathways. Immune infiltration analysis showed that ADAM12 expression was related to immune cell infiltration, PD-1, PD-L1, and CTLA4. These results suggest that ADAM12 may be a potential diagnostic and prognostic biomarker for ccRCC.

Enhancing astaxanthin accumulation in Xanthophyllomyces dendrorhous by a phytohormone: metabolomic and gene expression profiles.

Xanthophyllomyces dendrorhous is a promising source of natural astaxanthin due to its ability to accumulate high amounts of astaxanthin. This study showed that 6-benzylaminopurine (6-BAP) is an effective substrate that enhances cell biomass and astaxanthin accumulation in X. dendrorhous. In the current study, the biomass and astaxanthin content in X. dendrorhous were determined to be improved by 21.98% and 24.20%, respectively, induced by 6-BAP treatments. To further understand the metabolic responses of X. dendrorhous to 6-BAP, time-course metabolomics and gene expression levels of X. dendrorhous cultures with and without 6-BAP feeding were investigated. Metabolome analysis revealed that 6-BAP facilitated glucose consumption, promoted the glycolysis, suppressed the TCA cycle, drove carbon flux of acetyl-CoA into fatty acid and mevalonate biosynthesis, and finally facilitated the formation of astaxanthin. ROS analysis suggested that the antioxidant mechanism in X. dendrorhous can be induced by 6-BAP. Additionally, the process of 6-BAP significantly upregulated the expression of six key genes involved in pathways related to astaxanthin biosynthesis. This research demonstrates the metabolomic mechanism of phytohormone stimulation of astaxanthin production iNn X. dendrorhous and presents a new strategy to improve astaxanthin production to prevent the dilemma of choosing between accumulation of astaxanthin and cell biomass.

The role of fluconazole in the regulation of fatty acid and unsaponifiable matter biosynthesis in Schizochytrium sp. MYA 1381.

BACKGROUND: Schizochytrium has been widely used in industry for synthesizing polyunsaturated fatty acids (PUFAs), especially docosahexaenoic acid (DHA). However, unclear biosynthesis pathway of PUFAs inhibits further production of the Schizochytrium. Unsaponifiable matter (UM) from mevalonate pathway is crucial to cell growth and intracellular metabolism in all higher eukaryotes and microalgae. Therefore, regulation of UM biosynthesis in Schizochytrium may have important effects on fatty acids synthesis. Moreover, it is well known that UMs, such as squalene and ß-carotene, are of great commercial value. Thus, regulating UM biosynthesis may also allow for an increased valuation of Schizochytrium. RESULTS: To investigate the correlation of UM biosynthesis with fatty acids accumulation in Schizochytrium, fluconazole was used to block the sterols pathway. The addition of 60 mg/L fluconazole at 48 h increased the total lipids (TLs) at 96 h by 16% without affecting cell growth, which was accompanied by remarkable changes in UMs and NADPH. Cholesterol content was reduced by 8%, and the squalene content improved by 45% at 72 h, which demonstrated fluconazole's role in inhibiting squalene flow to cholesterol. As another typical UM with antioxidant capacity, the ß-carotene production was increased by 53% at 96 h. The increase of squalene and ß-carotene could boost intracellular oxidation resistance to protect fatty acids from oxidation. The NADPH was found to be 33% higher than that of the control at 96 h, which meant that the cells had more reducing power for fatty acid synthesis. Metabolic analysis further confirmed that regulation of sterols was closely related to glucose absorption, pigment biosynthesis and fatty acid production in Schizochytrium. CONCLUSION: This work first reported the effect of UM biosynthesis on fatty acid accumulation in Schizochytrium. The UM was found to affect fatty acid biosynthesis by changing cell membrane function, intracellular antioxidation and reducing power. We believe that this work provides valuable insights in improving PUFA and other valuable matters in microalgae.

Exploring biostimulation of plant hormones and nitrate supplement to effectively enhance biomass growth and lutein production with thermo-tolerant Desmodesmus sp. F51.

In this study, the interactive effect of plant hormone-salicylic acid and succinic acid on biomass growth, lutein content, and productivity of Desmodesmus sp. F51 were investigated. The results demonstrated that the synergistic action of salicylic acid and succinic acid could effectively enhance the assimilation of nitrate and significantly improve lutein production. The maximal lutein content 7.01 mg/g and productivity 5.11 mg/L/d could be obtained with a supplement of 100 µM salicylic acid and 2.5 mM succinic acid in batch culture. Furthermore, operation strategy of nitrate fed-batch coupled with supplementation for succinic acid and salicylic acid resulted in further enhancement of lutein content and productivity by 7.50 mg/g and 5.78 mg/L/d, respectively. The performance is better than most of the previously reported values.

Strategy and regulatory mechanisms of glutamate feeding to enhance astaxanthin yield in Xanthophyllomyces dendrorhous.

Xanthophyllomyces dendrorhous is an excellent industrial source for production of natural astaxanthin, but the yield of astaxanthin is relative low due to the contradiction between biomass weight and astaxanthin accumulation. Glutamate, a metabolite connecting nitrogen and carbon metabolisms, is probably a promising entry point to interfere cellular metabolisms. Thus, the effect of glutamate on cell growth and astaxanthin accumulation in X. dendrorhous was investigated. Results showed that glutamate feeding facilitated glucose consumption and further led to the increment of astaxanthin content with little influence of cell growth. A comparative proteomics study was applied to decipher the regulatory mechanisms of enhanced astaxanthin biosynthesis in X. dendrorhous as a response to the glutamate feeding. The expressions of proteins with the highest degree of fold change were involved in carbohydrate, amino acids, and carotenogenesis metabolisms as well as redox and stress-associated metabolisms. In addition, a possible regulatory model of enhanced astaxanthin accumulation in response to glutamate feeding in X. dendrorhous is also proposed.

Microencapsulation of Algal Oil Using Spray Drying Technology.

This work aims at developing a process of microencapsulation of algal oil containing ≥40% docosahexaenoic acid (DHA) using spray drying technology. Purity Gum® 2000 and Capsul®, both obtained from waxy corn starch, were chosen as the encapsulation materials. The effects of emulsification conditions on the droplet size, stability, viscosity and surface tension, and the effects of spraying conditions on the particle size, moisture content and surface oil content were investigated successively. The morphology of emulsion droplets and the microcapsules was observed by optical microscope and scanning electron microscopy. The results showed that the produced spherical microcapsules were smooth and free of pores, cracks, and surface indentation when shear velocity was 8.63 m/s in the first step of emulsification, homogenization pressure was 1.75·108 Pa and number of passes through homogenization unit was six for fine emulsification, rotational speed of spray disk was 400 s-1, and air inlet temperature was 170 °C. Therefore, it was concluded that the emulsification and encapsulation of algal oil containing DHA with above process was feasible.

Proteomic analysis of astaxanthin biosynthesis in Xanthophyllomyces dendrorhous in response to low carbon levels.

The ratio of carbon to nitrogen (C/N) in media plays a crucial role in the production of microbial carotenoids. However, the effects of a high C/N ratio on carotenoid production are ambiguous, and the mechanism of how C/N ratio affects astaxanthin accumulation in X. dendrorhous is unclear. In this study, the influence of C/N ratio on astaxanthin biosynthesis in X. dendrorhous at a fixed nitrogen concentration was investigated, and comparative proteomics were applied to address how C/N ratio affects cell growth and astaxanthin accumulation in X. dendrorhous. The results showed that cell growth and astaxanthin accumulation in X. dendrorhous were strongly related to the ratio of carbon to nitrogen with increasing C/N ratio in medium. However, the astaxanthin content per cell showed an inverse relationship, decreasing with an increasing C/N ratio. Differential proteomics showed the proteins with highest degree of change in expression under varying C/N ratios were mainly involved in carbohydrate metabolic pathways and carotenogenesis metabolism. In addition, several redox- and stress-associated proteins were up-regulated along with the carotenogenesis proteins, implying the environmental stress may affect metabolism and astaxanthin synthesis. A possible regulatory mechanism in response to glucose in X. dendrorhous is discussed.

Simple, effective protein extraction method and proteomics analysis from polyunsaturated fatty acids-producing micro-organisms.

Polyunsaturated fatty acids (PUFAs) are valuable ingredients in the food and pharmaceutical products due to their beneficial influence on human health. Most studies paid attention on the production of PUFAs from oleaginous micro-organisms but seldom on the comparative proteomics of cells. In the study, three methods (i.e., cold shock, acetone precipitation and ethanol precipitation) for lipid removal from crude protein extracts were applied in different PUFAs-producing micro-organisms. Among the selective strains, Schizochytrium was used as an oleaginous strain with high lipid of 60.3 (w/w%) in biomass. The Mortierella alpina and Cunninghamella echinulata were chosen as the low-lipid-content strains with 25.8 (w/w%) and 21.8 (w/w%) of lipid in biomass, respectively. The cold shock resulted as the most effective method for lipid removed, thus obtained higher protein amount for Schizochytrium. Moreover, from the comparative proteomics for the three PUFAs-producing strains, it showed more significant proteins of up or down-regulation were explored under cold shock treatment. Therefore, the essential proteins (i.e., polyunsaturated fatty acid synthase) and regulating proteins were observed. In conclusion, this study provides a valuable and practical approach for analysis of high PUFAs-producing strains at the proteomics level, and would further accelerate the understanding of the metabolic flux in oleaginous micro-organisms.