Proteomics Analysis of the Protective Effect of Polydeoxyribonucleotide Extracted from Sea Cucumber (Apostichopus japonicus) Sperm in a Hydrogen Peroxide-Induced RAW264.7 Cell Injury Model.

Sea cucumber viscera contain various naturally occurring active substances, but they are often underutilized during sea cucumber processing. Polydeoxyribonucleotide (PDRN) is an adenosine A2A receptor agonist that activates the A2A receptor to produce various biological effects. Currently, most studies on the activity of PDRN have focused on its anti-inflammatory, anti-apoptotic, and tissue repair properties, yet relatively few studies have investigated its antioxidant activity. In this study, we reported for the first time that PDRN was extracted from the sperm of Apostichopus japonicus (AJS-PDRN), and we evaluated its antioxidant activity using 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS), and hydroxyl radical scavenging assays. An in vitro injury model was established using H2O2-induced oxidative damage in RAW264.7 cells, and we investigated the protective effect of AJS-PDRN on these cells. Additionally, we explored the potential mechanism by which AJS-PDRN protects RAW264.7 cells from damage using iTRAQ proteomics analysis. The results showed that AJS-PDRN possessed excellent antioxidant activity and could significantly scavenge DPPH, ABTS, and hydroxyl radicals. In vitro antioxidant assays demonstrated that AJS-PDRN was cytoprotective and significantly enhanced the antioxidant capacity of RAW264.7 cells. The results of GO enrichment and KEGG pathway analysis indicate that the protective effects of AJS-PDRN pretreatment on RAW264.7 cells are primarily achieved through the regulation of immune and inflammatory responses, modulation of the extracellular matrix and signal transduction pathways, promotion of membrane repair, and enhancement of cellular antioxidant capacity. The results of a protein-protein interaction (PPI) network analysis indicate that AJS-PDRN reduces cellular oxidative damage by upregulating the expression of intracellular selenoprotein family members. In summary, our findings reveal that AJS-PDRN mitigates H2O2-induced oxidative damage through multiple pathways, underscoring its significant potential in the prevention and treatment of diseases caused by oxidative stress.

Anti-Tumor Effects of Astaxanthin by Inhibition of the Expression of STAT3 in Prostate Cancer.

Astaxanthin is a natural product gaining increasing attention due to its safety and anti-cancer properties. In this study, we investigated the mechanisms of the anti-cancer effects of astaxanthin on prostate cancer (PCa) cell lines using aggressive PCa DU145 cells. Also an instantaneous silenced cell line (si-STAT3) derived from DU145 and a control cell line (si-NK) were used for the MTT and colony formation assays to determine the role of astaxanthin in proliferation and colony formation abilities. Flow cytometry assays were used to detect the apoptosis of tumor cells. Migration and invasion assays detected the weakening of the respective abilities. Western blot and RT-PCR tests detected the levels of STAT3 protein and mRNA. Astaxanthin resulted in suppression of the proliferation of DU145 cells and the level of STAT3. The treatment of DU145 cells with astaxanthin decreased the cloning ability, increased the apoptosis percentage and weakened the abilities of migration and invasion of the cells. Furthermore, astaxanthin reduced the expression of STAT3 at protein and mRNA levels. The effects were enhanced when astaxanthin and si-STAT3 were combined. The results of animal experiments were consistent with the results in cells. Thus, astaxanthin inhibits the proliferation of DU145 cells by reducing the expression of STAT3.

Cost-effective optimization of gellan gum production by Sphingomonas paucimobilis using corn steep liquor.

Gellan gum, produced by Sphingomonas paucimobilis, is increasingly used in food and pharmaceutical industries as stabilizing, emulsifying, texturing and gelling agents. However, its high production costs may limit its full commercial potential. Therefore, in this study, we investigated ways to reduce gellan gum production costs and improve yields. We first revealed corn steep liquor (CSL) as a cost-effective nutrient source that can improve gellan gum yields. We then systematically optimized culture conditions even further, and revealed that the addition of Triton X-100 surfactant and selected inorganic nitrogen sources improved gellan gum production. Under our optimized conditions (glucose 33.75 g/L, CSL 10 g/L, urea 2.5 g/L, MgSO4 1.08 g/L, KH2PO4 3.24 g/L, K2SO4 1 g/L and Triton X-100 0.75 g/L), we yielded a maximum concentration of 14.41 g/L, which was about 1.5-fold higher than non-optimized CSL-based medium. Our findings highlight the use of CSL as a cost effective and promising nutrient source for industrial production of gellan gum.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production by Haloarchaeon Halogranum amylolyticum.

Haloarchaea is an important group of polyhydroxyalkanoate (PHA)-accumulating organisms. However, few promising haloarchaeal species for economical and efficient PHA production have been reported. Here, we first discovered that Halogranum amylolyticum TNN58 could efficiently accumulate poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with a high 3-hydroxyvalerate (3HV) fraction using glucose as carbon source. Briefly, transmission electron microscopy (TEM) analysis revealed the presence of a large number of PHA granules in the cells. Gas chromatography-mass spectrometry (GC-MS) and proton nuclear magnetic resonance ((1)H NMR) analyses showed that PHAs synthesized from glucose was PHBV. Moreover, the 3HV content reached 20.1 mol%, which is the highest 3HV fraction thus far reported, as for PHBV produced by the wild-type strains grown on unrelated carbon courses. Fermentation experiments suggested that nitrogen-limited MG medium was better than nutrient-rich NOMG and AS168 medium for PHBV production. Additionally, glucose was the most suitable carbon source among the tested carbon sources. Interestingly, PHBV accumulation was almost paralleled by cell growth and glucose consumption. By applying the fed-batch process in fermentor, the PHBV production and cell dry weight were increased by approximately eight and four times, respectively, as compared with those of the batch process in shaking flasks. The classical PHA synthase genes were successfully cloned via consensus-degenerate hybrid oligonucleotide primers (CODEHOPs) and high-efficiency thermal asymmetric interlaced (hiTAIL) PCR methods. This finding suggested that H. amylolyticum shows promising potential in the low-cost biotechnological production of PHBV after further process optimization.

[Breeding of L-tryptophan high-yield strain by ARTP mutagenesis and high-throughput screening].

L-tryptophan is an indispensable essential amino acid with a wide range of applications, which leads to a high demand. Accordingly, the production of L-tryptophan becomes a much-anticipated direction in research and industrial development. While irrational mutagenesis is an effective means to breed industrial strains, how to screen the strains with desirable phenotypes is still a major challenge. In order to improve the efficiency and accuracy of screening L-tryptophan high-yield strains, we used atmospheric and room temperature plasma mutagenesis to construct a random mutant library and then combined it with high-throughput screening in deep-well plates. Using a pseudo-fluorescent protein sensor capable of responding specifically to L-tryptophan, we successfully screened out a strain producing L-tryptophan at a high yield from a random mutagenesis library. The fermentation with the strain in shake flasks produced L-tryptophan at a yield of 1.99 g/L, which was 41.77% higher than that of the starting strain. Finally, the mechanism of high yield of the strain was deciphered by comparative genomics and transcriptomics. The above strategies provide a solid research foundation for further selection and development of high quality L-tryptophan producing strains.

Prevention of STAT3-related pathway in SK-N-SH cells by natural product astaxanthin.

PURPOSE: Neuroblastoma (NB) is the most common solid malignancy in children. Despite current intensive treatment, the long-term event-free survival rate is less than 50% in these patients. Thus, patients with NB urgently need more valid treatment strategies. Previous research has shown that STAT3 may be an effective target in high-risk NB patients. However, there are no effective inhibitors in clinical evaluation with low toxicity and few side effects. Astaxanthin is a safe and natural anticancer product. In this study, we investigated whether astaxanthin could exert antitumor effects in the SK-N-SH neuroblastoma cancer cell line. METHOD: MTT and colony formation assays were used to determine the effect of astaxanthin on the proliferation and colony formation of SK-N-SH cells. Flow cytometry assays were used to detect the apoptosis of SK-N-SH cells. The migration and invasion ability of SK-N-SH cells were detected by migration and invasion assays. Western blot and RT-PCR were used to detect the protein and mRNA levels. Animal experiments were carried out and cell apoptosis in tissues were assessed using a TUNEL assay. RESULT: We confirmed that astaxanthin repressed proliferation, clone formation ability, migration and invasion and induced apoptosis in SK-N-SH cells through the STAT3 pathway. Furthermore, the highest inhibitory effect was observed when astaxanthin was combined with si-STAT3. The reason for this may be that the combination of astaxanthin and si-STAT3 can lower STAT3 expression further than astaxanthin or si-STAT3 alone. CONCLUSION: Astaxanthin can exert anti-tumor effect on SK-N-SH cells. The inhibitory effect was the higher when astaxanthin was combined with si-STAT3.

Spo0A can efficiently enhance the expression of the alkaline protease gene aprE in Bacillus licheniformis by specifically binding to its regulatory region.

The expression of enzymes in Bacillus licheniformis, such as the valuable extracellular alkaline protease AprE, is highly regulated by a complex transcriptional regulation mechanism. Here, we found that the transcript abundance of aprE varies >343-fold in response to the supply of nutrients or to environmental challenges. To identify the underlying regulatory mechanism, the core promoter of aprE and several important upstream regulatory regions outside the promoter were firstly confirmed by 5'-RACE and mutagenesis experiments. The specific proteins that bind to the identified sequences were subsequently captured by DNA pull-down experiments, which yielded the transcriptional factors (TFs) Spo0A, CggR, FruR, YhcZ, as well as fragments of functionally unassigned proteins. Further electrophoretic mobility shift assay (EMSA) and DNase I foot-printing experiments indicated that Spo0A can directly bind to the region from -92 to -118 nucleotides upstream of the transcription start site, and the deletion of this specific region drastically decreased the production of AprE. Taken together, these results indicated that the expression of aprE was mainly regulated by the interplay between Spo0A and its cognate DNA sequence, which was successfully applied to overproduce AprE in a genetically modified host harboring three aprE expression cassettes. The DNA binding proteins may serve to increase the efficiency of transcription by creating an additional binding site for RNA polymerase. The discovery of this mechanism significantly increases our understanding of the aprE transcription mechanism, which is of great importance for AprE overproduction.

Hydroxypropyl ß-cyclodextrin improving multiple stresses tolerance of Lactococcus lactis subsp. lactis.

L. lactis is known as industrial starter in the fermentation of dairy and meat products, and it plays an important role in human health as an edible probiotic. During industrial production, L. lactis often experiences different stresses that delay the growth and decrease the survival in some serious conditions. In this study, the protective effects of hydroxypropyl ß-cyclodextrin (HP ß-CD) on L. lactis under multiple stresses were investigated. The microbial cells were treated with different stresses including heat, NaCl, cold, and H2 O2 stresses, and the results were showed by measuring the OD600 or spot plating method. The growth and tolerance were improved when HP ß-CD was added during different stress conditions, better than that of trehalose. Besides, the scanning electron microscopic and fluorescence spectrum studies showed that HP ß-CD could combine with L. lactis to protect the cell structure, suggesting that HP ß-CD may act as a protective agent of L. lactis. Therefore, HP ß-CD could be considered as a potential protective agent to be applied in food industry, and its protective mechanism on L. lactis still needs further investigation.

EGCG enhances cancer cells sensitivity under 60Coγ radiation based on miR-34a/Sirt1/p53.

Ionizing radiation (IR) resistance and toxicity to normal cells are the main problems in radiotherapy for cancer. In this study, we demonstrated that epigallocatechin gallate (EGCG) could inhibit effectively IR-induced damage to mouse normal hepatic cells AML-12, and improve dramatically the radiosensitivity of mouse hepatoma cells H22 to 60Coγ. In addition, the different effects of EGCG and underlying molecular mechanisms based on microRNA-34a (miR-34a) and apoptosis-related proteins were investigated by cells viability analysis, quantitative realtime PCR (qRT-PCR), Western blot and cells transfection. The results indicated EGCG played the key role of radiosensitization on H22 cells by activating the miR-34a/Sirt1/p53 signaling pathway. Besides, EGCG could down-regulate the expression of anti-apoptotic protein Bcl-2, and up-regulate the expression of pro-apoptotic proteins Bax and Caspase-3 in H22 cells. Interestingly, EGCG showed contrary results on AML-12 cells. Therefore, radiation protection and radiosensitization of EGCG were associated with apoptosis regulated by miR-34a/Sirt1/p53 signaling pathway.