A disulfide bond mutant of Pseudoalteromonas porphyrae κ-carrageenase conferred improved thermostability and catalytic activity and facilitated its utilization in κ-carrageenan industrial waste residues recycling.

In this study, Discovery Studio was employed to predict the potential disulfide bond mutants of the catalytic domain of Pseudoalteromonas porphyrae κ-carrageenase to improve the catalytic activity and thermal stability. The mutant N205C-G239C was identified with significantly increased catalytic activity toward κ-carrageenan substrate, with activity 4.28 times that of WT. The optimal temperature of N205C-G239C was 55 °C, 15 °C higher than that of WT. For N205C-G239C, the t1/2 value at 50 °C was 52 min, 1.41 times that of WT. The microstructural analysis revealed that the introduced disulfide bond N205C-G239C could create a unique catalytic environment by promoting favorable interactions with κ-neocarratetraose. This interaction impacted various aspects such as product release, water molecule network, thermodynamic equilibrium, and tunnel size. Molecular dynamics simulations demonstrated that the introduced disulfide bond enhanced the overall structure rigidity of N205C-G239C. The results of substrate tunnel analysis showed that the mutation led to the widening of the substrate tunnel. The above structure changes could be the possible reasons responsible for the simultaneous enhancement of the catalytic activity and thermal stability of mutant N205C-G239C. Finally, N205C-G239C exhibited the effective hydrolysis of the κ-carrageenan industrial waste residues, contributing to the recycling of the oligosaccharides and perlite.

Revealing the crucial roles of suppressive immune microenvironment in cardiac myxoma progression.

Cardiac myxoma is a commonly encountered tumor within the heart that has the potential to be life-threatening. However, the cellular composition of this condition is still not well understood. To fill this gap, we analyzed 75,641 cells from cardiac myxoma tissues based on single-cell sequencing. We defined a population of myxoma cells, which exhibited a resemblance to fibroblasts, yet they were distinguished by an increased expression of phosphodiesterases and genes associated with cell proliferation, differentiation, and adhesion. The clinical relevance of the cell populations indicated a higher proportion of myxoma cells and M2-like macrophage infiltration, along with their enhanced spatial interaction, were found to significantly contribute to the occurrence of embolism. The immune cells surrounding the myxoma exhibit inhibitory characteristics, with impaired function of T cells characterized by the expression of GZMK and TOX, along with a substantial infiltration of tumor-promoting macrophages expressed growth factors such as PDGFC. Furthermore, in vitro co-culture experiments showed that macrophages promoted the growth of myxoma cells significantly. In summary, this study presents a comprehensive single-cell atlas of cardiac myxoma, highlighting the heterogeneity of myxoma cells and their collaborative impact on immune cells. These findings shed light on the complex pathobiology of cardiac myxoma and present potential targets for intervention.

Efficiency enhancement in Aspergillus niger α-L-rhamnosidase reverse hydrolysis by using a tunnel site rational design strategy.

There has been ongoing interest in improving the efficiency of glycoside hydrolase for synthesizing glycoside compounds through protein engineering, given the potential applications of glycoside compounds. In this study, a strategy of modifying the substrate access tunnel was proposed to enhance the efficiency of reverse hydrolysis catalyzed by Aspergillus niger α-L-rhamnosidase. Analysis of the tunnel dynamics identified Tyr299 as a key modifiable residue in the substrate access tunnel. The location of Tyr299 was near the enzyme surface and at the outermost end of the substrate access tunnel, suggested its role in substrate recognition and throughput. Based on the properties of side chains, six mutants were designed and expressed by Pichia pastoris. Compared to WT, the reverse hydrolysis efficiencies of mutants Y299P and Y299W were increased by 21.3 % and 11.1 %, respectively. The calculation results of binding free energy showed that the binding free energy was inversely proportional to the reverse hydrolysis efficiency. Further, when binding free energy levels were comparable, the mutants with shorter side chains displayed a higher reverse hydrolysis efficiency. These results proved that substrate access tunnel modification was an effective method to improve the reverse hydrolysis efficacy of α-L-rhamnosidase and also provided new insights for modifying other glycoside hydrolases.

A comparative study of two α-L-rhamnosidases with high sequence identity.

The GH78 α-L-rhamnosidase from Aspergillus tubingensis (AT-Rha) was proved to be a new clade of Aspergillus α-L-rhamnosidases in the previous study. A putative α-L-rhamnosidase from A. kawachii IFO 4308 (AK-Rha) has 92 % identity in amino acid sequence with AT-Rha. In this study, AK-Rha was expressed in P. pastoris and characterized. Similar to AT-rRha, the recombinant AK-Rha (AK-rRha) showed a narrow substrate specificity to naringin. Interestingly, the enzyme activity of AK-rRha was 0.816 U/mg toward naringin, significantly lower than 125.142 U/mg of AT-rRha. Their large differences in catalytic efficiency was mainly due to their differences in kcat values between AK-rRha (0.67 s-1) and AT-rRha (4.89 × 104 s-1). The molecular dynamics simulation exhibited that the overall conformation of AK-Rha was rigid and that of AT-Rha was flexible; the Loop Y-L located above the catalytic domain formed different steric hindrances to naringin, and interacted with the flavonoid matrices at different strengths. The polar solvation energy analysis implied that the glycosidic bond was more easily hydrolysed in AT-Rha. The comparative study verified that the main feature of AK-Rha and AT-Rha represented Aspergillus α-L-rhamnosidase was the narrow substrate specificity toward naringin, and provided an insight of the relationships between their catalytic abilities and structures.

New strategies to study in depth the metabolic mechanism of astaxanthin biosynthesis in Phaffia rhodozyma.

Astaxanthin, a ketone carotenoid known for its high antioxidant activity, holds significant potential for application in nutraceuticals, aquaculture, and cosmetics. The increasing market demand necessitates a higher production of astaxanthin using Phaffia rhodozyma. Despite extensive research efforts focused on optimizing fermentation conditions, employing mutagenesis treatments, and utilizing genetic engineering technologies to enhance astaxanthin yield in P. rhodozyma, progress in this area remains limited. This review provides a comprehensive summary of the current understanding of rough metabolic pathways, regulatory mechanisms, and preliminary strategies for enhancing astaxanthin yield. However, further investigation is required to fully comprehend the intricate and essential metabolic regulation mechanism underlying astaxanthin synthesis. Specifically, the specific functions of key genes, such as crtYB, crtS, and crtI, need to be explored in detail. Additionally, a thorough understanding of the action mechanism of bifunctional enzymes and alternative splicing products is imperative. Lastly, the regulation of metabolic flux must be thoroughly investigated to reveal the complete pathway of astaxanthin synthesis. To obtain an in-depth mechanism and improve the yield of astaxanthin, this review proposes some frontier methods, including: omics, genome editing, protein structure-activity analysis, and synthetic biology. Moreover, it further elucidates the feasibility of new strategies using these advanced methods in various effectively combined ways to resolve these problems mentioned above. This review provides theory and method for studying the metabolic pathway of astaxanthin in P. rhodozyma and the industrial improvement of astaxanthin, and provides new insights into the flexible combined use of multiple modern advanced biotechnologies.

Improvement of thermostability by increasing rigidity in the finger regions and flexibility in the catalytic pocket area of Pseudoalteromonas porphyrae κ-carrageenase.

Poor thermostability reduces the industrial application value of κ-carrageenase. In this study, the PoPMuSiC algorithm combined with site-directed mutagenesis was applied to improve the thermostability of the alkaline κ-carrageenase from Pseudoalteromonas porphyrae. The mutant E154A with improved thermal stability was successfully obtained using this strategy after screening seven rationally designed mutants. Compared with the wild-type κ-carrageenase (WT), E154A improved the activity by 29.4% and the residual activity by 51.6% after treatment at 50 °C for 30 min. The melting temperature (Tm) values determined by circular dichroism were 66.4 °C and 64.6 °C for E154A and WT, respectively. Molecular dynamics simulation analysis of κ-carrageenase showed that the flexibility decreased within the finger regions (including F1, F2, F3, F5 and F6) and the flexibility improved in the catalytic pocket area of the mutant E154A. The catalytic tunnel dynamic simulation analysis revealed that E154A led to enlarged catalytic tunnel volume and increased rigidity of the enzyme-substrate complex. The increasing rigidity within the finger regions and more flexible catalytic pocket of P. porphyrae κ-carrageenase might be a significant factor for improvement of the thermostability of the mutant κ-carrageenase E154A. The proposed rational design strategy could be applied to improve the enzyme kinetic stability of other industrial enzymes. Moreover, the hydrolysates of κ-carrageenan digested by the mutant E154A demonstrated increased scavenging activities against hydroxyl (OH) radicals and 2,2'-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) radicals compared with the undigested κ-carrageenan.

Improving the thermostability of Pseudoalteromonas Porphyrae κ-carrageenase by rational design and MD simulation.

The industrial applications of the κ-carrageenases have been restricted by their poor thermostability. In this study, based on the folding free energy change (ΔΔG) and the flexibility analysis using molecular dynamics (MD) simulation for the alkaline κ-carrageenase KCgCD from Pseudoalteromonas porphyrae (WT), the mutant S190R was identified with improved thermostability. After incubation at 50 °C for 30 min, the residual activity of S190R was 63.7%, 25.7% higher than that of WT. The Tm values determined by differential scanning calorimetry were 66.2 °C and 64.4 °C for S190R and WT, respectively. The optimal temperature of S190R was 10 °C higher than that of WT. The κ-carrageenan hydrolysates produced by S190R showed higher xanthine oxidase inhibitory activity compared with the untreated κ-carrageenan. MD simulation analysis of S190R showed that the residues (V186-M194 and P196-G197) in F5 and the key residue R150 in F3 displayed the decreased flexibility, and residues of T169-N173 near the catalytic center displayed the increased flexibility. These changed flexibilities might be the reasons for the improved thermostability of mutant S190R. This study provides a useful rational design strategy of combination of ΔΔG calculation and MD simulation to improve the κ-carrageenase's thermostability for its better industrial applications.

Fermentation of waste water from agar processing with Bacillus subtilis by metabolomic analysis.

Fungal infection has become a major threat to crop loss and affects food safety. The waste water from agar processing industries extraction has a number of active substances, which could be further transformed by microorganisms to synthesize antifungal active substances. In this study, Bacillus subtilis was used to ferment the waste water from agar processing industries extraction to analyze the antifungal activity of the fermentation broth on Alternaria alternata and Alternaria spp. Results showed that 25% of the fermentation broth was the most effective in inhibited A. alternata and Alternaria spp., with fungal inhibition rates of 99.9% and 96.1%, respectively, and a minimum inhibitory concentration (MIC) was 0.156 µg/mL. Metabolomic analysis showed that flavonoid polyphenols such as coniferyl aldehyde, glycycoumarin, glycitin, and procyanidin A1 may enhance the inhibitory activity against the two pathogenic fungal strains. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that polyphenols involved in the biosynthesis pathways of isoflavonoid and phenylpropanoid were upregulated after fermentation. The laser confocal microscopy analyses and cell conductivity showed that the cytoplasm of fungi treated with fermentation broth was destroyed. This study provides a research basis for the development of new natural antifungal agents and rational use of seaweed agar waste. KEY POINTS: • Bacillus subtilis fermented waste water has antifungal activity • Bacillus subtilis could transform active substances in waste water • Waste water is a potential raw material for producing antifungal agents.

In vitro-simulated intestinal flora fermentation of Porphyra haitanensis polysaccharides obtained by different assisted extractions and their fermented products against HT-29 human colon cancer cells.

Herein, we studied the in vitro-simulated intestinal flora fermentation of Porphyra haitanensis polysaccharides (PHPs) with microwave, ultrasonic, ultra-high pressure-assisted extraction and the protective effect of their fermented products against HT-29 human colon cancer cells. The results showed that PHPs were largely degraded at the 18 h stage of ascending colon fermentation, further greatly increasing the contents of reducing sugars and short-chain fatty acids (p < 0.05). Particularly, the PHPs subjected to ultra-high pressure-assisted extraction (UHP-PHP) showed the highest reducing sugar content of 1.68 ± 0.01 mg mL-1 and butyric acid content of 410.77 ± 7.99 mmol mL-1. Moreover, UHP-PHP showed a better effect in increasing the ratio of Bacteroidetes/Firmicutes and decreasing the abundance of Proteobacteria and Escherichia coli. PHPs could protect against HT-29 cells by increasing the ROS levels in a concentration-dependent manner, especially UHP-PHP fermented in a descending colon for 24 h. This was related to the up-regulated apoptosis-related genes (Bax and Bak), down-regulated protein expression of Bcl-2 and activation of the p-AKT protein, thereby promoting the apoptosis of HT-29 cells. Our results can facilitate the modification of PHPs and their practical application in the development of intestinal health improving products.

Wrapping gastroduodenal artery stump with the teres hepatis ligament to prevent postpancreatectomy hemorrhage after pancreaticoduodenectomy.

BACKGROUND: Gastroduodenal artery (GDA) stump erosion hemorrhage is a fatal complication after pancreaticoduodenectomy. This study aimed to determine whether GDA stump wrapping with the teres hepatis ligament during pancreaticoduodenectomy decreased the incidence of postpancreatectomy hemorrhage (PPH). METHODS: We reviewed 307 patients who had undergone pancreaticoduodenectomy between March 2019 and June 2022. The patients were divided into two groups according to application of GDA stump wrapping with the teres hepatis ligament: GDA wrapping group (165 patients) and no-wrapping group (142 patients). The perioperative data were compared between the groups. RESULTS: The clinical characteristics were balanced between the two groups. Grades B and C PPH and GDA-stump-related hemorrhage were significantly reduced in the GDA wrapping group compared with the no-wrapping group (PPH B/C, 13.4% vs 6.1%, P = 0.029; GDA hemorrhage, 5.6% vs 0.6%, P = 0.014). No difference was observed in the incidence of clinically relevant postoperative pancreatic fistula, biliary leak, intra-abdominal abscess, delayed gastric emptying, 90-day mortality, and postoperative hospital stay between the two groups. CONCLUSION: Wrapping GDA stump with the teres hepatis ligament reduced the incidence of GDA-stump-related PPH. Therefore, the wrapping technique is a simple and effective strategy to prevent PPH. Prospective studies are needed to confirm the benefit of this procedure.

LMP2-mRNA lipid nanoparticle sensitizes EBV-related tumors to anti-PD-1 therapy by reversing T cell exhaustion.

BACKGROUND: Targeting EBV-proteins with mRNA vaccines is a promising way to treat EBV-related tumors like nasopharyngeal carcinoma (NPC). We assume that it may sensitize tumors to immune checkpoint inhibitors. RESULTS: We developed an LMP2-mRNA lipid nanoparticle (C2@mLMP2) that can be delivered to tumor-draining lymph nodes. C2@mLMP2 exhibited high transfection efficiency and lysosomal escape ability and induced an increased proportion of CD8 + central memory T cells and CD8 + effective memory T cells in the spleen of the mice model. A strong synergistic anti-tumor effect of C2@mLMP2 in combination with αPD-1 was observed in tumor-bearing mice. The mechanism was identified to be associated with a reverse of CD8 + T cell exhaustion in the tumor microenvironment. The pathological analysis further proved the safety of the vaccine and the combined therapy. CONCLUSIONS: This is the first study proving the synergistic effect of the EBV-mRNA vaccine and PD-1 inhibitors for EBV-related tumors. This study provides theoretical evidence for further clinical trials that may expand the application scenario and efficacy of immunotherapy in NPC.

Transcriptomics analysis and fed-batch regulation of high astaxanthin-producing Phaffia rhodozyma/Xanthophyllomyces dendrorhous obtained through adaptive laboratory evolution.

Astaxanthin has high utilization value in functional food because of its strong antioxidant capacity. However, the astaxanthin content of Phaffia rhodozyma is relatively low. Adaptive laboratory evolution is an excellent method to obtain high-yield strains. TiO2 is a good inducer of oxidative stress. In this study, different concentrations of TiO2 were used to domesticate P. rhodozyma, and at a concentration of 1000 mg/L of TiO2 for 105 days, the optimal strain JMU-ALE105 for astaxanthin production was obtained. After fermentation, the astaxanthin content reached 6.50 mg/g, which was 41.61% higher than that of the original strain. The ALE105 strain was fermented by batch and fed-batch, and the astaxanthin content reached 6.81 mg/g. Transcriptomics analysis showed that the astaxanthin synthesis pathway, and fatty acid, pyruvate, and nitrogen metabolism pathway of the ALE105 strain were significantly upregulated. Based on the nitrogen metabolism pathway, the nitrogen source was adjusted by ammonium sulphate fed-batch fermentation, which increased the astaxanthin content, reaching 8.36 mg/g. This study provides a technical basis and theoretical research for promoting industrialization of astaxanthin production of P. rhodozyma. ONE-SENTENCE SUMMARY: A high-yield astaxanthin strain (ALE105) was obtained through TiO2 domestication, and its metabolic mechanism was analysed by transcriptomics, which combined with nitrogen source regulation to further improve astaxanthin yield.

Mechanism of melanogenesis inhibition by Keggin-type polyoxometalates.

Abnormal melanin overproduction can result in hyperpigmentation syndrome in human skin diseases and enzymatic browning of fruits and vegetables. Recently, our group found that Keggin-type polyoxometalates (POMs) can efficiently inhibit tyrosinase activity. However, it remains unclear whether Keggin-type POMs exhibit optimal effects in vivo. Additionally, the inhibitory effect and mechanism of action of POMs on cellular tyrosinase activity and melanogenesis have been rarely reported. Here we demonstrate that our screened and synthesised PMo11Zn and GaMo12 show superior inhibitory effects on melanin formation as well as inhibition of cellular tyrosinase activity compared to other Keggin-type POMs. Intriguingly, we reveal that Keggin-type POMs competitively bind to tyrosinase mainly through more interactions with Cu2+ ions and the amino acid residue is capable of forming van der Waals, cation-π and hydrogen bonds, resulting in a reversible non-covalent complex formation. Our findings provide valuable insights into the design, synthesis and screening of polyoxometalates as multifunctional metallodrugs and food preservatives against hyperpigmentation.

Enzymatic hydrolysates of κ-carrageenan by κ-carrageenase-CLEA immobilized on amine-modified ZIF-8 confer hypolipidemic activity in HepG2 cells.

κ-Carrageenase can degrade κ-carrageenan to produce bioactive κ-carrageenan oligosaccharides (KCOs) that have potential applications in pharmaceutical, food, agricultural, and cosmetics industries. Immobilized enzymes gain their popularity due to their good reusability, enhanced stability, and tunability. In this study, the previously characterized catalytic domain of Pseudoalteromonas purpurea κ-carrageenase was covalently immobilized on the synthesized amine-modified zeolitic imidazolate framework-8 nanoparticles with the formation of cross-linked enzyme aggregates, and the immobilized κ-carrageenase was further characterized. The immobilized κ-carrageenase demonstrated excellent pH stability and good reusability, and exhibited higher optimal reaction temperature, better thermostability, and extended storage stability compared with the free enzyme. The KCOs produced by the immobilized κ-carrageenase could significantly decrease the TC, TG, and LDL-C levels in HepG2 cells, increase the HDL-C level in HepG2 cells, and reduce the free fatty acids level in Caco-2 cells. Biochemical assays showed that the KCOs could activate AMPK activity, increase the ratios of p-AMPK/AMPK and p-ACC/ACC, and downregulate the expression of the lipid metabolism related proteins including SREBP1 and HMGCR in the hyperlipidemic HepG2 cells. This study provides a novel and effective method for immobilization of κ-carrageenase, and the KCOs produced by the immobilized enzyme could be a potential therapeutic agent to prevent hyperlipidemia.

Corrigendum: In vivo total or partial hepatectomy followed by ex vivo liver resection and autotransplantation for malignant tumors: a single center experience.

[This corrects the article DOI: 10.3389/fonc.2023.1214451.].

Targeting lymph node delivery with nanovaccines for cancer immunotherapy: recent advances and future directions.

Although cancer immunotherapy is a compelling approach against cancer, its effectiveness is hindered by the challenge of generating a robust and durable immune response against metastatic cancer cells. Nanovaccines, specifically engineered to transport cancer antigens and immune-stimulating agents to the lymph nodes, hold promise in overcoming these limitations and eliciting a potent and sustained immune response against metastatic cancer cells. This manuscript provides an in-depth exploration of the lymphatic system's background, emphasizing its role in immune surveillance and tumor metastasis. Furthermore, it delves into the design principles of nanovaccines and their unique capability to target lymph node metastasis. The primary objective of this review is to provide a comprehensive overview of the current advancements in nanovaccine design for targeting lymph node metastasis, while also discussing their potential to enhance cancer immunotherapy. By summarizing the state-of-the-art in nanovaccine development, this review aims to shed light on the promising prospects of harnessing nanotechnology to potentiate cancer immunotherapy and ultimately improve patient outcomes.

In vivo total or partial hepatectomy followed by ex vivo liver resection and autotransplantation for malignant tumors: a single center experience.

Background: Ex vivo liver resection and autotransplantation (ELRAT) may provide an opportunity for R0 resection of conventionally unresectable hepatobiliary cancers and hepatic metastases. To date, few studies of the surgery for malignant tumors have been conducted and there are no known reports of in vivo partial hepatectomy followed by ELRAT (IPH-ELRAT) for malignant tumors. Methods: Between December 2021 and November 2022, ten patients with malignant hepatobiliary primary cancers or hepatic metastases underwent ELRAT at our institution. We shared the surgical skills and postoperative prognoses of these patients were assessed. Results: The types of tumors were biliary tract cancer (BTC, n=8), hepatic metastasis of colonic carcinoma (n=1), and hepatic metastasis of small-bowel stromal tumor (n=1). Five patients underwent in vivo total hepatectomy followed by ex vivo liver resection and autotransplantation (ITH-ELRAT), The other five received in vivo partial hepatectomy followed by ex vivo liver resection and autotransplantation (IPH-ELRAT). Four patients underwent inferior vena cava replacement using artificial blood vessels. The survival rate of all ten patients one month after surgery was 100%. Nine patients (90%) are currently alive, with a median follow-up of 8.5 months (range 6-16.5 months). To date, seven of the nine surviving patients have had no cancer recurrence, including six with BTC. Conclusions: We report the world first five cases that received IPH-ELRAT for malignancies. We also demonstrated relatively favorable outcomes in patients who underwent ELRAT. ELRAT may be a recommendable surgical option for selected patients with conventionally unresectable hepatobiliary malignant tumors.

Metabolomics of astaxanthin biosynthesis and corresponding regulation strategies in Phaffia rhodozyma.

Astaxanthin is a valuable carotenoid and is used as antioxidant and health care. Phaffia rhodozyma is a potential strain for the biosynthesis of astaxanthin. The unclear metabolic characteristics of P. rhodozyma at different metabolic stages hinder astaxanthin's promotion. This study is conducted to investigate metabolite changes based on quadrupole time-of-flight mass spectrometry metabolomics method. The results showed that the downregulation of purine, pyrimidine, amino acid synthesis, and glycolytic pathways contributed to astaxanthin biosynthesis. Meanwhile, the upregulation of lipid metabolites contributed to astaxanthin accumulation. Therefore, the regulation strategies were proposed based on this. The addition of sodium orthovanadate inhibited the amino acid pathway to increase astaxanthin concentration by 19.2%. And the addition of melatonin promoted lipid metabolism to increase the astaxanthin concentration by 30.3%. It further confirmed that inhibition of amino acid metabolism and promotion of lipid metabolism were beneficial for astaxanthin biosynthesis of P. rhodozyma. It is helpful in understanding metabolic pathways affecting astaxanthin of P. rhodozyma and provides regulatory strategies for metabolism.