The clinical association between coagulation indexes, platelet-related parameters, and bone metastasis of newly diagnosed prostate cancer.

BACKGROUND: At present, much evidence shows that many cancers have a high risk of thrombosis. Several studies have shown the prognostic value of platelet-related parameters and coagulation indexes in prostate cancer (PCa). However, the association between platelet-related parameters, coagulation indexes and bone metastasis of Pca is unclear. METHODS: A total of 234 pathologically diagnosed patients with Pca were consecutively collected and stratified into the bone metastasis group and non-bone metastasis group according to the results of the bone scan. ROC curve analysis was used to explore the auxiliary predictive value of single and combined parameters for bone metastasis in Pca patients. Univariate and multivariate Logistic regression analyses were used to determine the relationship between platelet-related parameters, coagulation indexes, and bone metastasis of Pca. RESULTS: Platelet count (PLT), fibrinogen (Fib), prostate-specific antigen (PSA), and D-dimer (DD) levels of the bone metastasis group were significantly higher than the non-bone metastasis group (P = 0.010, P < 0.001, P < 0.001, and P < 0.001, respectively). This study confirmed that PLT, PSA, DD and Fib have auxiliary predictive value for prostate cancer bone metastasis. After the combination of PLT, PSA, DD and Fib, the area under the curve, sensitivity and specificity increased significantly. The univariate logistic analysis demonstrated that PLT (OR: 1.008, P = 0.011), DD (OR: 2.690, P < 0.001), PSA (OR: 1.073, P < 0.001), Gleason score (OR: 7.060, P < 0.001), and Fib (OR: 2.082, P < 0.001) were significantly positively correlated with bone metastasis of Pca. Multivariate analysis showed that PSA (OR: 1.075, P < 0.001), DD (OR: 2.152, P < 0.001), Gleason score (OR: 2.904, P < 0.001), and Fib (OR: 1.706, P < 0.001) were independent risk factors for bone metastasis of Pca after adjusting for Age, BMI and other confounding factors. CONCLUSIONS: Higher platelet, D-dimer, prostate-specific antigen, Gleason score, and fibrinogen levels may predict a worse prognosis in patients with Pca. PLT, DD, and Fib, as readily available and relatively inexpensive indicators, help predict bone metastasis of Pca. It is suggested that PLT, DD and Fib may be helpful in the risk stratification of Pca.

Impact of intestinal dysbiosis on breast cancer metastasis and progression.

Breast cancer has a high mortality rate among malignant tumors, with metastases identified as the main cause of the high mortality. Dysbiosis of the gut microbiota has become a key factor in the development, treatment, and prognosis of breast cancer. The many microorganisms that make up the gut flora have a symbiotic relationship with their host and, through the regulation of host immune responses and metabolic pathways, are involved in important physiologic activities in the human body, posing a significant risk to health. In this review, we build on the interactions between breast tissue (including tumor tissue, tissue adjacent to the tumor, and samples from healthy women) and the microbiota, then explore factors associated with metastatic breast cancer and dysbiosis of the gut flora from multiple perspectives, including enterotoxigenic Bacteroides fragilis, antibiotic use, changes in gut microbial metabolites, changes in the balance of the probiotic environment and diet. These factors highlight the existence of a complex relationship between host-breast cancer progression-gut flora. Suggesting that gut flora dysbiosis may be a host-intrinsic factor affecting breast cancer metastasis and progression not only informs our understanding of the role of microbiota dysbiosis in breast cancer development and metastasis, but also the importance of balancing gut flora dysbiosis and clinical practice.

Application of enzymes as a feed additive in aquaculture.

Modern aquaculture must be sustainable in terms of energy consumption, raw materials used, and environmental impact, so alternatives are needed to replace fish feed with other raw materials. Enzyme use in the agri-food industry is based on their efficiency, safety, and protection of the environment, which aligns with the requirements of a resource-saving production system. Enzyme supplementation in fish feed can improve digestibility and absorption of both plant- and animal-derived ingredients, increasing the growth parameters of aquacultural animals. Herein we summarized the recent literature that reported the use of digestive enzymes (amylases, lipases, proteases, cellulases, and hemicellulases) and non-digestive enzymes (phytases, glucose oxidase, and lysozyme) in fish feed. In addition, we analyzed how critical steps of the pelleting process, including microencapsulation and immobilization, can interfere with enzyme activity in the final fish feed product. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-022-00128-z.

Improvement of the Catalytic Ability of a Thermostable and Acidophilic ß-Mannanase Using a Consensus Sequence Design Strategy.

In order to improve the catalytic efficiency of a thermostable and acidophilic ß-mannanase (ManAK; derived from marine Aspergillus kawachii IFO 4308), three mutants were designed by amino acid sequence consensus analysis with a second ß-mannanase (ManCbs), which also belongs to the glycoside hydrolase family 5 (GH5) and has excellent catalytic efficiency. Three mutants were constructed and their biochemical characteristics were measured after heterologous expression in Pichia pastoris. The results revealed that the kcat/Km values of the three recombinant mannanases ManAKC292V, ManAKL293V, and ManAKL294H were enhanced by 303.0, 280.4, and 210.1%, respectively. Furthermore, ManAKL293V showed greater thermostability than ManAK, retaining 36.5% of the initial enzyme activity after incubation at 80°C for 5min. This study therefore provides a rational design strategy based on consensus sequence analysis to develop industrially valuable ß-mannanase for future applications in marine aquafeed.

Surface charged amino acid-based strategy for rational engineering of kinetic stability and specific activity of enzymes: Linking experiments with computational modeling.

A rational workflow for engineering kinetically stable enzymes with good specific activity by surface charged amino acids engineering was proposed based on systematically analyzing the results of mutating 44 negatively charged surface amino acids of a thermophilic ß-mannanase (ManAK). Computational data, combined with experimental results indicated that percentage side-chain solvent accessibility (PSSA), changes in Gibbs free energy of unfolding (∆∆Gmut) and root-mean-square fluctuations (RMSF) could be suitable for screening kinetically stable mutants. A combinational standard (∆∆Gmut < -0.5 kJ/mol and RMSF >0.68 Å) resulted a decrease in the proportion of destabilizing mutants to 12.5%. The perturbations of substrate affinity and specific activity caused by mutation were weakened as the shortest distance from Cα of mutated site to Cα of catalytic sites (DsCα-Cα) increased. Results indicated that hotspot zones contributing to the local stability and integrity of catalytic motif at elevated temperatures might be widely distributed across spatial structure of the protein, while the mutation perturbation on enzyme specific activity demonstrated a gradually weakening trend from the catalytic core to the protein surface. These findings further our understanding of the structural-functional relationships of protein and highlight a deduced workflow to engineering industrially useful enzymes.

A New Cold-Active Glucose Oxidase From Penicillium: High-Level Expression and Application in Fish Preservation.

Glucose oxidase (GOx) with high enzyme activity at low temperature (4°C) is potentially useful for food preservation, especially for aquatic products preservation. A cold-active GOx with approximately 83% similarity to known protein sequences, was isolated from Penicillium sp. MX3343 and expressed in Pichia pastoris X33. Through high cell density fermentation, the yield of recombinant enzyme (named GOxP5) reached 458.6 U/mL. GOxP5 showed optimal activity at 30°C and pH 5.5, and was stable at a broad pH range from pH 2-6. Moreover, GOxP5 could maintain 72% maximum activity at 4°C, suggesting its application for the preservation of aquatic products at low-temperatures. Importantly, GOxP5 showed a good antimicrobial effect against common fish pathogenic bacteria (Listeria monocytogenes and Vibrio parahaemolyticus). Moreover, sensory, microbiological (total bacterial count), and physicochemical (total volatile basic nitrogen and pH) systematic analyses proved GOxP5 to be an excellent freshness preserving agent in the context of the grass carp. These favorable enzymatic properties of GOxP5 make it potentially useful in food biopreservation, and the effect was better compared to the commonly used chemical preservatives.

A thermostable glucose oxidase from Aspergillus heteromophus CBS 117.55 with broad pH stability and digestive enzyme resistance.

In this study, the heterologous expression of an engineered thermostablle glucose oxidase from Aspergillus heteromophus CBS 117.55 was achieved in P. pastoris. This recombinant GoxAh was thermostable, with an optimal temperature range 25 °C-65 °C, and it was capable of retaining greater than 90% of its initial activity following a 10-min incubation at 75 °C. This enzyme had an optimum pH of 6.0, and it could retain above 80% of its initial activity following a 2-h incubation at a broad pH range (2.0-8.0). Moreover, GoxAh displayed excellent pepsin and trypsin resistance, and highly resistant to a range of tested metal ions and chemical reagents. These good properties make GoxAh a promising candidate for feed additive. The Km and kcat/Km values of GoxAh were 187 mM and 1.09/mM/s, which limited its widespread application to some degree. However, due to its excellent characteristics, GoxAh is still of potential economic value for high value-added areas, as well as a good initial enzyme for developing applicable feed enzyme by protein engineering.

High-efficiency expression of a superior ß-mannanase engineered by cooperative substitution method in Pichia pastoris and its application in preparation of prebiotic mannooligosaccharides.

ß-mannanase with high specific activity is a prerequisite for the industrial preparation of prebiotic mannooligosaccharides. Three mutants, namely MEI, MER, and MEIR, were constructed by cooperative substitution based on three predominant single-point site mutations (K291E, L211I, and Q112R, respectively). Heterologous expression was facilitated in Pichia pastoris and the recombinase was characterized completely. The specific activities of MER (7481.9 U mg-1) and MEIR (9003.1 U mg-1) increased by 1.07- and 1.29-fold from the initial activity of ME (6970.2U mg-1), respectively. MEIR was used for high-cell-density fermentation to further improve enzyme activity, and the expression levels achieved in the 10-L fermenter were significantly high (105,836 U mL-1). The prebiotic mannooligosaccharides (<2000 Da) were prepared by hydrolyzing konjac gum and locust bean gum with MEIR, with 100% and 76.40% hydrolysis rates, respectively. These characteristics make MEIR highly attractive for prebiotic development in food and related industries.

High-level expression of a thermophilic and acidophilic ß-mannanase from Aspergillus kawachii IFO 4308 with significant potential in mannooligosaccharide preparation.

An engineered thermophilic and acidophilic ß-mannanase (ManAK) from Aspergillus kawachii IFO 4308 was highly expressed in Pichia pastoris. Through high cell density fermentation, the maximum yield reached 11,600 U/mL and 15.5 g/L, which is higher than most extreme ß-mannanases. The recombinant ManAK was thermostable with a temperature optimum of 80 °C, and acid tolerant with a pH optimum of 2.0. ManAK could efficiently degrade locust bean gum, konjac gum, and guar gum into small molecular mannooligosaccharide (<2000 Da), even at high initial substrate concentration (10%), and displayed different Mw distributions in their end products. Docking analysis demonstrated that the catalytic pocket of ManAK could only accommodate a galactopyranosyl residue in subsite -1, which might be responsible for the distinct hydrolysis product compositions from locust bean gum and guar gum. These superior properties of ManAK strongly facilitate mannooligosaccharide preparation and application in food and feed area.

Improving catalytic efficiency and maximum activity at low pH of Aspergillus neoniger phytase using rational design.

A preferable phytase for use in animal feeds for industrial applications should have high, optimal activity at low pH in the monogastric gut environment and high thermostability. To obtain enzymes with enhanced catalytic efficiency (pH 5.5) and excellent activity in acidic pH range, we performed structure-based rational design of a thermostable phytase (PhyAn). For this, six mutants based on different rational design strategies were constructed and heterologously expressed in Pichia pastoris. Particularly, the extracellular enzymatic activity was assessed to ensure that the produced enzymes met requirements of further analyses. Several positive mutants with enhanced catalytic efficiency or pH-profile shifts were carefully examined. Biochemical and kinetic investigations of purified mutants revealed that E79K, E80K, E79K + E80K and D68K had higher catalytic efficiency than the parent enzyme by approximately 49%, 67%, 86% and 15%, respectively. Moreover, the optimum pH of mutant Y65H was shifted from 5.0 to 3.0, and the peak of D68K shifted to pH 5.5. Analysis of the structural-functional relationships revealed that changes in amino acid charges, structural flexibility and space hindrance could significantly influence certain enzyme characteristics. Our results illustrate the feasibility and present a structural foundation for enhancing the phytase-catalytic efficiency and acid resistance by assembling mutations derived using rational design.

Production and characterization of cellulolytic enzymes from the thermoacidophilic fungal Aspergillus terreus M11 under solid-state cultivation of corn stover.

The production of extracellular cellulases by a newly isolated thermoacidophilic fungus, Aspergillus terreus M11, on the lignocellulosic materials was studied in solid-state fermentation (SSF). The results showed that the high-level cellulase activity was produced at 45 degrees C pH 3 and moisture 80% with corn stover and 0.8% yeast extract as carbon and nitrogen sources. 581 U endoglucanase activity, 243 U filter paper activity and 128 U beta-glucosidase activity per gram of carbon source were obtained in the optimal condition. Endoglucanase and beta-glucosidase exhibited their maximum activity at pH 2 and pH 3, respectively, and both of them showed remarkable stability in the range of pH 2-5. The activities of endoglucanase and beta-glucosidase were up to the maximum at 70 degrees C and maintained about 65% and 53% of their original activities after incubation at 70 degrees C for 6h. The enzyme preparations from this strain were used to hydrolyze Avicel. Higher hydrolysis yields of Avicel were up to 63% on 5% Avicel (w/v) for 72 h with 20 U FPase/g substrate.