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In this study, oral supplementation of stachyose significantly improved HFD-induced MetS symptom, including overweight, hyperlipidemia, hepatic steatosis and system-wide inflammation. The subsequent analysis revealed that stachyose supplement significantly enhanced the integrity of the intestinal epithelial barrier and effectively reversed the gut microbiota dysbiosis, as evidenced by improvements in gut microbial gene richness, microbiota composition, and functional characteristics. The abundance of butyrate-producing strains, such as Bacteroides faecis, Butyomonas faecalis, Parabacteroides distasonis and Phocaeicola coprophilus is significantly increased, concurrently with the activation of the PPAR-γ signaling pathway. The findings of our study suggest that stachyose exhibits potential as a prebiotic agent for the prevention of gut microbiota dysbiosis and the intestinal epithelial barrier disruption in obese. The results of our study suggest that stachyose demonstrates potential as a prebiotic agent for mitigating gut microbiota dysbiosis and preserving the integrity of the intestinal epithelial barrier in individuals with MetS.

Oral drug delivery systems (ODDSs) have various advantages of simple operation and few side effects. ODDSs are highly desirable for colon-targeted therapy (e.g. ulcerative colitis and colorectal cancer), as they improve therapeutic efficiency and reduce systemic toxicity. Chitosan/alginate nanoparticles (CANPs) show strong electrostatic interaction between the carboxyl group of alginates and the amino group of chitosan which leads to shrinkage and gel formation at low pH, thereby protecting the drugs from the gastrointestinal tract (GIT) and aggressive gastric environment. Meanwhile, CANPs as biocompatible polymer, show intestinal mucosal adhesion, which could extend the retention time of drugs on inflammatory sites. Recently, CANPs have attracted increasing interest as colon-targeted oral drug delivery system for intestinal diseases. The purpose of this review is to summarize the application and treatment of CANPs in intestinal diseases and insulin delivery. And then provide a future perspective of the potential and development direction of CANPs as colon-targeted ODDSs.

AbstractHigh intensity focused ultrasound (HIFU) has demonstrated its safety, efficacy and noninvasiveness in the ablation of solid tumor. However, its further application is limited by its inherent deficiencies, such as postoperative recurrence caused by incomplete ablation and excessive intensity affecting surrounding healthy tissues. Recent research has indicated that the integration of nanomaterials with HIFU exhibits a promising synergistic effect in tumor ablation. The concurrent utilization of nanomaterials with HIFU can help overcome the limitations of HIFU by improving targeting and ablation efficiency, expanding operation area, increasing operation accuracy, enhancing stability and bio-safety during the process. It also provides a platform for multi-therapy and multi-mode imaging guidance. The present review comprehensively expounds upon the synergistic mechanism between nanomaterials and HIFU, summarizes the research progress of nanomaterials as cavitation nuclei and drug carriers in combination with HIFU for tumor ablation. Furthermore, this review highlights the potential for further exploration in the development of novel nanomaterials that enhance the synergistic effect with HIFU on tumor ablation.

Breast cancer (BC) is the most common malignancy in women worldwide, and the standard treatment is chemotherapy or radiotherapy after surgery. In order to reduce the side effects of chemotherapy, various nanoparticles (NPs) have been discovered and synthesized, which has become a promising treatment for BC. In this study, a co-delivery nanodelivery drug system (Co-NDDS) was designed and synthesized with 2,3-dimercaptosuccinic acid (DMSA) coated Fe3O4 NPs as core encapsulated into chitosan/alginate nanoparticles (CANPs) shell, doxorubicin (DOX) and hydroxychloroquine (HCQ) as loading drugs. Smaller NPs carrying DOX (FeAC-DOX NPs) were loaded into larger NPs containing HCQ (FeAC-DOX@PC-HCQ NPs) by ionic gelation and emulsifying solvent volatilization methods. The physicochemical properties of this Co-NDDS were characterised, followed by in vitro studies of the anticancer effects and mechanisms using two different BC cell lines, MCF-7 cells and MDA-MB-231 cells. The results indicated that the Co-NDDS showcases exemplary physicochemical qualities and encapsulation capacity, facilitating accurate intracellular release through pH-sensitive attributes. Importantly, NPs can significantly increase the in vitro cytotoxicity of co-administered drugs and effectively inhibit the autophagy level of tumour cells. The Co-NDDS constructed in this study provides a promising strategy for the treatment of BC.

Cordyceps exopolysaccharide (CEP) has shown emerging potential in adjustment of gut microbiota and immune cell function. In this study, a water-soluble CEP with a molecular weight of 58.14 kDa was extracted from the fermentation broth of Paecilomyces hepiali, an endophytic fungus of Cordyceps sinensis. Our results indicated that Paecilomyces hepiali polysaccharide (PHP) showed significantly preventive potential on dextran sulfate sodium (DSS)-induced colitis in mice, which can prevent colon shortening, reduce intestinal epithelial cell (IEC) destruction, suppress inflammatory cell infiltration, and regulate the balance between regulatory T (Treg) cells and T helper type 17 (Th17) cells. Meanwhile, the disturbed gut microbiota was partially restored after PHP treatment. Further Pearson correlation coefficient analyses exhibited that the alteration of the gut microbiota was significantly related to adjustment of the IEC barrier and Treg/Th17 balance. In conclusion, all findings proposed that purified PHP has the potential to develop into a promising agent for colitis prevention and adjuvant therapy via maintaining intestinal homeostasis of gut microbiota and immune system.

BackgroundLactic acid, as a product of glycolysis, increases tumor cell migration and the invasion of tumor cells in the tumor microenvironment. Besides this, lactic acid promotes the expression of programmed death-1 expression (PD-1) in regulatory T cells, which could cause the failure of PD-1 blockade therapy. However, the implications of lactic acid in the tumor microenvironment of lung adenocarcinoma (LUAD) remain largely unclear.MethodsWe performed unsupervised consensus clustering to identify lactic-associated subtypes using expression profile of lactate regulators in LUAD. Differentially expressed genes (DEGs) associated with lactic-associated subtypes was used to construct lactate signature (LaSig) using LASSO regression algorithm. Immune infiltration analysis was conducted by ESTIMATER and drug sensitivity was estimated by R package called “pRRophetic”. The difference between two groups was calculated using Wilcox rank sum test and correlation analysis was calculated using Pearson correlation coefficient.ResultsIn this study, we evaluated DNA methylation and the mutation frequency of lactate regulators and found lactate regulators showed low mutation frequency in the TCGA-LUAD cohort, except TP53. At the RNA level, the expression level of lactate regulators was significantly associated with the immune cell component. In particular, expression of LDHA was positively correlated with CD4 T cell, CD8 T cell, M1 macrophages, and the enrichment score of multiple immune pathways. Two clusters were defined using the gene expression level of lactate regulators, and LDHA was significantly upregulated in cluster 1 with poor overall survival. A lactate signature (LaSig) had a robust performance in predicting the survival rate and immunotherapy response of LUAD patients. Moreover, patients in the high LaSig group may be more likely to benefit from these drugs (Cisplatin, Erlotinib, Gemcitabine, and Vinblastine) than those in the low LaSig group.ConclusionIn summary, our study explores the role of lactate regulators in guiding the clinical treatment of lung adenocarcinoma and provides additional help to supplement traditional molecular subtypes.

Ulcerative colitis (UC) is a chronic inflammatory bowel disease (IBD). Its etiology is unclear. Much evidence suggests that the death of abnormal intestinal epithelial cells (IECs) leads to intestinal barrier disruption, and the subsequent inflammatory response plays a vital role in UC. Pyroptosis is a form of programmed inflammatory cell death, and the role of pyroptosis in UC etiology remains to be explored. This study identified 10 hub genes in pyroptosis by gene expression profiles obtained from the GSE87466 dataset. Meanwhile, the biomarkers were screened based on gene significance (GS) and module membership (MM) through the Weighted Gene Co-Expression Network Analysis (WGCNA). The following analysis indicated that hub genes were closely associated with the UC progression and therapeutic drug response. The single-cell RNA (scRNA) sequencing data from UC patients within the GSE162335 dataset indicated that macrophages were most related to pyroptosis. Finally, the expression of hub genes and response to the therapeutic drug [5-aminosalicylic acid (5-ASA)] were verified in dextran sulfate sodium (DSS)-induced colitis mice. Our study identified IL1B as the critical pyroptosis-related biomarker in UC. The crosstalk between macrophage pyroptosis and IEC pyroptosis may play an essential role in UC, deserving further exploration.

Ulcerative colitis (UC) is an inflammatory bowel disease caused by mucosal immune system disorder, which has increased steadily all over the world. Previous studies have shown that collagen peptide (CP) has various beneficial biological activities, it is not clear whether the effect of CP on UC is positive or negative. In this study, 2.5% dextran sulfate sodium (DSS) was used to establish acute colitis in mice. Our results suggested that CP supplementation (200, 400 mg/kg/day) promoted the progression of colitis, increased the expression of inflammatory factors and the infiltration of colonic lamina propria macrophages. Gut microbiota analysis showed the composition changed significantly and inflammation promoted bacteria was after CP treatment. Meanwhile, the effect of CP on macrophage polarization was further determined in Raw264.7 cell line. The results showed that CP treatment could increase the polarization of M1 macrophages and promote the expression of inflammatory factors. In conclusion, our results showed that CP treatment could disrupt the gut microbiota of host, promote macrophage activation and aggravate DSS-induced colitis. This may suggest that patients with intestinal inflammation should not take marine derived CP.

Oral route colon-targeted drug delivery systems (CDDSs) are desirable for the treatment of ulcerative colitis (UC). However, CDDSs are challenging owing to the physiological and anatomical barriers associated with the gastrointestinal tract (GIT). In this study, we developed an effective enzyme-triggered controlled release system using curcumin–cyclodextrin (CD–Cur) inclusion complex as core and low molecular weight chitosan and unsaturated alginate resulting nanoparticles (CANPs) as shell. The formed CD–Cur–CANPs showed a narrow particle-size distribution and a compact structure. In vitro drug release determination indicated that CD–Cur–CANPs showed pH-sensitive and α-amylase-responsive release characteristics. Furthermore, in vivo experiments demonstrated that oral administration of CD–Cur–CANPs had an efficient therapeutic efficacy, strong colonic biodistribution and accumulation, rapid macrophage uptake, promoted colonic epithelial barrier integrity and modulated production of inflammatory cytokines, reshaped the gut microbiota in mice with dextran sodium sulfate (DSS)-induced colitis. Taken together, our synthetic CD–Cur–CANPs are a promising synergistic colon-targeted approach for UC treatment.

In order to control the release of mesalazine (MSZ) in the gastrointestinal tract to achieve better pharmacological effects in the colon, in this study, MSZ was added to hydroxypropyl-β-cyclodextrin (HP-β-CD) to form a water-soluble HP-β-CD/MSZ inclusion complex. Then, the inclusion compound was loaded into the structure of the bilayer polyelectrolyte complex microsphere formed by alginate (Alg), chitosan (Cs), and kappa carrageenan (κ-Car) as the hydrogel carrier, and the hydrogel beads with colon-specific release MSZ after oral administration were formed. The formed hydrogel beads have different swelling capabilities in different pH media and have the greatest swelling degree under pH 7.4. The encapsulation efficiency and drug loading of hydrogel beads can reach up to 83.23 and 18.31%, respectively, and the size of hydrogel beads can be reduced to less than 1 mm after drying, so that the size of oral administration can be reached. In vivo experiments also showed that the formed hydrogel beads had a better therapeutic effect on colitis than free drugs, and the microspheres were biodegradable, so the double-layer pH-sensitive microspheres could be effectively used in colon-targeting drug delivery.

β-Galactosidase plays an important role in medicine and dairy industry. In this study, a new glycoside hydrolase family 42 (GH42) β-galactosidase-encoding gene, gal42, was cloned from a newly isolated marine bacterium Bacillus sp. BY02 and expressed in Escherichia coli. Structural characterization indicated that the encoding β-galactosidase, Gal42, is a homotrimer in solution, and homology modeling indicated that it retains the zinc binding sites of the Cys cluster. The reaction activity of Gal42 was significantly increased by Zn2+ (229.6%) and other divalent metal ions (Mn2+, Mg2+, and Co2+), while its activity was inhibited by EDTA (53.9%). Meanwhile, the thermo-stability of the Gal42 was also significantly enhanced by 5 and 10 mM of zinc ion supplement, which suggested that the “Cys-Zn” motif played important roles in both structural stability and catalytic function. Furthermore, Gal42 showed effective lactose hydrolysis activity, which makes the enzyme hydrolyze the lactose in milk effectively. These properties make Gal42 a potential candidate in food technology.

Ulcerative colitis (UC) is highly correlated with the perturbation in gut microbiota. In our previous study, unsaturated alginate oligosaccharides (UAOS), enzymatic degradation products of alginate, showed great effect on obesity prevention through regulation of gut microbiota. The aim of this study was to investigate the ameliorative effect of UAOS in UC induced by dextran sulfate sodium (DSS) on mice models and further investigated its underlying mechanism. Herein, the results showed a dramatic alleviation of inflammatory reaction after UAOS supplement. Subsequent analyses showed that the community structure of gut microbiota changed. The abundance of phylum Firmicutes and Actinobacteria increased, while that of phylum Bacteroidetes decreased. Moreover, the balance renovation of the anti-inflammatory flora and pro-inflammatory flora is highly correlated with the alteration of host immune reaction. These results indicate that the protective effects of UAOS treatment attributes to the maintenance of the mucosal barrier function and inhibition of immune injury by regulating the gut microbiota.

Oligoalginate lyases catalyze the degradation of alginate polymers and oligomers into monomers, a prerequisite for biotechnological utilizing alginate. In this study, we report the cloning, expression and biochemical characterization of a new polysaccharide lyase (PL) family 17 oligoalginate lyase, OalV17, from the marine bacterium Vibrio sp. SY01. The recombinant OalV17 showed metal ion independent and detergent resistant properties. Furthermore, OalV17 is an exo-type enzyme that yields alginate monomers as the main product and recognizes alginate disaccharides as the minimal substrate. Site-directed mutagenesis followed by kinetic analysis indicates that the residue Arg231 plays a key role in substrate specificity. Furthermore, a rapid and efficient alginate monomer-producing method was developed directly from Laminaria japonica. These results suggest that OalV17 is a potential candidate for saccharification of alginate.

(1) Background: In the treatment of ulcerative colitis (UC), accurate delivery and release of anti-inflammatory drugs to the site of inflammation can reduce systemic side effects. (2) Methods: We took advantage of this goal to prepare resveratrol-loaded PLGA nanoparticles (RES-PCAC-NPs) by emulsification solvent volatilization. After layer-by-layer self-assembly technology, we deposited chitosan and alginate to form a three-layer polyelectrolyte film. (3) Results: It can transport nanoparticles through the gastric environment to target inflammation sites and slowly release drugs at a specific pH. The resulting RES-PCAC-NPs have an ideal average diameter (~255 nm), a narrow particle size distribution and a positively charged surface charge (~13.5 mV). The Fourier transform infrared spectroscopy showed that resveratrol was successfully encapsulated into PCAC nanoparticles, and the encapsulation efficiency reached 87.26%. In addition, fluorescence imaging showed that RES-PCAC-NPs with positive charges on the surface can effectively target and accumulate in the inflammation site while continuing to penetrate downward to promote mucosal healing. Importantly, oral RES-PCAC-NPs treatment in DSS-induced mice was superior to other results in significantly improved inflammatory markers of UC. (4) Conclusions: Our results strongly prove that RES-PCAC-NPs can target the inflamed colon for maximum efficacy, and this oral pharmaceutical formulation can represent a promising formulation in the treatment of UC.

As prebiotics, galacto-oligosaccharides (GOSs) can improve the intestinal flora and have important applications in medicine. β-galactosidases could promote the synthesis of GOSs in lactose and catalyze the hydrolysis of lactose. In this study, a new β-galactosidase gene (gal2A), which belongs to the glycoside hydrolase family 2, was cloned from marine bacterium Alteromonas sp. QD01 and expressed in Escherichia coli. The molecular weight of Gal2A was 117.07 kDa. The optimal pH and temperature of Gal2A were 8.0 and 40 °C, respectively. At the same time, Gal2A showed wide pH stability in the pH range of 6.0–9.5, which is suitable for lactose hydrolysis in milk. Most metal ions promoted the activity of Gal2A, especially Mn2+ and Mg2+. Importantly, Gal2A exhibited high transglycosylation activity, which can catalyze the formation of GOS from milk and lactose. These characteristics indicated that Gal2A may be ideal for producing GOSs and lactose-reducing dairy products.

The recent emergence of antibiotic-resistant bacteria requires the development of new antibiotics or new agents capable of enhancing antibiotic activity. Lysozyme degrades bacterial cell wall without involving antibiotic resistance and has become a new antibacterial strategy. However, direct use of native, active proteins in clinical settings is not practical as it is fragile under various conditions. In this study, lysozyme was integrated into chitosan nanoparticles (CS-NPs) by the ionic gelation technique to obtain lysozyme immobilized chitosan nanoparticles (Lys-CS-NPs) and then characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM), which showed a small particle size (243.1 ± 2.1 nm) and positive zeta potential (22.8 ± 0.2 mV). The immobilization significantly enhanced the thermal stability and reusability of lysozyme. In addition, compared with free lysozyme, Lys-CS-NPs exhibited superb antibacterial properties according to the results of killing kinetics in vitro and measurement of the minimum inhibitory concentration (MIC) of CS-NPs and Lys-CS-NPs against Pseudomonas aeruginosa (P. aeruginosa), Klebsiella pneumoniae (K. pneumoniae), Escherichia coli (E. coli), and Staphylococcus aureus (S. aureus). These results suggest that the integration of lysozyme into CS-NPs will create opportunities for the further potential applications of lysozyme as an anti-bacterium agent.

Chitooligosaccharide (COS) has been recognized to exhibit efficient anti-oxidant activity. Enzymatic hydrolysis using chitosanases can retain all the amino and hydroxyl groups of chitosan, which are necessary for its activity. In this study, a new chitosanase encoding gene, csnQ, was cloned from the marine Bacillus sp. Q1098 and expressed in Escherichia coli. The recombinant chitosanase, CsnQ, showed maximal activity at pH 5.31 and 60 °C. Determination of CsnQ pH-stability showed that CsnQ could retain more than 50% of its activity over a wide pH, from 3.60 to 9.80. CsnQ is an endo-type chitosanase, yielding chitodisaccharide as the main product. Additionally, in vitro and in vivo analyses indicated that chitodisaccharide possesses much more effective anti-oxidant activity than glucosamine and low molecular weight chitosan (LMW-CS) (~5 kDa). Notably, to our knowledge, this is the first evidence that chitodisaccharide is the minimal COS fragment required for free radical scavenging.

Chitosanase plays an important role in the production of chitooligosaccharides (CHOS), which possess various biological activities. Herein, a glycoside hydrolase (GH) family 46 chitosanase-encoding gene, csnB, was cloned from marine bacterium Bacillus sp. BY01 and heterologously expressed in Escherichia coli. The recombinant chitosanase, CsnB, was optimally active at 35 °C and pH 5.0. It was also revealed to be a cold-adapted enzyme, maintaining 39.5% and 40.4% of its maximum activity at 0 and 10 °C, respectively. Meanwhile, CsnB showed wide pH-stability within the range of pH 3.0 to 7.0. Then, an improved reaction condition was built to enhance its thermostability with a final glycerol volume concentration of 20%. Moreover, CsnB was determined to be an endo-type chitosanase, yielding chitosan disaccharides and trisaccharides as the main products. Overall, CsnB provides a new choice for enzymatic CHOS production.

As an important medical enzyme, β-galactosidases catalyze transgalactosylation to form prebiotic Galacto-Oligosaccharides (GOS) that assist in improving the effect of intestinal flora on human health. In this study, a new glycoside hydrolase family 2 (GH2) β-galactosidase-encoding gene, galA, was cloned from the Antarctic bacterium Alteromonas sp. ANT48 and expressed in Escherichia coli. The recombinant β-galactosidase GalA was optimal at pH 7.0 and stable at pH 6.6−7.0, which are conditions suitable for the dairy environment. Meanwhile, GalA showed most activity at 50 °C and retained more than 80% of its initial activity below 40 °C, which makes this enzyme stable in normal conditions. Molecular docking with lactose suggested that GalA could efficiently recognize and catalyze lactose substrates. Furthermore, GalA efficiently catalyzed lactose degradation and transgalactosylation of GOS in milk. A total of 90.6% of the lactose in milk could be hydrolyzed within 15 min at 40 °C, and the GOS yield reached 30.9%. These properties make GalA a good candidate for further applications.

Bacterial biofilm causes severe antibiotic resistance. An extracellular polymeric substance (EPS) is the main component in the bacterial biofilm. Alginate is a key EPS component in the biofilm of Pseudomonas aeruginosa and responsible for surface adhesion and stabilization of biofilm. Alginate lyase has emerged as an efficient therapeutic strategy targeting to degrade the alginate in the biofilm of P. aeruginosa. However, the application of this enzyme is limited by its poor stability. In this study, chitosan nanoparticles (CS-NPs) were synthesized using low molecular weight chitosan and alginate lyase Aly08 was immobilized on low molecular weight chitosan nanoparticles (AL-LMW-CS-NPs). As a result, the immobilization significantly enhanced the thermal stability and reusability of Aly08. In addition, compared with free Aly08, the immobilized AL-LMW-CS-NPs exhibited higher efficiency in inhibiting biofilm formation and interrupting the established mature biofilm of P. aeruginosa, which could reduce its biomass and thickness confirmed by confocal microscopy. Moreover, the biofilm disruption greatly increased the antibiotic sensitivity of P. aeruginosa. This research will contribute to the further development of alginate lyase as an anti-biofilm agent.

Obesity and its related complications have become one of the leading problems affecting human health. However, current anti-obesity treatments are limited by high cost and numerous adverse effects. In this study, we investigated the use of a non-toxic green food additive, known as unsaturated alginate oligosaccharides (UAOS) from the enzymatic degradation of Laminaria japonicais, which showed effective anti-obesity effects in a high-fat diet (HFD) mouse model. Compared with acid hydrolyzed saturated alginate oligosaccharides (SAOS), UAOS significantly reduced body weight, serum lipid, including triacylglycerol (TG), total cholesterol (TC) and free fatty acids (FFA), liver weight, liver TG and TC, serum alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels, adipose mass, reactive oxygen species (ROS) formation, and accumulation induced in HFD mice. Moreover, the structural differences in β-d-mannuronate (M) and its C5 epimer α-l-guluronate (G) did not cause significant functional differences. Meanwhile, UAOS significantly increased both AMP-activated protein kinase α (AMPKα) and acetyl-CoA carboxylase (ACC) phosphorylation in adipocytes, which indicated that UAOS had an anti-obesity effect mainly through AMPK signaling. Our results indicate that UAOS has the potential for further development as an adjuvant treatment for many metabolic diseases such as fatty liver, hypertriglyceridemia, and possibly diabetes.

Alginate oligosaccharides (AOS) show versatile bioactivities. Although various alginate lyases have been characterized, enzymes with special characteristics are still rare. In this study, a polysaccharide lyase family 7 (PL7) alginate lyase-encoding gene, aly08, was cloned from the marine bacterium Vibrio sp. SY01 and expressed in Escherichia coli. The purified alginate lyase Aly08, with a molecular weight of 35 kDa, showed a specific activity of 841 U/mg at its optimal pH (pH 8.35) and temperature (45 °C). Aly08 showed good pH-stability, as it remained more than 80% of its initial activity in a wide pH range (4.0−10.0). Aly08 was also a thermo-tolerant enzyme that recovered 70.8% of its initial activity following heat shock treatment for 5 min. This study also demonstrated that Aly08 is a polyG-preferred enzyme. Furthermore, Aly08 degraded alginates into disaccharides and trisaccharides in an endo-manner. Its thermo-tolerance and pH-stable properties make Aly08 a good candidate for further applications.

Chitosanases play an important role in chitosan degradation, forming enzymatic degradation products with several biological activities. Although many chitosanases have been discovered and studied, the enzymes with special characteristics are still rather rare. In this study, a new chitosanase, CsnM, with an apparent molecular weight of 28 kDa was purified from the marine bacterium Pseudoalteromonas sp. SY39. CsnM is a cold-adapted enzyme, which shows highest activity at 40 °C and exhibits 30.6% and 49.4% of its maximal activity at 10 and 15 °C, respectively. CsnM is also a thermo-tolerant enzyme that recovers 95.2%, 89.1% and 88.1% of its initial activity after boiling for 5, 10 and 20 min, respectively. Additionally, CsnM is an endo-type chitosanase that yields chitodisaccharide as the main product (69.9% of the total product). It’s cold-adaptation, thermo-tolerance and high chitodisaccharide yield make CsnM a superior candidate for biotechnological application to produce chitooligosaccharides.

Chitooligosaccharides (CHOS) have gained increasing attention because of their important biological activities. Enhancing the efficiency of CHOS production essentially requires screening of novel chitosanase with unique characteristics. Therefore, a rapid and efficient one-step affinity purification procedure plays important roles in screening native chitosanases. In this study, we report the design and synthesis of affinity resin for efficient purification of native chitosanases without any tags, using chitodisaccharides (CHDS) as an affinity ligand, to couple with Sepharose 6B via a spacer, cyanuric chloride. Based on the CHDS-modified affinity resin, a one-step affinity purification method was developed and optimized, and then applied to purify three typical glycoside hydrolase (GH) families: 46, 75, and 80 chitosanase. The three purified chitosanases were homogeneous with purities of greater than 95% and bioactivity recovery of more than 40%. Moreover, we also developed a rapid and efficient affinity purification procedure, in which tag-free chitosanase could be directly purified from supernatant of bacterial culture. The purified chitosanases samples using such a procedure had apparent homogeneity, with more than 90% purity and 10⁻50% yield. The novel purification methods established in this work can be applied to purify native chitosanases in various scales, such as laboratory and industrial scales.

Metalloproteases are emerging as useful agents in the treatment of many diseases including arthritis, cancer, cardiovascular diseases, and fibrosis. Studies that could shed light on the metalloprotease pharmaceutical applications require the pure enzyme. Here, we reported the structure-based design and synthesis of the affinity medium for the efficient purification of metalloprotease using the 4-aminophenylboronic acid (4-APBA) as affinity ligand, which was coupled with Sepharose 6B via cyanuric chloride as spacer. The molecular docking analysis showed that the boron atom was interacting with the hydroxyl group of Ser176 residue, whereas the hydroxyl group of the boronic moiety is oriented toward Leu175 and His177 residues. In addition to the covalent bond between the boron atom and hydroxyl group of Ser176, the spacer between boronic acid derivatives and medium beads contributes to the formation of an enzyme-medium complex. With this synthesized medium, we developed and optimized a one-step purification procedure and applied it for the affinity purification of metalloproteases from three commercial enzyme products. The native metalloproteases were purified to high homogeneity with more than 95% purity. The novel purification method developed in this work provides new opportunities for scientific, industrial and pharmaceutical projects.

Unsaturated alginate disaccharides (UADs), enzymatically derived from the degradation of alginate polymers, are considered powerful antioxidants. In this study, a new high UAD-producing alginate lyase, AlySY08, has been purified from the marine bacterium Vibrio sp. SY08. AlySY08, with a molecular weight of about 33 kDa and a specific activity of 1070.2 U/mg, showed the highest activity at 40 °C in phosphate buffer at pH 7.6. The enzyme was stable over a broad pH range (6.0–9.0) and retained about 75% activity after incubation at 40 °C for 2 h. Moreover, the enzyme was active in the absence of salt ions and its activity was enhanced by the addition of NaCl and KCl. AlySY08 resulted in an endo-type alginate lyase that degrades both polyM and polyG blocks, yielding UADs as the main product (81.4% of total products). All these features made AlySY08 a promising candidate for industrial applications in the production of antioxidants from alginate polysaccharides.

Breast cancer (BC) is the most common malignancy in women worldwide, and the standard treatment is chemotherapy or radiotherapy after surgery. In order to reduce the side effects of chemotherapy, various nanoparticles (NPs) have been discovered and synthesized, which has become a promising treatment for BC. In this study, a co-delivery nanodelivery drug system (Co-NDDS) was designed and synthesized with 2,3-dimercaptosuccinic acid (DMSA) coated Fe3O4 NPs as core encapsulated into chitosan/alginate nanoparticles (CANPs) shell, doxorubicin (DOX) and hydroxychloroquine (HCQ) as loading drugs. Smaller NPs carrying DOX (FeAC-DOX NPs) were loaded into larger NPs containing HCQ (FeAC-DOX@ PC-HCQ NPs) by ionic gelation and emulsifying solvent volatilization methods. The physicochemical properties of this Co-NDDS were characterised, followed by in vitro studies of the anticancer effects and mechanisms using two different BC cell lines, MCF-7 cells and MDA-MB-231 cells. The results indicated that the Co-NDDS showcases exemplary physicochemical qualities and encapsulation capacity, facilitating accurate intracellular release through pH-sensitive attributes. Importantly, NPs can significantly increase the in vitro cytotoxicity of co-administered drugs and effectively inhibit the autophagy level of tumour cells. The Co-NDDS constructed in this study provides a promising strategy for the treatment of BC. [ABSTRACT FROM AUTHOR]

Ulcerative colitis (UC) is a chronic inflammatory bowel disease (IBD). Its etiology is unclear. Much evidence suggests that the death of abnormal intestinal epithelial cells (IECs) leads to intestinal barrier disruption, and the subsequent inflammatory response plays a vital role in UC. Pyroptosis is a form of programmed inflammatory cell death, and the role of pyroptosis in UC etiology remains to be explored. This study identified 10 hub genes in pyroptosis by gene expression profiles obtained from the GSE87466 dataset. Meanwhile, the biomarkers were screened based on gene significance (GS) and module membership (MM) through the Weighted Gene Co-Expression Network Analysis (WGCNA). The following analysis indicated that hub genes were closely associated with the UC progression and therapeutic drug response. The single-cell RNA (scRNA) sequencing data from UC patients within the GSE162335 dataset indicated that macrophages were most related to pyroptosis. Finally, the expression of hub genes and response to the therapeutic drug [5-aminosalicylic acid (5-ASA)] were verified in dextran sulfate sodium (DSS)-induced colitis mice. Our study identified IL1B as the critical pyroptosis-related biomarker in UC. The crosstalk between macrophage pyroptosis and IEC pyroptosis may play an essential role in UC, deserving further exploration. [ABSTRACT FROM AUTHOR]