Enzymatic Digestion Research Articles

Prophylactic Effect and Antiulcerogenic Potential of Probiotic Lactiplantibacillus plantarum E1K2R2 and its Exopolysaccharide Against Ibuprofen-Induced Acute Gastric Ulcer.

Lactiplantibacillus plantarum (Lpb. plantarum), as a safe probiotic microorganism, has been documented for its production of multiple bioactive compounds, such as exopolysaccharides (EPS), which have been used in the treatment of many gastrointestinal diseases, including gastric ulcers. The present study aims to investigate the prophylactic and antiulcerogenic effects of the potential probiotic Lbp. plantarum E1K2R2 and its EPS against ibuprofen-induced gastric ulcer. A gastric ulcer model was established by feeding fasted rats with ibuprofen at a single dose (200 mg/kg body weight). The Lpb. plantarum E1K2R2 (109 CFU), its EPS (200 mg/kg bw), and the anti-ulcer reference drug (omeprazole) (20 mg/kg bw) were separately used to feed rats for seven consecutive days before ibuprofen administration. The mechanisms were meticulously examined, focusing on the anti-secretory activity and mucus production as well as the anti-inflammatory and antioxidant activities. The findings revealed that the gastro-preventive effect of Lbp. plantarum E1K2R2 (88.43%) was higher than that of the EPS (66.26%) and close to that of omeprazole (89.87%). This effect was achieved through similar mechanisms, including regulation of the secretory activity, augmentation of mucus production, mitigation of inflammation, and enhancement of the gastric mucosa's antioxidant capacity. Moreover, it was found that Lbp. plantarum E1K2R2 and its EPS induce the activities of gastric antioxidant enzymes: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and S-transferase (GST); enhance glutathione (GSH) content; and reduce mucosal nitric oxide (NO), myeloperoxidase (MPO), and malondialdehyde (MDA) levels. Furthermore, histopathological and hematological examinations confirmed that both pre-treatments could effectively maintain the structural integrity of the gastric mucosa and improve some hematological parameters, respectively. This implies that Lpb. plantarum E1K2R2 and its EPS possess the potential to counteract ibuprofen-associated gastric ulcers, leveraging a variety of protective mechanisms.

Nerve Regeneration Through Differentiation of Endometrial-Derived Mesenchymal Stem Cells into Nerve-Like Cells Using Polyacrylonitrile/Chitosan Conduit and Berberine in a Rat Sciatic Nerve Injury Model.

Nervous injuries are common in humans. One of the most advanced treatment methods is neural tissue engineering. This research aims to utilize nerve-like cells (NLCs) derived from endometrial mesenchymal stem cells (EnMSCs) on a polyacrylonitrile/chitosan (PAN/CS) scaffold, along with berberine, for the reconstruction of a rat sciatic nerve injury model. In this experimental study, EnMSCs were obtained through enzymatic digestion and identified using flow cytometry and their differentiation into adipocyte and osteoblast. PAN nanofiber scaffolds were produced through electrospinning, and EnMSCs were neurally differentiated on these scaffolds for grafting into an animal model. The expression of Nestin, Map-2, Tuj-1, and NF genes in NLCs was confirmed through RT-PCR and immunocytochemistry. Twenty-five adult male rats were used in this study, divided into 5 groups: (1) Scaffold/Cells/Berberine, (2) Scaffold/Cells, (3) Scaffold, (4) Berberine, and (5) Control. The animals were maintained for 8 weeks, and their sciatic nerve function (SFI) was assessed. Additionally, histological examinations were performed using hematoxylin/eosin, luxol fast blue staining, and immunohistochemistry. According to the results, extraction, identification, and differentiation of EnMSCs and fabrication of PAN conduit and its transplantation were successfully performed. The best behavioral performance and histology were observed in the Scaffold/Cells/Berberine group. The SFI test results were -24.08 for the Scaffold/Cells/Berberine group and -39.27 for the control group. The nerve diameter in these two groups was 591 µm and 80 µm, respectively, and the percentage of new nerve formation was 18.5% in the Scaffold/Cells/Berberine group and 0.2% in the control group. The immunohistochemistry results demonstrated that the intensity of the green color was higher in the groups with cells compared to the groups without cells. Furthermore, in the luxol staining results, all groups showed a significant improvement compared to the control group. In the Scaffold/Cells/Berberine group, fibers, and axons appeared denser, more organized, and displayed a higher intensity of blue staining. According to the results of this study, EnMSCs demonstrated efficient differentiation into NLCs. With the assistance of PAN/CS scaffolds and simultaneous administration of berberine, EnMSCs have the potential for nerve regeneration and recovery from sciatic nerve injury in the rat animal model.

Cholesterol-lowering effect of Pediococcus strains isolated from mother’s milk in mice

Purpose The Pediococcus strains belong to the group of lactic acid bacteria (LAB). This study aims to isolate and identify the Pediococcus spp. from mother’s milk, and investigate their tolerance to low pH and bile salts, antibacterial activity, attachment to HT-29 cells and effect on cholesterol-lowering and digestive enzymes in mice. Design/methodology/approach Pediococcus species were isolated from mother’s milk, then tested for tolerance to acid and bile salts, their antibacterial effect and attachment to HT-29 cell line. In mice experiment, the levels of triglyceride (TG), total cholesterol (TC), HDL cholesterol (HDL-C), LDL cholesterol (LDL-C) and digestive enzymes such as amylase and lipase in serum of mice were measured after administration of 108 CFU/mL bacterial suspension. Findings A total of 24 Pediococcus spp. including 16 isolates of Pediococcus pentosaceus and 8 isolates of Pediococcus acidilactici were isolated. Fifteen isolates (62.5%) were able to survive in low pH and bile salt concentration. Ten isolates (41.6%) exhibited the most powerful inhibitory effects against both Gram-positive and Gram-negative bacterial pathogens. A total of four Pediococcus isolates (16.67%) showed strong attachment to HT-29 cells. In contrast to HDL-C and amylase, the serum levels of TC, TG and LDL-C were reduced and lipase was elevated significantly following administration of mother milk-derived Pediococcus isolates. Originality/value The Pediococcus isolates demonstrated good probiotic properties in particular cholesterol-lowering ability in mice. Thus, after more studies they can be considered as probiotic strain(s).

A closed-loop negative feedback model for the pancreas: A new paradigm and pathway to a cure.

To develop a model that describes how the pancreas functions, how the rate of synthesis of digestive enzymes is regulated, and finally what puts the pancreas to rest between meals. We applied the principals of control theory to previously published canine data to develop a model for how the canine pancreas functions. Using this model, we then describe the steps needed to apply this model to the human pancreas. This new closed-loop negative feedback model describes what regulates digestive enzyme synthesis. This model is based on basolateral exocytosis of butyrylcholinesterase (BCHE) into the interstitial space. It is this level of BCHE * BCHE activity that controls the rate of canine pancreas digestive enzyme synthesis, and in the absence of stimulation from the vagus nerve, puts the pancreas to rest between meals. Finding secretagogue-specific inhibitory enzymes in the human pancreas that are analogous to BCHE in the canine, and blocking its associated receptors, may lead to a cure for human pancreatitis.

Cross-linking affecting properties and in-vitro digestibility of starch-sucrose ester complexes

This study investigated the effects of cross-linking on the characteristics and in-vitro digestibility of starch-sucrose ester (SE) complexes. To achieve this, corn starch (CS) was cross-linked with various concentrations of sodium trimetaphosphate /sodium tripolyphosphate (5 %, 10 %, and 15 %). Subsequently, cross-linked starches (CLS) were complexed with SE through hydrothermal treatment. X-ray diffraction analysis revealed that V-type amylose-lipid complexes formed by the interaction between CS and SE. The resultant CS-SE complex significantly reduced CS digestibility, increasing its resistant starch (RS) content from 10.19 % to 22.71 %. The cross-linking modification did not alter the crystalline pattern of the CS-SE complex. Several CLS-SE complexes demonstrated higher enzymatic resistance compared to the CS-SE complex. The CLS10-SE complex exhibited the highest RS content of 39.37 % when the cross-linking agent concentration was 10 %. This phenomenon may be attributable to the cross-linking reaction having enhanced the interaction between starch molecular chains, reducing the solubility and swelling power, thereby hindering the accessibility of starch chains to digestive enzymes. These findings indicate that cross-linking modification is a practical approach to improving the anti-digestion performance of starch-lipid complexes.

Alleviative effects of C60 fullerene nanoparticles on arsenate transformation and toxicity to Danio rerio

Widely-used C60 fullerene nanoparticles (C60) result in their release into the aquatic environment, which may affect the distribution and toxicity of pollutants such as arsenic (As), to aquatic organism. In this study, arsenate (As(V)) accumulation, speciation and subcellular distribution was determined in Danio rerio (zebrafish) intestine, head and muscle tissues in the presence of C60. Meanwhile we compared how single-walled carbon nanotubes (SWCNTs), multi-walled carbon nanotubes (MWCNTs), graphene oxide (GO) and graphene (GN) nanoparticles alter the behaviors of As(V). Results showed that C60 significantly inhibited As accumulation and toxicity in D. rerio, due to a decrease in total As and monomethylarsonic acid (MMA) and As(V) species concentrations, a lower relative distribution in the metal-sensitive fraction (MSF). It was attributed that C60 may coat As(V) ion channels and consequently, affect the secretion of digestive enzymes in the gut, favoring As excretion and inhibiting As methylation. Similarly, MWCNTs reduced the species concentration of MMA and As(V) in the intestines, low GSH (glutathione) contents in the intestine. Due to the disparity of other carbon-based nanomaterial morphologies, SWCNTs, GO and GN exhibited the various effects on the toxicity of As(V). In addition, the possible pathway of arsenobetaine (AsB) biosynthesis included migration from the intestine to muscle in D. rerio, with the precursor of AsB likely to be 2-dimethylarsinylacetic acid (DMAA). The results of this study suggest that C60 is beneficial for controlling As(V) pollution and reducing the impact of As(V) biogeochemical cycles throughout the ecosystem.

Valorization of Lignocellulose with One-Step Acidified Monophasic Phenoxyethanol Fractionation.

Effective fractionation of lignocelluosic biomass and subsequent valorization of all three major components under mild conditions were achieved. Pretreatment with acidified monophasic phenoxyethanol (EPH) efficiently removed 92.6 % lignin and 80 % xylan from poplar at 110 °C in 60 min, yielding high-value EPH-xyloside, EPH-modified lignin (EPHL), and a solid residue nearly purely composed of carbohydrates. After removing the grafted acetyl groups using 1 % NaOH at 50 °C, the highest enzymatic digestibility reached 92.3 %. EPHL could be recovered in high yield and purity with an uncondensed structure, while xylose was converted to EPH-xyloside, a potential precursor in biomedical industries. Additionally, the acidified monophasic EPH solvent could effectively fractionate biomass from species other than hardwood, achieving over 70 % delignification from recalcitrant pinewood under the same mild conditions, demonstrating the high potential of monophasic EPH pretreatment.

Quenching Trypsin Is Unnecessary in Filter-Based Bottom-Up Proteomics.

Quenching digestions in proteomics prior to analysis is routine in order to eliminate residual protease activity. Residual activity leads to overdigestion, nonspecific star-activity, and back-exchange in isotopic 18O quantitation. Chemical and isobaric labeling (e.g., TMT/iTRAQ) of proteins or peptides for mass spectrometry-based proteomics is generally incompatible with ubiquitous postdigestion acidification. This necessitates buffer exchange and pH adjustments. We demonstrate that quenching is unnecessary with peptides generated from protein filter-traps, as trypsin activity and intact trypsin are negligible in the eluate from these preparations. Labeling can be directly performed on enzymatic digests from these methods, improving recovery, throughput, and ease of automation.

Quenching Trypsin Is Unnecessary in Filter-Based Bottom-Up Proteomics.

Quenching digestions in proteomics prior to analysis is routine in order to eliminate residual protease activity. Residual activity leads to overdigestion, nonspecific star-activity, and back-exchange in isotopic 18O quantitation. Chemical and isobaric labeling (e.g., TMT/iTRAQ) of proteins or peptides for mass spectrometry-based proteomics is generally incompatible with ubiquitous postdigestion acidification. This necessitates buffer exchange and pH adjustments. We demonstrate that quenching is unnecessary with peptides generated from protein filter-traps, as trypsin activity and intact trypsin are negligible in the eluate from these preparations. Labeling can be directly performed on enzymatic digests from these methods, improving recovery, throughput, and ease of automation.

Integrated biofloc technology in red tilapia aquaculture: Salinity-dependent effects on water quality, parental stock physiology, reproduction, and immune responses

AbstractThe study examines the impact of integrated biofloc technology (BFT), different salinity levels, and their combined effects over 90 days on various physiological parameters. The investigation includes growth performance and feed utilization, water quality, the chemical composition of biofloc and fish, digestive enzymes, reproductive performance, stress and biochemical indicators, and antioxidant-immune responses in red tilapia (Oreochromis spp.) broodstock. The fish were initially weighed (males: n = 270; 104 ± 0.96 g; females: n = 270; 93.2 ± 0.66 g) and subsequently divided into 12 treatment groups (6 for males and 6 for females) spread across 36 separate tanks (3 tanks per treatment; 45 fish per treatment; 15 fish/tank). The treatments involved three salinity levels (18, 28, and 36 ppt) in both clear water (CW) and BFT systems. The outcomes demonstrated that fish in the 36 ppt salinity with BFT treatment demonstrated significant improvements (P < 0.05) in growth parameters (final body weight, weight gain, and specific growth rate, feed intake, and feed conversion ratio). The condition factor in BFT groups increased in all salinity situations. The survival rates of broodstock were consistently high in all experimental conditions The study found that BFT and salinity significantly impacted (P < 0.05) whole body contents (moisture, protein, lipid, and ash) in both males and females. Water quality parameters showed variations between BFT and CW, with notable impacts (P < 0.05) on dissolved oxygen and pH. The BFT and salinity influenced digestive enzyme activities (protease, amylase, and lipase) and reproductive performance (males) and the 36 ppt salinity with BFT recorded the highest values. The hemato-biochemical and antioxidant-immune responses were also impacted by BFT and salinity exposure. The study highlights the potential benefits of incorporating BFT into red tilapia aquaculture systems, particularly in optimizing growth, health, and reproductive performance under various salinity conditions, which can enhance sustainable intensification, disease control, and environmental stewardship.

Spatiotemporal proteolytic susceptibility of allergens: positive or negative effects on the allergic sensitization?

From their expression in their respective allergenic source to their processing by antigen presenting cells, allergens continuously encounter proteases. The ability of allergens to resist to proteolysis by digestive enzymes or host-cell/microbial proteases is considered as an important property that influences their allergenic potential. However, the relationship between proteolytic stability and allergenicity is much more complex and depends on various factors, such as the protein structure dynamics, the exposure level, the route of sensitization, and their respective protease susceptibility. In this review, we summarize and discuss the current knowledge on several aspects of allergen proteolytic stability in different environments including the allergenic sources, routes of sensitization (skin, respiratory tract, gastrointestinal tract) and endolysosomal compartment of antigen-presenting cells. Proteolytic stability alone cannot represent a definitive criterion to allergenicity. The proteolytic susceptibility of allergens in processed extracts can affect allergy diagnosis and immunotherapy. Furthermore, the fine tuning of allergen stability during antigen processing can be exploited for the development of novel immunotherapeutic strategies.

Characterization of BrGH3A, a bovine rumen-derived glycoside hydrolase family 3 β-glucosidase with a permuted domain arrangement.

The bovine rumen contains a large consortium of residential microbes that release a variety of digestive enzymes for feed degradation. However, the utilization of these microbial enzymes is still limited because these rumen microorganisms are mostly anaerobes and are thus unculturable. Therefore, we applied a sequence-based metagenomic approach to identify a novel 2,445-bp glycoside hydrolase family 3 β-glucosidase gene known as BrGH3A from the metagenome of bovine ruminal fluid. BrGH3A β-glucosidase is a 92-kDa polypeptide composed of 814 amino acid residues. Unlike most glycoside hydrolases in the same family, BrGH3A exhibited a permuted domain arrangement consisting of an (α/β)6 sandwich domain, a fibronectin type III domain and a (β/α)8 barrel domain. BrGH3A exhibited greater catalytic efficiency toward laminaribiose than cellobiose. We proposed that BrGH3A is an exo-acting β-glucosidase from Spirochaetales bacteria that is possibly involved in the intracellular degradation of β-1,3-/1,4-mixed linkage glucans that are present in grass cell walls. BrGH3A exhibits rich diversity in rumen hydrolytic enzymes and may represent a member of a new clan with a permuted domain topology within the large family.

The effects of β-mannanase supplementation on growth performance, digestive enzyme activity, cecal microbial communities, and short-chain fatty acid production in broiler chickens fed diets with different metabolizable energy levels.

This study assessed the effects of β-mannanase (BM) supplementation on growth performance, digestive enzyme activity, cecal microbial communities, and short-chain fatty acid (SCFA) production in broiler chickens fed diets with different metabolizable energy (ME) levels. A total of 1,296 male one-d-old Cobb 500 broilers were randomly distributed in a 3 × 2 factorial arrangement (three ME levels × 0 or 200g/ton BM), with 6 replicates per treatment combination. The three ME levels were 3,000 (ME1), 2,930 (ME2), and 2,860 (ME3) kcal/kg, respectively, during the 0-3 w-old stages and 3,150 (ME1), 3,080 (ME2), and 3,010 (ME3) kcal/kg, respectively, during the 3-6 w-old stages. Reducing ME levels increased broiler feed intake (P = 0.036) and decreased average daily gain (ADG, P = 0.002) during the entire period. While BM supplementation increased ADG (P = 0.002) and improved the feed conversion ratio (P = 0.001) during the 0-3 w-old stages, with no effect during the 3-6 w-old stages. Overall, reducing ME levels increased pancreatic lipase (P = 0.045) and amylase (P = 0.013) activity and duodenal amylase activity (P = 0.047). Notably, BM supplementation significantly increased pancreatic lipase activity (P = 0.015) and increased lipase (P = 0.029) and amylase (P = 0.025) activities in the jejunal chyme. Although diet or enzyme supplementation did not affect microbial diversity, significant differences in microbial communities were observed. At the genus level, decreasing ME levels significantly affected the average abundances of Tyzzerella (P = 0.028), Candidatus_Bacilloplasma (P = 0.001), Vibrio (P = 0.005) and Anaerotruncus (P = 0.026) among groups, whereas BM supplementation reduced the average abundances of Escherichia-Shigella (P = 0.048) and increased the average abundances of Barnesiella (P = 0.047), Ruminococcus (P = 0.020), Alistipes (P = 0.050), and Lachnospiraceae_unclassified (P = 0.009). SCFA concentrations strongly depended on bacterial community composition, and BM supplementation increased acetic acid (P = 0.004), propionic acid (P = 0.016), and total SCFA concentrations. In conclusion, BM supplementation improved the performance of younger broilers, and both enzyme supplementation and reduced ME levels positively affected digestive enzyme activity and intestinal microflora.

The effect of various dietary MUFA content and n-6/n-3 ratios on rainbow trout (Oncorhynchus mykiss) fed on a whole plant protein based diet: growth performance, digestive enzymes activity, intestinal morphometry and fatty acids profile of muscle and liver.

The effect of diets with various dietary MUFA content and n-6/n-3 ratios, diets 1 (55.33, 1.00), 2 (25.30, 1.00), 3 (55.13, 2.05), 4 (24.92, 2.03), 5 (54.94, 8.06) and 6 (24.91, 8.06) and a control diet with fish oil (FO), diet 7 (33.60, 5.97), was studied on growth and digestive physiology of rainbow trout, Oncorhynchus mykiss. Juveniles, 14.65g ± 0.17, were fed the experimental diets for eight weeks. Those fish fed diet 1 (55.33, 1.00) had the lowest growth and nutritional indices, while fish fed diet 6 (24.91, 8.06) possessed the highest indices. However, fish fed D7 (33.60, 5.97) was intermediate in this regard. Proximate body composition results revealed no significant differences among various experimental groups (P > 0.05). Fish fed on diet 1 possessed the highest alkaline protease activity, while fish raised on diet 2 showed the lowest enzyme activity (P < 0.05). Fish fed on diets 1, 3 and 4 had the highest lipase activity (P > 0.05). Amylase activity and intestinal parameters did not differ among groups (P > 0.05). MUFA contents of liver and muscle reflected the MUFA contents of the diets; however, liver PUFA contents was not affected by dietary PUFA contents (P < 0.05). However, 18:3n-3 contents of liver decreased as dietary n-6/n-3 ratio increased (P < 0.05). Furthermore, the liver 20:4n-6 contents considerably increased as dietary n-6/n-3 increased to 8.06 (P < 0.05). Muscle 22:6n-3 content of groups 1 to 6 was lower than group 7 (P < 0.05). In conclusion, rainbow trout was considerably capable of de novo synthesis of 22:6n-3. Meanwhile, increasing dietary n-6/n-3 ratio to 8.06 decreased the muscle 22:6n-3 content which requires further considerations regarding fish immune competence and nutritional quality for human consumption.

Complementary and Alternative Medicine for Autism - A Systematic Review.

Complementary and Alternative Medicine (CAM) is a therapeutic option currently used by autistic people with continued interest and uptake. There remains limited evidence regarding the efficacy of CAM use in autism. The aim of this systematic review is to comprehensively review published clinical trials to explore the efficacy of CAM in autism. A systematic literature review of available research published from June 2013 to March 2023 was conducted. Our literature search identified 1826 eligible citations, and duplications removed (n = 694) with 102 articles eligible for title/abstract screening. After full text review, 39 studies were included. The results of this systematic review identified that for autistic people, vitamin and mineral supplements may only be of benefit if there is a deficiency. The results also found that the main interventions used were dietary interventions and nutraceuticals, including targeted supplements, vitamins and minerals, omega 3s and prebiotics, probiotics and digestive enzymes. The evidence does not support some of the most frequently utilised dietary interventions, such as a Gluten Free Casein Free (GFCF) diet, and the use of targeted nutraceutical supplements may be of benefit, but more conclusive research is still required to direct safe and effective treatment.

Response of growth and physiological enzyme activities in Eriogyna pyretorum to various host plants.

Morphological attributes and chemical composition of host plants shape growth and development of phytophagous insects via influences on their behavior and physiological processes. This research delves into the relationship between Eriogyna pyretorum and various host plants through studuying how feeding on different host tree species affect growth, development, and physiological enzyme activities. We examined E. pyretorum response to three distinct host plants: Camphora officinarum, Liquidambar formosana and Pterocarya stenoptera. Notably, larvae feeding on C. officinarum and L. formosana displayed accelerated development, increased pupal length, and higher survival rates compared to those on P. stenoptera. This underlines the pivotal role of host plant selection in shaping the E. pyretorum's life cycle. The activities of a-amylase, lipase and protective enzymes were the highest in larvae fed on the most suitable host L.formosana which indicated that the increase of these enzyme activities was closely related to growth and development. Furthermore, our investigation revealed a relationship between enzymatic activities and host plants. Digestive enzymes, protective enzymes, and detoxifying enzymes exhibited substantial variations contingent upon the ingested host plant. Moreover, the total phenolics content in the host plant leaves manifested a noteworthy positive correlation with catalase and lipase activities. In contrast, a marked negative correlation emerged with glutathione S-transferase and α-amylase activities. The total developmental duration of larvae exhibited a significant positive correlation with the activities of GST and CarE. The survival rate of larvae showed a significant positive correlation with CYP450. These observations underscore the insect's remarkable adaptability in orchestrating metabolic processes in accordance with available nutritional resources. This study highlights the interplay between E.pyretorum and its host plants, offering novel insights into how different vegetation types influence growth, development, and physiological responses. These findings contribute to a deeper comprehension of insect-plant interactions, with potential applications in pest management and ecological conservation.

A single dose of VEGF-A circular RNA sustains in situ long-term expression of protein to accelerate diabetic wound healing

Diabetic foot ulcer (DFU), which is characterised by damage to minute blood vessels or capillaries around wounds, is one of the most serious and dreaded complications of diabetes. It is challenging to repair chronic non-healing DFU wounds. Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis and promotes wound healing in DFU. However, it is difficult to sustainably deliver VEGF to the wound site owing to its poor stability and easy degradation. To overcome this challenge, lipid nanoparticles (LNP) encapsulating circular RNA (circRNA) encoding VEGF-A have been developed to continuously generate and release VEGF-A and accelerate diabetic wound healing. First, VEGF-A circRNA was synthesized using group I intron autocatalysis strategy and confirmed by enzyme digestion, polymerase chain reaction, and sequencing assay. VEGF-A circRNA was encapsulated in ionizable lipid U-105-derived LNP (U-LNP) using microfluidic technology to fabricate U-LNP/VEGF-A circRNA. For comparison, a commercially ionizable lipid ALC-0315-derived LNP (A-LNP) encapsulating circRNA (A-LNP/circRNA) was used. Dynamic light scattering and transmission electron microscopy characterization indicated that U-LNP/circRNA had spherical structure with an average diameter of 108.5 nm, a polydispersity index of 0.22, and a zeta potential of −3.31 mV. The messenger RNA (mRNA) encapsulation efficiency (EE%) of U-LNP was 87.12%. In vitro transfection data confirmed better stability and long-term VEGF-A expression of circRNA compared with linear mRNA. Assessment of cytotoxicity and innate immunity further revealed that U-LNP/circRNA was biocompatible and induced a weak congenital immune response. Cell scratch and angiogenesis tests demonstrated the bioactivity of U-LNP/VEGF-A circRNA owing to its VEGF-A expression. In situ bioluminescence imaging of firefly luciferase (F-Luc) probe and ELISA demonstrated that circRNA had long-term and strong expression of VEGF-A in the first week, and a gradual decrease in the next week at the wound site and surrounding areas. Finally, a diabetic mouse model was used to validate the healing effect of U-LNP/VEGF-A circRNA formulation. The results showed that a single dose of U-LNP/VEGF-A circRNA administered by dripping resulted in almost complete wound recovery on day 12, which was significantly superior to that of U-LNP/VEGF-A linear mRNA, and it also outperformed recombinant human vascular endothelial growth factor (rhVEGF) injection and A-LNP/circRNA dripping. Histological analysis confirmed the healing efficiency and low toxicity of U-LNP/VEGF-A circRNA formulation. Together, VEGF-A circRNA delivered by U-105-derived LNP showed good performance in wound healing, which was ascribed to the long-term expression and continuous release of VEGF-A, and has potential applications for the treatment of diabetic foot ulcer wounds.

Methionine Source and Level Modulate Gut pH, Amino Acid Transporters and Metabolism Related Genes in Broiler Chickens.

This study determined the effects of two methionine (Met) sources at three total sulfur amino acids (TSAA) to lysine ratios (TSAA/Lys) on gut pH, digestive enzyme activity, amino acid transporter expression, and Met metabolism of broilers. The birds were randomly assigned to a 2 × 3 factorial arrangement with Met sources (dl-Met and dl-2-hydroxy-4-(methylthio)-butanoic acid (OH-Met)) and TSAA/Lys (0.58, 0.73, and 0.88) from 1 to 21 days. The results demonstrated that dl-Met and OH-Met supported the same growth performance, but high TSAA/Lys ratio reduced the feed intake and body weight (P < 0.05). OH-Met reduced the crop chyme pH and enhanced the jejunal lipase activity (P < 0.05). ATB0,+ expression decreased with increased dl-Met levels in the duodenum; the low TSAA/Lys ratio induced a stronger mRNA expression of basolateral Met transporters. OH-Met resulted in an increase of cystathionine β-synthase expression in the liver and a decrease in serum homocysteine levels at middle TSAA/Lys ratio compared with dl-Met treatment (P < 0.05). In conclusion, two Met sources support the same growth, but OH-Met acidified the crop chyme. The investigated transporter transcripts differed significantly along the small intestine. At the middle TSAA/Lys ratio, OH-Met showed a higher metabolic tendency of the trans-sulfuration pathway compared with dl-Met.

Supplementation of Clostridium butyricum LV1 lyophilised powder to cottonseed protein concentrate basal diets improves growth, intestinal microbiota, and immunity of Litopenaeus vannamei

Clostridium butyricum (CB) is a strictly anaerobic gram-positive probiotic. In this study, CB strain (LV1) was extracted from the intestine of Litopenaeus vannamei for preparing CB lyophilised powder (CBLP, 5.6 × 108 CFU/g) by freeze-drying method, with the aim to explore the effect of CB LV1 lyophilised powder on the growth, intestinal microbiota, and immunity of Litopenaeus vannamei after substitution of fishmeal with cottonseed protein concentrate (CPC). Six isonitrogenous and isolipid diets were formulated with 25% fishmeal in the positive control (FM) and 40% fishmeal replaced by CPC in the negative control (CBLP 0). CBLP was added to the CBLP 0 group at 3 × 105 CFU/g (CBLP 1), 1.2 × 106 CFU/g (CBLP 2), 4.8 × 106 CFU/g (CBLP 3), and 1.92 × 107 CFU/g (CBLP 4), respectively. The results showed that the CBLP 3 group had the highest weight gain rate and specific growth rate (P > 0.05). Serum superoxide dismutase and acid phosphatase activities were remarkably enhanced in the CBLP 2, CBLP 3, and CBLP 4 groups (P < 0.05). CBLP supplementation improved the intestinal digestive enzyme activity. The addition of CBLP optimized the structure of the intestinal microbiota, increased the beneficial bacteria, and reduced the potentially pathogenic bacteria. Chao1, ACE, and Sob indices were elevated in the CBLP 2, CBLP 3, and CBLP 4 groups (P < 0.001). Relish, PO, eIF4E1α, and IGF1 gene expressions were remarkably elevated in the CBLP 3 group (P < 0.01). CBLP further improved the intestinal morphological structure and enhanced the disease resistance. Muscle phenylalanine, histidine, and tyrosine contents were remarkably elevated in the CBLP 1 and CBLP 2 groups (P < 0.05). In conclusion, supplementation of CBLP in CPC basal diets improved the growth, intestinal microbiota, and immunity of Litopenaeus vannamei. Binary regression analysis of SGR indicated that the appropriate addition level of CBLP was 8.79 × 106 CFU/g.

Effects of dietary ginger (Zingiber officinale) polysaccharide on the growth, antioxidant, immunity response, intestinal microbiota, and disease resistance to Aeromonas hydrophila in crucian carp (Carassius auratus)

Ginger polysaccharides (GP) promote growth and development in fish. However, the effects of GP on crucian carp remain unclear. The present study investigated the effects of GP on the growth performance, immunity, intestinal microbiota, and disease resistance in crucian carp. Four treatment groups were established with different concentrations of GP (0.1 %, 0.2 %, 0.4 %, and 0.8 %). GP was not added as the control group, and the feeding period lasted for 56 d, followed by a 96-h anti-infection treatment using Aeromonas hydrophila. The results showed that dietary GP significantly improved growth performance, especially in the 0.4 % GP group. Furthermore, GP administration notably increased serum lysozyme (LMZ) activity, digestive enzyme performance, and antioxidant capacity of crucian carp. Moreover, dietary inclusion of GP up-regulated the expression of tumour necrosis factor-α (TNF-α), interleukin-8 (IL-8), interferon-γ (IFN-γ), and nuclear factor kappa-B (NF-κB) genes while down-regulating IL-10 and transforming growth factor-β (TGF-β) gene expressions, thus promoting liver health in crucian carp. Additionally, incorporating GP into the diet regulated both the diversity and composition of the intestinal microbiota in crucian carp, explicitly enhancing the relative abundance of beneficial bacteria, such as Fusobacteriota and Firmicutes. Therefore, GP reduces the mortality of crucian carp infected with A. hydrophila. In conclusion, this study provides novel insights into the application of dietary GP in cultured fish and evaluates the value of traditional Chinese medicinal polysaccharides against pathogenic bacteria.