Perspective: A proposal on solutions of modern supply chain construction for lactoferrin.

Lactoferrin is an iron-binding glycoprotein of the transferrin family that is found in most bodily fluids of mammals and has a variety of biological and beneficial functions, with great importance in health enhancement as a supplement for humans and other animals. More than 300 t of lactoferrin were produced in 2021, and this number is expected to grow yearly by 10% to 12%, to over 580 t in 2030. With new and important functions of lactoferrin being revealed and studied, focus on its industrial production and application is increasing accordingly. However, lactoferrin is mainly sourced from cheese whey or skim milk by cation-exchange column chromatography, which is a costly and low-quality method. A potential solution for lactoferrin global supply chain construction is proposed in this article as a complement to traditional routes of purification from whey or skim milk. The large-scale production of lactoferrin, mainly by recombinant yeast, mammal, and grain systems, as well as the market niche and product design, are discussed.

Effects of dietary supplementation of bovine lactoferrin on growth performance, immune function and intestinal health in weaning piglets.

Weaning is a crucial period in the pig's life cycle, which is frequently followed by gastrointestinal (GI) infections, diarrhea and even death. This study focused on the impact of bovine lactoferrin (bLF) supplementation on the intestinal health of weaning piglets. Weaning piglets (Duroc × Landrace × Yorkshire, 23 days) were randomly allocated into four groups, which included negative control group (CON): basic diet; positive control group (ANT): basic diet + 20 mg/kg flavomycin + 100 mg/kg aureomycin; treatment group bLF-A: basic diet + 1 g/kg bLF; treatment group bLF-B: basic diet + 3 g/kg bLF. The result showed that dietary supplementation of bLF can improve growth performance and reduce diarrhea, which exhibits dose-dependency (P < 0.05). Compared with CON group, supplementation with bLF significantly improved immunity, and increased villus height and ratio of villus height/crypt depth at the small intestinal mucosa (P < 0.05). The mRNA expression of claudin-1, occludin and ZO-1 was greatly increased in the ileum of bLF group on days 7 and 14 (P < 0.05). Furthermore, the supplementation of bLF increased the abundance of Lactobacillus and Bifidobacterium and decreased the abundance of Escherichia coli in the cecum on day 7 (P < 0.05). The dietary supplementation of bLF enhanced the growth performance, reduced diarrhea rate in weaning piglets by improving intestinal immunity, morphology and barrier function, balancing intestinal microbiota. And bLF can be a promising feed additive in relieving stress situation of weaning piglets.

Exploration of the Pharmacological Mechanism of Vitexicarpin against Triple-Negative Breast Cancer in Network Pharmacology.

BACKGROUND: Vitexicarpin (VIT), an isoflavone derived from various medicinal herbs, has shown promising anti-tumor activities against multiple cancer cells. However, the understanding of the mechanisms and potential targets of VIT in treating triple-negative breast cancer (TNBC) remains limited. METHODS: The potential VIT targets were searched for in the Super-PRED online database, while the TNBC targets were acquired in the DisGeNET database, and the Veeny database was used to identify the VIT and TNBC targets that overlapped. Then, GO and KEGG enrichment analyses were carried out in the DAVID database. The protein-protein interaction (PPI) network was constructed to acquire the hub targets in the STRING database, and the overall survival analysis of the hub targets was examined in the Kaplan-Meier plotter database. Afterward, molecular docking was performed to evaluate the binding capabilities between VIT and the hub targets. In order to measure the effect of VIT on proliferation, apoptosis, and cell cycle arrest in the TNBC cell lines-MDA-MB-231 and HCC-1937-the Cell Counting Kit-8 (CCK-8) assay and flow cytometry analysis were performed. The Western blot and pull-down assays were used to verify the molecular mechanisms by modulating the hub targets. RESULTS: The network pharmacology results identified a total of 37 overlapping genes that were shared by VIT and TNBC. The results of the PPI network and molecular docking analyses showed that HSP90AA1, CREBBP, and HIF-1A were key targets of VIT against TNBC. However, the pull-down results suggested that VIT could directly bind to HSP90AA1 and HIF-1A, yet not to CREBBP. The results of the in vitro tests showed that VIT decreased proliferation and induced apoptosis in MDA-MB-231 and HCC-1937 cells, in a dose-dependent manner, while the cell cycle arrest occurred at the G2 phase. Mechanistically, the Western blot assay demonstrated that VIT decreased the expression of HSP90AA1, CREBBP, and HIF-1A. CONCLUSIONS: VIT inhibited growth and induced apoptosis of TNBC cells by modulating HIF-1A, HSP90AA1, and CREBBP expression. Our findings suggest that VIT is a potential drug for TNBC therapy.

Fatty acid modified-antimicrobial peptide analogues with potent antimicrobial activity and topical therapeutic efficacy against Staphylococcus hyicus.

There is an urgent need to explore new antimicrobial agents due to the looming threat of bacteria resistance. Bovine lactoferricin (LfcinB), as a multifunctional peptide, has the potential to be a new active drug in the future. In this study, it aims to investigate the effect of fatty acid conjugation on antimicrobial peptide activity and topical therapeutic efficacy in a mouse model infected with Staphylococcus hyicus. Both Lfcin4 and Lfcin5 were conjugated with the unsaturated fatty acid linoleic acid (18-C) at their N-terminus and modified by acylation at the C-terminus. The derived peptides of Lin-Lf4NH2 and Lin-Lf5NH2 showed better antibacterial activity (MICs of 3.27 to 6.64 µM) than their parent peptides (MICs of 1.83 to 59.57 µM). Lin-Lf4NH2 (63.2%, 5 min) and Lin-Lf5NH2 (35.8%, 5 min) could more rapidly penetrate bacterial membrane than Lf4NH2 (2.34%, 5 min) and Lf5NH2 (1.94%, 5 min), which further confirmed by the laser scanning confocal microscopy (LSCM). Electron microscopy observations showed Lin-Lf4NH2 and Lin-Lf5NH2 disrupted S. hyicus cell membranes and led to the leakage of contents. Furthermore, after treatment with Lin-Lf4NH2 and Lin-Lf5NH2, the abscess symptoms of mice were significantly alleviated; the recovery rate of abscesses scope of Lin-Lf4NH2 (73.25%) and Lin-Lf5NH2 (71.71%) were 38.8 and 37.9-fold higher than that of untreated group (1.89%), respectively, and superior to Lf4NH2 (46.87%) and Lf5NH2 (58.75%). They significantly reduced the bacterial load and the levels of the pro-inflammatory cytokines (TNF-α, IL-6, and IL-1ß) and chemokine (MCP-1) in S. hyicus skin lesions. This study provides evidence that conjugation of a fatty acid to antimicrobial peptides can improve the activity and have potential for topical therapeutic of S. hyicus skin infections. KEY POINTS: • Lin-Lfcin4NH2/Lfcin5NH2 showed stronger antimicrobial activity than parent peptides. • Lin-Lfcin4NH2/Lfcin5NH2 had a more effective ability to destroy bacterial membranes. • Lin-Lfcin4NH2/Lfcin5NH2 showed a topically higher efficacy than parent peptides.

Arsenic Trioxide Combining Leflunomide Activates Nrf2-ARE-HO-1 Signaling Pathway and Protects Heart Xenografts.

OBJECTIVE: To investigate the molecular mechanisms underlying the effects of arsenic trioxide (As2O3) in combination with leflunomide on the hamster-to-rat heart xenotransplant. METHODS: Transplantation of LVG hamster hearts to Lewis rats was performed by anastomosis of vessels in the neck using end-to-end anastomosis with a non-suture cuff technique. Four groups of recipient rats (n=6 in each) were treated with normal saline (control), As2O3 [5 mg/(kg·day) intraperitoneally], leflunomide [5 mg/(kg·d) orally], or leflunomide [5 mg/(kg·d)+As2O3 [5 mg/(kg·d)] in combination. Donor hearts and/or rat spleens were harvested and analyzed 4 days after transplantation. Quantitative reverse-transcription polymerase chain reaction and Western blot analysis were performed to detect the expression of the nuclear factor erythroid-derived factor 2-related factor (Nrf2) and its target gene heme oxygenase-1 (HO-1), Treg cell marker fork-head Box P3 (FOXP3), apoptosis-associated proteins Bcl-2, Bax, and cleaved caspase-3. Immunohistochemical staining was used to detect the levels of inflammatory natural killer cell and macrophage infiltration, intercellular cell adhesion molecule-1 (ICAM-1) and complement C3. RESULTS: Expression of Nrf2-ARE-HO-1 signaling pathway was upregulated in heart xenografts in rats treated with As2O3 plus leflunomide compared with control rats or rats treated with either drug alone (P<0.01), and this was accompanied by an increased Treg cells in the recipient rat spleen (P<0.01). In contrast, the expressions of Bax, cleaved caspase-3, ICAM-1, and complement C3, and infiltration of inflammatory cells in the xenografts were inhibited by As2O3 plus leflunomide treatment (P<0.01). CONCLUSION: Combination treatment with As2O3 and leflunomide protected hamster heart-xenografts in recipient rats.

Literature Research on Origin and Application of Classical Prescription Xiebaisan / 中国实验方剂学杂志

Xiebaisan is one of the 100 classical prescriptions published by the state， and the research and development of its compound preparation has gradually become an upsurge. The research and development of classical prescriptions should start from the theory of traditional Chinese medicine and clinical practice， laying a solid foundation for research and development. Based on the above thinking， this study adopts the methods of traceability and bibliometrics to analyze the source， pathogenesis， efficacy， Fangyi and clinical application of Xiebaisan in order to further promote its literature research work. The results show that the origin of Xiebaisan can be traced back to the prescription of Xiefeitang in Yinhai Jingwei（Volume 1）. Although there are more than 20 prescriptions with the same name of Xiebaisan developed in later generations， they either reflect the inheritance and development of Qian Yi's thought of composing prescriptions， or only the meaning of "Xiebai" in the name of the prescription. The main pathogenesis of Xiebaisan is adverse lung Qi of heat. Mori Cortex and Lycii Cortex are sweet and cold， which can clear away lung heat and reduce adverse Qi， and then cough and asthma can stop. Supplemented with Glycyrrhizae Radix et Rhizoma and japonica rice， which are endowed with the meaning of nourishing earth and generating gold， to invigorate spleen and replenish Qi. The compatibility of Xiebaisan can clear away lung heat， relieve cough and asthma， and norish erath and generate gold to treat lung heat， asthma and cough. Although the main treatment of Xiebaisan recorded in ancient books involves various diseases and syndromes of internal， external， gynecology and pediatrics， the pathogenesis of Xiebaisan is "adverse lung Qi of heat". Modern clinical application of Xiebaisan focuses on the respiratory system， skin and subcutaneous tissue system， ear， nose and throat system， digestive system， ophthalmic system， etc.， and it has more advantages in the treatment of pneumonia， cough， bronchitis， epistaxis， acne， bronchiectasis， postinfectious cough， constipation and other diseases. The syndrome differentiation of the above diseases is in accordance with the relevant syndrome types with lung heat as the main pathological factor， so we can modify and apply the prescription appropriately.

Treatment Effect of Tuftsin and Antigen Peptide Combined with Immune Cells on Colorectal Cancer.

BACKGROUND The aim of this study was to investigate the effect of antigenic peptides on dendritic cell maturation and activation as well as the role of dendritic cell induced cell function. The tumor-specific cytotoxic T lymphocytes induced by activation of the dendritic cells were also evaluated. MATERIAL AND METHODS SW-480 cell lysate and peptide antigens were selected as adjuvants in dendritic cell sensitization, and tuftsin was used to induce the phagocytosis of dendritic cells. Immature dendritic cells were stimulated with the antigen and adjuvant as follows: group A was negative control; group B was SW-480 (20 µg/mL); group C was SW-480 (20 µg/mL)+tumor necrosis factor (TNF)-alpha (10 µg/mL); group D was SW-480 (20 µg/mL)+tuftsin (20 µg/mL); group E was antigen peptide (2 µg/mL); group F was antigen peptide (2 µg/mL)+TNF-alpha (10 µg/mL); group G was antigen peptide (2 µg/mL)+tuftsin (20 µg/mL). Cytotoxic T lymphocytes activation and in vitro anti-tumor effects were examined by detecting the maturation marks of dendritic cells as well as interleukin (IL)-10 and IL-12 levels secreted by dendritic cells. Cells with the strongest immunizing effects were injected into nude mice and tumor suppression status was evaluated. RESULTS Group D (SW-480+tuftsin), group E (antigen peptides), group F (antigen peptide+TNF-alpha), and group G (antigen peptides+tuftsin) displayed significant differences compared to the control group (P<0.05). Group G (antigen peptides+tuftsin) could also promote the secretion of cytokines IL-12, as well as inhibit cytokine IL-10 secretion, compared to the other experimental groups (P<0.05). In the in vivo experiments of tumor inhibitions, antigenic polypeptide+tuftsin was the most effective (P<0.05). CONCLUSIONS Combination of cytotoxic T lymphocytes and T peptide therapy in treating human colorectal cancer might be used as a new treatment strategy based on adoptive cellular immunotherapy.

Combined Systems Approaches Reveal a Multistage Mode of Action of a Marine Antimicrobial Peptide against Pathogenic Escherichia coli and Its Protective Effect against Bacterial Peritonitis and Endotoxemia.

A marine arenicin-3 derivative, N4, displayed potent antibacterial activity against Gram-negative bacteria, but its antibacterial mode of action remains elusive. The mechanism of action of N4 against pathogenic Escherichia coli was first researched by combined cytological and transcriptomic techniques in this study. The N4 peptide permeabilized the outer membrane within 1 min, disrupted the plasma membrane after 0.5 h, and localized in the cytoplasm within 5 min. Gel retardation and circular dichroism (CD) spectrum analyses demonstrated that N4 bound specifically to DNA and disrupted the DNA conformation from the B type to the C type. N4 inhibited 21.1% of the DNA and 20.6% of the RNA synthesis within 15 min. Several hallmarks of apoptosis-like cell death were exhibited by N4-induced E. coli, such as cell cycle arrest in the replication (R) and division(D) phases, reactive oxygen species production, depolarization of the plasma membrane potential, and chromatin condensation within 0.5 h. Deformed cell morphology, disappearance of the plasma membrane, leakage of the contents, and ghost cell formation were demonstrated by transmission electron microscopy, and nearly 100% of the bacteria were killed by N4. A total of 428 to 663 differentially expressed genes are involved in the response to N4, which are associated mainly with membrane biogenesis (53.9% to 56.7%) and DNA binding (13.3% to 14.9%). N4-protected mice that were lethally challenged with lipopolysaccharide (LPS) exhibited reduced levels of interleukin-6 (IL-6), IL-1ß, and tumor necrosis factor alpha (TNF-α) in serum and protected the lungs from LPS-induced injury. These data facilitate an enhanced understanding of the mechanisms of marine antimicrobial peptides (AMPs) against Gram-negative bacteria and provide guidelines in developing and applying novel multitarget AMPs in the field of unlimited marine resources as therapeutics.

Potential protective effect of angiopoietin-1 on the leakage of rat choroidal neovascularization.

OBJECTIVES: To observe the potential protective effect of angiopoietin-1 (Ang-1) on rat choroidal neovascularization (CNV) leakage. METHODS: The study was conducted at the Eye Institute of Shandong University of Traditional Chinese Medicine, Jinan, China from June 2012 to June 2013. Thirty CNV model rats were induced by laser. In vivo, fluorescein fundus angiography and pathological techniques were applied to detect the effect of vascular endothelial growth factor (VEGF) and Ang-1 intravitreous injection. In vitro, 3-(4, 5-dimethylthiazole-2-yl)-2, 5-biphenyl tetrazolium bromide (MTT) assay was applied to detect the proliferation of cultured bovine retinal endothelial cells (BRECs) after treatment with VEGF and Ang-1. Transmission electron microscopy (TEM) was used to detect the morphological changes under VEGF and Ang-1. RESULTS: In the CNV rat model, less late leakage was found in the Ang-1 group than the vehicle control or the VEGF group. The MTT assay showed Ang-1 administration inhibited the proliferation of BRECs. The VEGF promoted proliferation at low concentrations and inhibited the proliferation when its concentration reached 50 ng/ml. The administration of VEGF+Ang-1 rescued the inhibition effect of Ang-1 alone. The TEM results showed that there were less intercellular junctions in the VEGF group compared with the vehicle control. In the VEGF + Ang-1 group, the intercellular junctions were nearly normal. CONCLUSION: The Ang-1 can induce intercellular junction formation and decrease the CNV leakage.

Construction of a standard reference plasmid for detecting GM cottonseed meal.

A plasmid was constructed for quantification of genetically modified (GM) cottonseed meal in the gene-specific level. The Cry1Ab/c gene was connected with the Sad1 gene by fusion PCR. The fusion gene was cloned into the pMD®19-T Simple Vector. The plasmid DNA was then digested with a restriction endonuclease SmaI to reduce the characteristic differences between the plasmid DNA and genomic DNA. For a rough quantitative analysis of GM cotton meal contents, a rapid method for measurement of the copy numbers of the transgenic Cry and cotton endogenous Sad1 gene using a real-time PCR system with the plasmid DNA as a calibrator was established. The inter-run and intra-run coefficients of variation were less than 1.48% and 2.36%, respectively. The limits of detection and quantitation of the Cry and Sad1 genes were 9 and 91 copies of pMDCS, respectively. These results prove that the standard plasmid represents a valuable alternative to genomic DNA as a certified reference material for the quantification of GM cotton and is a useful tool to establish a feasible identification management for GM cottonseed meal content in the feed industry.