Unlocking the power of Lactoferrin: Exploring its role in early life and its preventive potential for adult chronic diseases.

Nutrition during the early postnatal period exerts a profound impact on both infant development and later-life health. Breast milk, which contains lactoferrin, a dynamic protein, plays a crucial role in the growth of various biological systems and in preventing numerous chronic diseases. Based on the relationship between early infant development and chronic diseases later in life, this paper presents a review of the effects of lactoferrin in early life on neonates intestinal tract, immune system, nervous system, adipocyte development, and early intestinal microflora establishment, as well as the preventive and potential mechanisms of early postnatal lactoferrin against adult allergy, inflammatory bowel disease, depression, cancer, and obesity. Furthermore, we summarized the application status of lactoferrin in the early postnatal period and suggested directions for future research.

[Structure and function of human-derived lysozyme: a review].

Human-derived lysozyme is a general term for a group of naturally occurring alkaline proteins in the human body that are capable of lysing bacterial cell walls. Its action is characterized by its ability to cleave the ß-(1,4)-glycosidic bond between N-acetylglucosamine and N-acetylmuramic acid in peptidoglycan. Human-derived lysozyme has a variety of properties such as antibacterial, anti-inflammatory, antiviral and immune enhancing, and is therefore widely used in the domestic and international pharmaceutical markets. This review summarizes the structural features, expression sites, biological functions of human-derived lysozymes and its market applications.

Lactoferrin deficiency during lactation increases the risk of depressive-like behavior in adult mice.

BACKGROUND: Lactoferrin is an active protein in breast milk that plays an important role in the growth and development of infants and is implicated as a neuroprotective agent. The incidence of depression is currently increasing, and it is unclear whether the lack of lactoferrin during lactation affects the incidence of depressive-like behavior in adulthood. RESULTS: Lack of lactoferrin feeding during lactation affected the barrier and innate immune functions of the intestine, disrupted the intestinal microflora, and led to neuroimmune dysfunction and neurodevelopmental delay in the hippocampus. When exposed to external stimulation, adult lactoferrin feeding-deficient mice presented with worse depression-like symptoms; the mechanisms involved were activation of the LPS-TLR4 signalling pathway in the intestine and hippocampus, reduced BDNF-CREB signaling pathway in hippocampus, increased abundance of depression-related bacteria, and decreased abundance of beneficial bacteria. CONCLUSIONS: Overall, our findings reveal that lactoferrin feeding deficient during lactation can increase the risk of depressive-like behavior in adults. The mechanism is related to the regulatory effect of lactoferrin on the development of the "microbial-intestinal-brain" axis.

Versatile generation of precise gene edits in bovines using SEGCPN.

BACKGROUND: Gene knockout and knock-in have been widely performed in large farm animals based on genome editing systems. However, many types of precise gene editing, including targeted deletion, gene tagging, and large gene fragment replacement, remain a challenge in large farm animals. RESULTS: Here, we established versatile self-excising gene-targeting technology in combination with programmable nucleases (SEGCPN) to efficiently generate various types of precise gene editing in bovine. First, we used this versatile method to successfully generate bovine embryos with point mutations and 11-bp deletions at the MSTN locus. Second, we successfully generated bulls with EGFP labeling at the SRY locus. Finally, we successfully generated humanized cows in which the endogenous 18-kb α-casein gene was replaced with a 2.6-kb human α-lactalbumin gene. CONCLUSIONS: In summary, our new SEGCPN method offers unlimited possibilities for various types of precise gene editing in large animals for application both in agriculture and disease models.

De novo genome assembly depicts the immune genomic characteristics of cattle.

Immunogenomic loci remain poorly understood because of their genetic complexity and size. Here, we report the de novo assembly of a cattle genome and provide a detailed annotation of the immunogenomic loci. The assembled genome contains 143 contigs (N50 ~ 74.0 Mb). In contrast to the current reference genome (ARS-UCD1.2), 156 gaps are closed and 467 scaffolds are located in our assembly. Importantly, the immunogenomic regions, including three immunoglobulin (IG) loci, four T-cell receptor (TR) loci, and the major histocompatibility complex (MHC) locus, are seamlessly assembled and precisely annotated. With the characterization of 258 IG genes and 657 TR genes distributed across seven genomic loci, we present a detailed depiction of immune gene diversity in cattle. Moreover, the MHC gene structures are integrally revealed with properly phased haplotypes. Together, our work describes a more complete cattle genome, and provides a comprehensive view of its complex immune-genome.

Efficient Editing of the ZBED6-Binding Site in Intron 3 of IGF2 in a Bovine Model Using the CRISPR/Cas9 System.

BACKGROUND: Insulin-like growth factor 2 is a growth-promoting factor that plays an important role in the growth and development of mammals. A nucleotide substitution in intron 3 of IGF2-which disrupts the ZBED6-binding site-affects muscle mass, organ size, and fat deposition in pigs. The ZBED6-binding site is also conserved in cattle. METHODS: In the present study, we introduced mutations in the ZBED6-binding site in intron3 of IGF2 in bovine fetal fibroblasts using the CRISPR/Cas9 system, and investigated the effect of disruption of ZBED6 binding on IGF2 expression. RESULTS: Eleven biallelic-mutant single-cell clones were established, three of which contained no foreign DNA residues. Single-cell clones 93 and 135 were used to produce cloned embryos. Dual-luciferase reporter assay in C2C12 cells demonstrated that the mutation in the ZBED6-binding site increases the promoter 3 activity of bovine IGF2. A total of 49 mutant cloned embryos were transplanted into surrogate cows. Unfortunately, all cloned embryos died before birth. IGF2 was found to be hypomethylated in the only fetus born (stillborn), which may have been due to the incomplete reprogramming. CONCLUSIONS: We efficiently constructed IGF2-edited cell lines and cloned embryos, which provided a theoretical basis and experimental materials for beef cattle breeding.

Lactoferrin Deficiency Impairs Proliferation of Satellite Cells via Downregulating the ERK1/2 Signaling Pathway.

Lactoferrin (Ltf), a naturally active glycoprotein, possesses anti-inflammatory, anti-microbial, anti-tumor, and immunomodulatory activities. Many published studies have indicated that Ltf modulates the proliferation of stem cells. However, the role of Ltf in the proliferation of satellite cells, an important cell type in muscle regeneration, has not yet been reported. Here, by using Ltf systemic knockout mice, we illustrate the role of Ltf in skeletal muscle. Results shows that Ltf deficiency impaired proliferation of satellite cells (SCs) and the regenerative capability of skeletal muscle. Mechanistic studies showed that ERK1/2 phosphorylation was significantly downregulated after Ltf deletion in SCs. Simultaneously, the cell cycle-related proteins cyclin D and CDK4 were significantly downregulated. Intervention with exogenous recombinant lactoferrin (R-Ltf) at a concentration of 1000 µg/mL promoted proliferation of SCs. In addition, intraperitoneal injection of Ltf effectively ameliorated the skeletal muscle of mice injured by 1.2% BaCl2 solution. Our results suggest a protective effect of Ltf in the repair of skeletal muscle damage. Ltf holds promise as a novel therapeutic agent for skeletal muscle injuries.

Lactoferrin, a Critical Player in Neonate Intestinal Development: RHLF may be a Good Choice in Formula.

Lactoferrin (LF) is a bioactive glycoprotein in human milk and has positive effects on neonates. The LF knockout mouse model was generated as a mother mouse that provided LF-free milk. The intestinal development of suckling neonates drinking normal milk and LF-free milk was studied. The results showed that the intestinal density, maturity, and barrier integrity of mice drinking LF-free milk were lower than those of mice drinking normal milk. Therefore, the importance of adding lactoferrin to the human formula is considered. Human lactoferrin (HLF), bovine lactoferrin (BLF), and recombinant HLF (RHLF) were used to compare their functional impact on Caco-2 cell lines. Cell proliferation, differentiation, the establishment of the intestinal barrier, and protective effects on lipopolysaccharide injury were detected. Our results showed that RHLF exhibited more similar functions to HLF than BLF and showed the combined advantages of HLF and BLF in promoting the establishment of the intestinal barrier. This study emphasizes the important role of LF in neonatal intestinal development and provides a theoretical basis for the availability of RHLF.

Efficient TALEN-mediated gene knockin at the bovine Y chromosome and generation of a sex-reversal bovine.

Functional elucidation of bovine Y-chromosome genes requires available genome editing technologies. Meanwhile, it has yet to be proven whether the bovine Sry gene is the main or single factor involved in the development of the male phenotype in bovine. Here, we efficiently knocked out four Y-linked genes (Sry, ZFY, DDX3Y, and EIF2S3Y) in bovine fetal fibroblasts (BFFs) with transcription activator-like effector nucleases (TALENs) individually. Furthermore, we used TALEN-mediated gene knockin at the Sry gene and generated a sex-reversal bovine by somatic cell nuclear transfer (SCNT). The resulting bovine had only one ovary and was sterile. We demonstrate, for the first time, that the Sry gene is an important sex-determining gene in bovine. Our method lays a solid foundation for detecting the biology of the bovine Y chromosome, as it may provide an alternative biological model system for the study of mammalian sex determination, and new methods for the practical application in agricultural, especially for sex predetermination.

miR-29a/b1 Inhibits Hair Follicle Stem Cell Lineage Progression by Spatiotemporally Suppressing WNT and BMP Signaling.

Hair follicle stem cells (HFSCs) and subsequent generations of matrix progeny make lineage choices by responding to spatiotemporal signals; however, the cues driving that specification are not well understood. Here, we demonstrate that the dynamics of microRNA (miR)-29 expression are inversely proportional to HFSC lineage progression. Furthermore, we show that sustained miR-29a/b1 overexpression in anagen or telogen in mice causes a short-hair phenotype and eventual hair loss by inhibiting the proliferation of HFSCs and matrix cells and likely preventing their differentiation. Conversely, in a loss-of-function in vivo model, miR-29a/b1 deficiency accelerates HFSC lineage progression in telogen. Mechanistically, miR-29a/b1 blocks HFSC lineage specification by spatiotemporally targeting Ctnnb1, Lrp6, Bmpr1a, and Ccna2. We further show that skin-specific Lrp6 or Bmpr1a ablation partially accounts for the short-hair phenotype. Overall, these synergistic targets reveal miR-29a/b1 as a high-fidelity antagonist of HFSC lineage progression and a potential therapeutic target for hair loss.

No imprinted XIST expression in pigs: biallelic XIST expression in early embryos and random X inactivation in placentas.

Dosage compensation, which is achieved by X-chromosome inactivation (XCI) in female mammals, ensures balanced X-linked gene expression levels between the sexes. Although eutherian mammals commonly display random XCI in embryonic and adult tissues, imprinted XCI has also been identified in extraembryonic tissues of mouse, rat, and cow. Little is known about XCI in pigs. Here, we sequenced the porcine XIST gene and identified an insertion/deletion mutation between Asian- and Western-origin pig breeds. Allele-specific analysis revealed biallelic XIST expression in porcine ICSI blastocysts. To investigate the XCI pattern in porcine placentas, we performed allele-specific RNA sequencing analysis on individuals from reciprocal crosses between Duroc and Rongchang pigs. Our results were the first to reveal that random XCI occurs in the placentas of pigs. Next, we investigated the H3K27me3 histone pattern in porcine blastocysts, showing that only 17-31.8% cells have attained XCI. The hypomethylation status of an important XIST DMR (differentially methylated region) in gametes and early embryos demonstrated that no methylation is pre-deposited on XIST in pigs. Our findings reveal that the XCI regulation mechanism in pigs is different from that in mice and highlight the importance of further study of the mechanisms regulating XCI during early porcine embryo development.

Production of hypoallergenic milk from DNA-free beta-lactoglobulin (BLG) gene knockout cow using zinc-finger nucleases mRNA.

The whey protein ß-lactoglobulin (BLG) is a major milk allergen which is absent in human milk. Here, we for the first time generated DNA-free BLG bi-allelic knockout cow by zinc-finger nuclease (ZFNs) mRNA and produced BLG-free milk. According to the allergenicity evaluation of BLG-free milk, we found it can trigger lower allergic reaction of Balb/c mice including the rectal temperature drop and the allergen-specific immunoglobulin IgE production; BLG free-milk was easily digested by pepsin at 2 min, while BLG in control milk was still not completely digested after 60 min, and the binding of IgE from cow's milk allergy (CMA) patients to BLG free-milk was significantly lower than that to the control milk. Meanwhile, the genome sequencing revealed that our animal is free of off-target events. Importantly, editing animal genomes without introducing foreign DNA into cells may alleviate regulatory concerns related to foods produced by genome edited animals. Finally, the ZFNs-mediated targeting in cow could be transmitted through the germline by breeding. These findings will open up unlimited possibilities of modifying milk composition to make it more suitable for human health and also improve the functional properties of milk.

A novel glycosylated anti-CD20 monoclonal antibody from transgenic cattle.

The monoclonal antibody (mAb) against CD20 known as Rituxan is widely used to treat autoimmune diseases and lymphomas. However, further application of Rituxan faces challenges of high production cost, which limits its availability in developing countries. Here, we report a new approach for large production of a recombinant anti-CD20 mAb in the milk of transgenic cattle (at a yield of up to ~6.8 mg/mL), with ~80% recovery rate and >99% purity. Crystallography study showed that our recombinant mAb is structurally nearly identical to Rituxan with only minor differences in N-linked glycosylation pattern. Functional study showed that, while our mAb shared similar target-cell binding capacities and complement-dependent cytotoxicity with Rituxan, our product exhibited a higher binding affinity for FcγRIIIα and a greater antibody-dependent cellular cytotoxicity. Accordingly, our recombinant mAb demonstrated a superior efficacy over Rituxan against B-cell lymphomas in severe combined immunodeficiency mice. Taken together, our data supports transgenic cattle as a novel model for cost-competitive, large-scale production of therapeutic antibodies.

Efficient targeted integration into the bovine Rosa26 locus using TALENs.

The genetic modification of cattle has many agricultural and biomedical applications. However, random integration often results in the unstable expression of transgenes and unpredictable phenotypes. Targeting genes to the "safe locus" and stably expressing foreign genes at a high level are desirable methods for overcoming these hurdles. The Rosa26 locus has been widely used to produce genetically modified animals in some species expressing transgenes at high and consistent levels. For the first time, we identified a bovine orthologue of the mouse Rosa26 locus through a genomic sequence homology analysis. According to 5' rapid-amplification of cDNA ends (5'RACE), 3' rapid-amplification of cDNA ends (3'RACE), reverse transcription PCR (RT-PCR) and quantitative PCR (Q-PCR) experiments, this locus encodes a long noncoding RNA (lncRNA) comprising two exons that is expressed ubiquitously and stably in different tissues. The bovine Rosa26 (bRosa26) locus appears to be highly amenable to transcription activator-like effector nucleases (TALENs)-mediated knock-in, and ubiquitous expression of enhanced green fluorescent protein (EGFP) inserted in the bRosa26 locus was observed in various stages, including cells, embryos, fetus and cattle. Finally, we created a valuable master bRosa26-EGFP fetal fibroblast cell line in which any gene of interest can be efficiently introduced and stably expressed using recombinase-mediated cassette exchange (RMCE). The new tools described here will be useful for a variety of studies using cattle.

Effects of Recombinant Human Lactoferrin on Osteoblast Growth and Bone Status in Piglets.

Lactoferrin (LF), an ~80 kDa iron-binding glycoprotein, modulates many biological effects, including antimicrobial and immunomodulatory activities. Recently, it was shown that LF also regulates bone cell activity, suggesting its therapeutic effect on postmenopausal bone loss. However, a minimal amount is known regarding the effects of recombinant human LF (rhLF) supplementation on bone status in young healthy infants. We found osteoblast cell differentiation was significantly promoted in vitro. Furthermore, treatment of human osteoblast cells with rhLF rapidly induced phosphorylation of p44/p42 mitogen-activated protein kinase (p44/p42 MAPK, ERK1/2). In order to investigate the effects of rhLF on bone status in vivo, we used a piglet model, which is a useful model for human infants. Piglets were supplemented with rhLF milk for 30 days. Bone formation markers, Serum calcium concentration, bone mineral density (BMD), bone mineral content (BMC), tibia bone strength, and the overall metabolite profile analysis showed that rhLF was advantageous to the bone growth in piglets. These findings suggest that rhLF supplementation benefits neonate bone health by modulating bone formation.

Melatonin Improves the Quality of Inferior Bovine Oocytes and Promoted Their Subsequent IVF Embryo Development: Mechanisms and Results.

The inferior oocytes (IOs), which are not suitable for embryo development, occupy roughly one-third or more of the collected immature bovine oocytes. The IOs are usually discarded from the in vitro bovine embryo production process. Improving the quality of the inferior oocytes (IOs) and make them available in in vitro embryo production would have important biological, as well as commercial, value. This study was designed to investigate whether melatonin could improve the quality of IOs and make them usable in the in vitro maturation (IVM) and subsequent (in vitro fertilization) IVF embryo development. The results indicated that: the maturation rate of IOs and their subsequent IVF embryo developments were impaired compared to cumulus-oocyte complexes and melatonin treatment significantly improved the quality of IOs, as well as their IVF and embryo developments. The potential mechanisms are that: (1) melatonin reduced reactive oxygen species (ROS) and enhanced glutathione (GSH) levels in the IOs, thereby protecting them from oxidative stress; (2) melatonin improved mitochondrial normal distribution and function to increase ATP level in IOs; and (3) melatonin upregulated the expression of ATPase 6, BMP-15, GDF-9, SOD-1, Gpx-4, and Bcl-2, which are critical genes for oocyte maturation and embryo development and downregulated apoptotic gene expression of caspase-3.

Bovine lineage specification revealed by single-cell gene expression analysis from zygote to blastocyst.

Preimplantation embryos undergo zygotic genome activation and lineage specification resulting in three distinct cell types in the late blastocyst. The molecular mechanisms underlying this progress are largely unknown in bovines. Here, we sought to analyze an extensive set of regulators at the single-cell level to define the events involved in the development of the bovine blastocyst. Using a quantitative microfluidics approach in single cells, we analyzed mRNA levels of 96 genes known to function in early embryonic development and maintenance of stem cell pluripotency in parallel in 384 individual cells from bovine preimplantation embryos. The developmental transitions can be distinguished by distinctive gene expression profiles and we identified NOTCH1, expressed in early developmental stages, while T-box 3 (TBX3) and fibroblast growth factor receptor 4 (FGFR4), expressed in late developmental stages. Three lineages can be segregated in bovine expanded blastocysts based on the expression patterns of lineage-specific genes such as disabled homolog 2 (DAB2), caudal type homeobox 2 (CDX2), ATPase H+/K+ transporting non-gastric alpha2 subunit (ATP12A), keratin 8 (KRT8), and transcription factor AP-2 alpha (TFAP2A) for trophectoderm; GATA binding protein 6 (GATA6) and goosecoid homeobox (GSC) for primitive endoderm; and Nanog homeobox (NANOG), teratocarcinoma-derived growth factor 1 (TDGF1), and PR/SET domain 14 (PRDM14) for epiblast. Moreover, some lineage-specific genes were coexpressed in blastomeres from the morula. The commitment to trophectoderm and inner cell mass lineages in bovines occurs later than in the mouse, and KRT8 might be an earlier marker for bovine trophectoderm cells. We determined that TDGF1 and PRDM14 might play pivotal roles in the primitive endoderm and epiblast specification of bovine blastocysts. Our results shed light on early cell fate determination in bovine preimplantation embryos and offer theoretical support for deriving bovine embryonic stem cells.

Large-scale production of recombinant human lactoferrin from high-expression, marker-free transgenic cloned cows.

Human lactoferrin (hLF) is a valuable protein for pharmaceutical products and functional foods, and worldwide demand for this protein has steadily increased. However, large-scale recombinant human lactoferrin (rhLF) production using current animal bioreactor techniques is limited by the low expression of foreign proteins, the use of antibiotic resistance genes and the down-regulation of endogenous milk proteins. Here, we generated a herd of marker-free, hLF bacterial artificial chromosome (BAC) transgenic cloned cows, as confirmed by Polymerase chain reaction, Southern blot and Western blot analyses. These transgenic cloned cows produced rhLF in milk at concentrations of 4.5-13.6 g/L. Moreover, the total protein content of the milk was increased. Over two hundred transgenic cloned cows were propagated by multiple ovulation and embryo transfer (MOET). A total of 400-450 g of rhLF protein, which shows similar enzymatic activity to natural hLF in iron binding and release, can be purified on a large scale from >100 L of milk per day. Our results suggested that transgenic bovine mammary bioreactors have the potential for large-scale protein production.

Purification and characterization of recombinant human bile salt-stimulated lipase expressed in milk of transgenic cloned cows.

Bile salt-stimulated lipase (BSSL) is a lipolytic digestive enzyme with broad substrate specificity secreted from exocrine pancreas into the intestinal lumen in all species and from the lactating mammary gland into the milk of some species, notably humans but not cows. BSSL in breast milk facilitates digestion and absorption of milk fat and promotes growth of small for gestational age preterm infants. Thus, purified recombinant human BSSL (rhBSSL) can be used for treatment of patients with fat malabsorption and expressing rhBSSL in the milk of transgenic cloned cows would therefore be a mean to meet a medical need. In the present study, a vector pBAC-hLF-hBSSL was constructed, which efficiently expressed active rhBSSL in milk of transgenic cloned cows to a concentration of 9.8 mg/ml. The rhBSSL purified from cow milk had the same enzymatic activity, N-terminal amino acid sequence, amino acid composition and isoelectric point and similar physicochemical characteristics as human native BSSL. Our study supports the use of transgenic cattle for the cost-competitive, large-scale production of therapeutic rhBSSL.