Dampening of ISGylation of RIG-I by ADAP regulates type I interferon response of macrophages to RNA virus infection.

While macrophage is one of the major type I interferon (IFN-I) producers in multiple tissues during viral infections, it also serves as an important target cell for many RNA viruses. However, the regulatory mechanism for the IFN-I response of macrophages to respond to a viral challenge is not fully understood. Here we report ADAP, an immune adaptor protein, is indispensable for the induction of the IFN-I response of macrophages to RNA virus infections via an inhibition of the conjugation of ubiquitin-like ISG15 (ISGylation) to RIG-I. Loss of ADAP increases RNA virus replication in macrophages, accompanied with a decrease in LPS-induced IFN-ß and ISG15 mRNA expression and an impairment in the RNA virus-induced phosphorylation of IRF3 and TBK1. Moreover, using Adap-/- mice, we show ADAP deficiency strongly increases the susceptibility of macrophages to RNA-virus infection in vivo. Mechanically, ADAP selectively interacts and functionally cooperates with RIG-I but not MDA5 in the activation of IFN-ß transcription. Loss of ADAP results in an enhancement of ISGylation of RIG-I, whereas overexpression of ADAP exhibits the opposite effect in vitro, indicating ADAP is detrimental to the RNA virus-induced ISGylation of RIG-I. Together, our data demonstrate a novel antagonistic activity of ADAP in the cell-intrinsic control of RIG-I ISGylation, which is indispensable for initiating and sustaining the IFN-I response of macrophages to RNA virus infections and replication.

Alginate-Mediated Mineralization for Ultrafine Hydroxyapatite Hybrid Nanoparticles.

The good bioactivity of hydroxyapatite (HA) makes it become a popular biomaterial, and so, the synthesis of HA has attracted much attention. However, a simple method to prepare well-dispersive and ultrafine HA nanoparticles (NPs) still needs to be explored. Here, needle-like hybrid HA NPs were synthesized in the presence of alginate (ALG) and the mechanism of ALG regulation on forming HA/ALG hybrid NPs was investigated. The size and crystallinity of HA/ALG NPs could be controlled simply by varying the amount of ALG. With a higher concentration of ALG, the size of HA/ALG NPs reduced and their dispersity was further improved. The assembly with ca. 3-6 nm thick nanoneedles was discernible in HA/ALG NPs. The results collected by Ca2+ concentration, pH, and conductometric measurements further provided the supportive data for ALG inhibition of calcium phosphate compound formation and thus modulation of HA crystallization. In addition, the good biocompatibility of synthesized HA/ALG NPs was indicated from the results of CCK-8 assays and CLSM observations after cultured with L929 cells. The needle-like HA NPs are promising for varied biomedical purposes, and their potential application as drug carriers would be further studied.

Expression and regulation of MMP1, MMP3, and MMP9 in the chicken ovary in response to gonadotropins, sex hormones, and TGFB1.

Matrix metalloproteinases (MMPs) are a specific class of proteolytic enzymes that play critical roles in follicular development and luteinization in mammals. However, the role of MMPs in avian ovary remains largely unknown. We found that three MMP genes (MMP1, MMP3, and MMP9) were significantly up-regulated in 23-wk-old (laying phase) chicken ovaries compared with 6-wk-old ovaries (prepubertal phase). In reproductively active chicken ovary, MMP1 expression (both mRNA and protein) remained low in prehierarchical and preovulatory follicles but increased in postovulatory follicles (POFs). Both MMP3 and MMP9 expression levels increased during follicular maturation. MMP3 reached maximal expression in the first largest follicle (F1), while MMP9 levels continued to rise in POF1 and POF2 after ovulation. Immunohistochemistry, Western blot analysis, and zymography experiments indicated that MMP1, MMP3, and MMP9 were synthesized and secreted by granulosa cells of different follicles in the chicken ovary. The mRNA expression of MMP1 and MMP3 in the granulosa cells was stimulated by follicle-stimulating hormone, luteinizing hormone, progesterone, and estrogen but not by transforming growth factor beta 1 (TGFB1). However, the mRNA of MMP9 was induced by TGFB1 but not follicle-stimulating hormone, luteinizing hormone, progesterone, or estrogen. Luciferase reporter and mutagenesis analysis indicated the AP1 and NFkappaB elements located in the promoter region from -1700 to -2400 bp were critical for both basal and TGFB1-induced MMP9 transcription. These data provide the first spatial-temporal expression analysis of MMP system in the chicken ovary.

Identification of miRNAs associated with sexual maturity in chicken ovary by Illumina small RNA deep sequencing.

BACKGROUND: MicroRNAs have been suggested to play important roles in the regulation of gene expression in various biological processes. To investigate the function of miRNAs in chicken ovarian development and folliculogenesis, two small RNA libraries constructed from sexually mature (162-day old) and immature (42-day old) ovary tissues of Single Comb White Leghorn chicken were sequenced using Illumina small RNA deep sequencing. RESULTS: In the present study, 14,545,100 and 14,774,864 clean reads were obtained from sexually mature (162-d) and sexually immature (42-d) ovaries, respectively. In total, 202 known miRNAs were identified, and 93 of them were found to be significantly differentially expressed: 42 miRNAs were up-regulated and 51 miRNAs were down-regulated in the mature ovary compared to the immature ovary. Among the up-regulated miRNAs, gga-miR-1a has the largest fold-change (6.405-fold), while gga-miR-375 has the largest fold-change (11.345-fold) among the down-regulated miRNAs. The three most abundant miRNAs in the chicken ovary are gga-miR-10a, gga-let-7 and gga-miR-21. Five differentially expressed miRNAs (gga-miR-1a, 21, 26a, 137 and 375) were validated by real-time quantitative RT-PCR (qRT-PCR). Furthermore, the expression patterns of the five miRNAs were analyzed in different developmental stages of chicken ovary and follicles of various sizes. CONCLUSION: The present study provides the first miRNA profile in sexually immature and mature chicken ovaries. Some miRNAs such as gga-miR-1a and gga-miR-21are expressed differentially in immature and mature chicken ovaries as well as among different sized follicles, suggesting an important role in the follicular growth or ovulation mechanism in the chicken.

Expression of CCT6A mRNA in chicken granulosa cells is regulated by progesterone.

CCT6A, the zeta subunit of the chaperonin containing TCP1 complex, is the only cytosolic chaperonin in eukaryotes and is estimated to assist in the folding of multiple proteins including actin, tubulin, cyclin E, myosin, transducin and the Von Hippel Lindau tumor suppressor. In this study, we examined the expression of CCT6A and progesterone receptor (PGR) mRNA in various tissues of chickens and the regulation of CCT6A and PGR mRNA in ovarian granulosa cells. Northern blot analysis revealed that CCT6A had one transcript and was highly expressed in the ovary tissues from chickens at both the sexually immature and mature stages. CCT6A mRNA expression was increased maximally from pre-hierarchy follicles to F5 follicles and subsequently declined in pre-ovulatory and post-ovulatory follicles. The expression of PGR mRNA exhibited the similar pattern to CCT6A. In granulosa cells isolated from pre-ovulatory follicles, follicle-stimulating hormone (FSH) inhibited the expression of CCT6A mRNA, whereas progesterone activated CCT6A and suppressed PGR expression in a time-dependent manner. We further investigated the regulation of CCT6A transcription by progesterone by constructing various progressive deletions and mutants and identified the core promoter element of CCT6A and the binding region of progesterone, which is located from -2056 to -2051. Taken together, our results indicate that CCT6A likely plays an important role in follicle growth, and in granulosa cells, progesterone activates CCT6A transcription via a progesterone response element (PRE) located in the distal promoter of CCT6A.

Identification of differentially expressed genes in ovaries of chicken attaining sexual maturity at different ages.

In poultry as well as in other birds, sexual maturity is one of the important factors influencing female reproduction and egg production. In this study, cDNA-amplified fragment length polymorphism (cDNA-AFLP) differential display approach was used to identify genes related to sexual maturity. Using 54 EcoR I/Mse I selective primer combinations, totally 403 differentially expressed transcript-derived fragments (TDFs) were isolated, 27 of which belong to 25 unigenes. By real-time quantitative PCR (qPCR), the expression pattern of 13 genes was confirmed; among them, four genes including ZNF183 (P < 0.01), KIAA0700, CCT6A, and 23e 15 (P < 0.05) are significantly up-regulated and one gene (Loc418883) is significantly down-regulated (P < 0.01) in sexually mature ovaries compared to immature ones. The mRNA expression dynamics of ZNF183, CCT6A, 23e 15 and Loc418883 were further investigated in ovaries of 70-, 300- and 500-day-old commercial egg-laying hens: the expression level of CCT6A was the highest in 300-day-old hens (P < 0.05), while that of Loc418883 in 500-day-old hens was significantly higher than the other two stages (P < 0.01). The expression levels of ZNF183 and 23e 15 in ovary increase significantly from 70-day-old hens (P < 0.01) and 300-day-old (P < 0.05) to 500-day-old hens, respectively. The consistence of CCT6A expression and egg-laying performance suggests that CCT6A likely plays important role in sexual maturity in hens.

ADAP restraint of STAT1 signaling regulates macrophage phagocytosis in immune thrombocytopenia.

Heightened platelet phagocytosis by macrophages accompanied by an increase in IFN-γ play key roles in the etiology of immune thrombocytopenia (ITP); however, it remains elusive how macrophage-mediated platelet clearance is regulated in ITP. Here, we report that adhesion and degranulation-protein adaptor protein (ADAP) restrains platelet phagocytosis by macrophages in ITP via modulation of signal transducer and activator of transcription 1 (STAT1)-FcγR signaling. We show that ITP was associated with the underexpression of ADAP in splenic macrophages. Furthermore, macrophages from Adap-/- mice exhibited elevated platelet phagocytosis and upregulated proinflammatory signaling, and thrombocytopenia in Adap-/- mice was mitigated by the depletion of macrophages. Mechanistically, ADAP interacted and competed with STAT1 binding to importin α5. ADAP deficiency potentiated STAT1 nuclear entry, leading to a selective enhancement of FcγRI/IV transcription in macrophages. Moreover, pharmacological inhibition of STAT1 or disruption of the STAT1-importin α5 interaction relieved thrombocytopenia in Adap-/- mice. Thus, our findings not only reveal a critical role for ADAP as an intracellular immune checkpoint for shaping macrophage phagocytosis in ITP but also identify the ADAP-STAT1-importin α5 module as a promising therapeutic target in the treatment of ITP.

Biocompatible, Antioxidant Nanoparticles Prepared from Natural Renewable Tea Polyphenols and Human Hair Keratins for Cell Protection and Anti-inflammation.

Excessive reactive oxygen species (ROS) can cause oxidative stress of tissues and adversely influence homeostasis of the body. Epigallocatechin gallate (EGCG) with an antioxidative effect can effectively eliminate the ROS, but an evident weakness associated with it is the relatively poor cytocompatibility. Combining with other biomacromolecules such as human hair keratin (KE) and using nanotechnology to prepare nanoparticles can improve this situation. By covalent bonding, we assembled KE and EGCG into KE-EGCG nanoparticles (NANO) with size of about 50 nm and characterized them by DLS, UV, FTIR, NMR, and XPS. Free radical scavenging experiments show that antioxidant properties of the obtained NANO are superior to that of vitamin C. Cell culture experiments also show that the NANO can effectively protect the proliferation of L929 cells and HUVEC cells. In addition, we also used RAW264.7 cells to establish a H2O2-induced cell injury model and an lipopolysaccharide-induced cellular inflammatory model to evaluate the antioxidant and anti-inflammatory properties of NANO. The results show that the NANO can effectively prevent cells from oxidative damage and reduce inflammatory expression of the cells, indicating that the NANO have a good antioxidative and anti-inflammatory effect on cells which can be applied to many diseases related to oxidative stress.

Preparation of Strong Antioxidative, Therapeutic Nanoparticles Based on Amino Acid-Induced Ultrafast Assembly of Tea Polyphenols.

Nanoformulations offer the opportunity to overcome the shortcomings of drug molecules, such as low solubility, side effects, insufficient stability, etc., but in most of the current nanomedicines, nanocarriers as excipients do not directly participate in the therapy procedure. Accordingly, it is promising to develop the nanotherapeutics composed entirely of pharmaceutically active molecules. Tea polyphenols, especially epigallocatechin gallate (EGCG), are a kind of natural antioxidants with various biological and health beneficial effects and are extensively investigated as nutrients and anticancer drugs. Here, the size-tunable and highly active polyphenol nanoparticles were conveniently synthesized in water and could be massively produced with a simple facility. Compared to the previous strategies, either molecular assembly via oxidative coupling or combination with other biomacromolecules, the present preparation was conducted by the amino acid-triggered Mannish condensation reactions, thus permitting the flexible molecular design of various polyphenol nanoparticles by selecting different amino acids. This straightforward and ultrafast method actually opens up a novel means to make use of naturally reproducible polyphenols. Moreover, inheriting the salient properties of EGCG, these nanoparticles show strong antioxidation capacity, 10-fold higher than the extensively investigated polydopamine nanoparticles, and they are biosafe but have therapeutic effects, according to the in vitro and in vivo assessments of anticancer activity, which is promising for various biomedical purposes.

Freeze-thaw cycles for biocompatible, mechanically robust scaffolds of human hair keratins.

The keratin-based scaffolds are getting more and more attention in the application of tissue engineering. Though various approaches have been considered to improve the physical properties of these scaffolds, few succeeded in achieving the enhanced properties of the pure keratin scaffolds. Due to the presence of -OH, -NH2 , >CO, and -SH on the extracted human hair keratin (HHK), the formation of hydrogen bonds and disulfide bridges could be triggered under certain conditions, leading to the self-cross-linking of HHK materials. Herein, a simple and green strategy was introduced, via freeze-thaw cycles of keratin solutions without addition of extraneous reagents, to obtain the mechanically robust HHK scaffolds. The comparative quantitation of residual -SH among the samples treated with 1, 5, and 9 cycles confirmed the oxidation in the thaw process for forming disulfide bonds. So, the equivalent thaw time was applied in this study, and three groups of the treated samples after 1, 5, and 9 cycles with an appropriate extension thaw time were prepared to solely investigate the effects of physical cross-linking networks, primarily by formation of hydrogen bonds, on the properties of the obtained scaffolds. The systematic assessments including swelling behavior, porosity, thermal analysis, compressive measurement, and microstructural observation confirmed that the repetitive freeze-thaw treatment contributed to mechanically robust scaffolds with good porous interconnectivity. The cell culturing experiments further verified that these HHK scaffolds had desirable cytocompatibility, permitting the proper proliferation, attachment, and infiltration. Accordingly, this study provided a simple and efficient method to obtain biocompatible, mechanically robust keratin scaffolds. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1452-1461, 2019.

Cellulose/keratin-catechin nanocomposite hydrogel for wound hemostasis.

Uncontrolled bleeding can lead to serious injury or even death, which often happens in some emergency situations. In previous studies, keratin was found to have a good hemostatic effect. However, the hemostatic use of pure keratin materials is limited due to keratin's poor physical properties. In the present study, a hemostatic hydrogel was prepared by adding keratin-catechin nanoparticles (KE-NPs) into cellulose hydrogel. Keratin extracted from human hair was firstly combined with EGCG, the primary constituent of catechins, to induce the self-assembly of nanoparticles. The prepared KE-NPs had a spherical shape with a particle size of around 40 nm. KE-NPs were subsequently added into cellulose hydrogel, giving rise to the cellulose/keratin-catechin composite hydrogel (KEC). The resultant KEC exhibited good adhesiveness and hemadsorption, and resulted in rapid blood coagulation. In the rat liver puncture model and the tail amputation model, KEC significantly reduced the amount of bleeding and the bleeding time. The results indicated that KEC could quickly adhere to the wound and accelerate blood coagulation. This KEC exhibited the capability of preventing blood loss and can serve as a novel type of hemostatic material.

Size-controlled, colloidally stable and functional nanoparticles based on the molecular assembly of green tea polyphenols and keratins for cancer therapy.

While intelligent nanoparticles with therapeutic effects provide a resolving strategy for low drug loading efficacy, poor metabolism and elimination of current nanoparticulate drug delivery systems, precise preparation of colloidally stable but stimuli-responsive nanocarriers with size tunability is still a challenging task. Here, we develop a facile and sustainable method through the use of naturally reproducible green tea polyphenols and hair keratins to prepare biocompatible, colloidally stable, stimuli-responsive nanoparticles with therapeutic effects. The present strategy simply involves covalent interactions of tea catechins and keratins, giving rise to the molecular assembly of size-controlled nanoparticles (30-230 nm) which are long-term colloidally stable at physiological media but are disassembled under pathological conditions, ideally for targeted delivery of anticancer drugs. The cell experiments confirmed that these nanoparticles are bio-safe, have the inherent bioactivity of tea catechins, and that the drug-loaded nanoparticles yield a higher cancer cell inhibition rate than free drugs. In addition, the nanoparticles are found to improve the bioavailability of tea polyphenols, according to animal studies, which further demonstrates that the use of nanoparticles as drug carriers results in enhanced anticancer efficacy with negligible systemic toxicity. Given that large-scale preparation of size-controlled nanoparticles could already be easily achieved, the present study actually provides an innovative nanotechnological approach to make good use of green tea polyphenols with beneficial health effects, potentially for therapeutic and preventive purposes.

RhBMP-2 and concomitant rapid material degradation synergistically promote bone repair and regeneration with collagen-hydroxyapatite nanocomposites.

The effective treatment of bone defects is still a great challenge in clinical practice. Synthetic bone-grafting substitutes of composition and structure analogous to bone as well as incorporated with growth factors are considered to be a promising solution. In this study, a collagen-hydroxyapatite (CHA) nanocomposite scaffold was developed by collagen self-assembly with simultaneous HA synthesis. The physicochemical properties such as morphology, inorganic phase, thermal decomposition, specific surface area and pore size distribution were characterized. The osteogenicity of CHA in the absence or presence of recombinant human bone morphogenetic protein-2 (rhBMP-2) was assessed both by cell culturing and animal implantation experiments. The gene expression results showed that the osteogenic differentiation capacity of rat bone mesenchymal stem cells (rBMSCs) has been enhanced both by CHA and rhBMP-2. The efficient bone regeneration of femoral defects in rabbits was achieved with CHA and CHA pre-absorbed rhBMP-2 (CHA/B), confirmed by micro-computed tomography measurements, histological observation and immunohistochemical analyses. The CHA nanocomposite was completely degraded within 8 weeks and replaced by new bone. It was found that rhBMP-2 not only accelerated and enhanced bone formation, but also expedited the degradation of CHA. It is believed that the rhBMP-2 and concomitant rapid material degradation synergistically promote bone repair and regeneration with CHA. The biodegradation behavior of CHA in the presence of rhBMP-2 can be further investigated to gain an in-depth understanding of the complex interplays among biomaterials, growth factors and their target cells. The relevant knowledge will facilitate the search for a reasonable, safe and efficient methodology for the introduction of growth factors to biomaterials so as to achieve satisfactory tissue regeneration.

Biologically inspired, catechol-coordinated, hierarchical organization of raspberry-like calcium phosphate nanospheres with high specific surface area.

The elaborate design of calcium phosphates (CaP) with a hierarchical structure for biomedical applications is severely restricted due to their instinctive pertinacious irregular agglomeration. Thus, the development of mesoporous CaP with large internal pores is far from satisfactory. In this study, catechol is selected as a modifier to intervene in the precipitation of CaP. Compared to pure CaP with smooth irregular aggregates with an average size of 110 nm, the catechol-intervened CaP (CaP/Cat) samples are raspberry-like nano-spherical assemblies of ca. 25 nm nanoparticles. The obtained spheres possess a mesoporous structure, large interior pores and high specific surface area, which endow them with a high protein adsorption capacity and high DNA delivery efficacy. Catechol is subjected to oxidative polymerization while CaP precipitation occurs. This simultaneous reaction is supposed to play a crucial role in directing the assembling of the CaP/Cat nanospheres. The flexible catechol moiety is shown to be a novel and promising modifier for manufacturing CaP with tunable structures and properties for various applications.

Tumor-targeted and nitric oxide-generated nanogels of keratin and hyaluronan for enhanced cancer therapy.

The development of safe and effective nano-drug delivery systems to deliver anticancer drugs to targeted cells and organs is crucial to enhance the therapeutic efficacy and overcome unwanted side effects of chemotherapy. Herein, we prepared CD44-targeted dual-stimuli responsive human hair keratin and hyaluronic acid nanogels (KHA-NGs) through a simple crosslinking method. KHA-NGs, which consisted of spheres 50 nm in diameter, were used as carriers to load the anticancer drug doxorubicin hydrochloride (DOX). The drug release, cellular uptake, cytotoxicity, and targeting ability of DOX-loaded KHA-NGs (DOX@KHA-NGs) were assessed in vitro and the anticancer effects were further evaluated in vivo. The DOX@KHA-NGs had a super-high drug loading capacity (54.1%, w/w) and were stable under physiological conditions (10 µM glutathione (GSH)), with the drug being rapidly released under a tumor cell microenvironment of trypsin and 10 mM GSH. Cellular uptake and in vitro cytotoxicity results indicated that DOX@KHA-NGs specifically targeted cancer cells and effectively inhibited their growth. Furthermore, KHA-NGs were capable of improving intracellular nitric oxide levels, which sensitizes the cells and enhances the anticancer efficacy of chemotherapeutic drugs. In vivo experiments showed that DOX@KHA-NGs had a better anti-tumor effect and lower side effects compared to free DOX. These results suggest that the bio-responsive KHA-NGs have potential applications for targeted cancer therapy.

Identification and Genetic Effect of Haplotypes in the Distal Promoter Region of Chicken CCT6A Gene Associated with Egg Production Traits.

The chaperonin containing TCP-1 complex protein 1 subunit zeta (CCT6A) is the only cytosolic chaperonin in eukaryotes assisting in the folding of cytoplasmic proteins. Previous study revealed that the mRNA expression of chicken CCT6A gene was remarkably elevated in the sexually mature ovaries. However, the mechanism underlying chicken CCT6A expression changes remains largely unknown. In this study, haplotypes caused by two single nucleotide polymorphisms (SNPs) of chicken CCT6A gene promoter (g.-2215 T>C and g.-1959 T>C) were identified and their associations with egg production traits as well as effects on gene expression were analyzed. Altogether four haplotypes including A (C-2215-T-1959), B (C-2215-C-1959), C (T-2215-T-1959) and D (T-2215-C-1959) were detected in all of the five chicken populations. Diplotypes AA, AD and DD were predominant in Xinyang brown hens, among which diplotype AD was associated with higher egg number at the age of 28 weeks old (E28) (P<0.05). In addition, diplotype AD was also predominant in Xinyang brown and Hy-line brown chicken populations with high egg production; whereas in Wenchang and Shouguang chicken populations which are Chinese indigenous chicken breeds and relatively lower in egg production, diplotype AA was predominant. Compared with diplotypes AA and DD, the mRNA expression of CCT6A in diplotype AD birds is the highest in F1, F5, and POF1 follicles of Hy-line brown hens (P<0.05). These results suggest that the two SNPs in chicken CCT6A promoter region are potential DNA marker for improving egg production trait.

Folate deficiency during early-mid pregnancy affects the skeletal muscle transcriptome of piglets from a reciprocal cross.

Folate deficiency (FD) during pregnancy can cause fetal intrauterine growth restriction in pigs, of which the skeletal dysplasia is a major manifestation. Factors influencing muscle development are very important in the formation of porcine meat quality trait. However, the effect of folate deficiency on skeletal muscle development and its molecular mechanisms are unknown. The objective of this study is to determine the effect of maternal folate deficiency on the skeletal muscle transcriptome of piglets from a reciprocal cross, in which full-sibling Landrace (LR) and full-sibling Chinese local breed Laiwu (LW) pigs were used for reciprocal cross matings, and sows were fed either a folate deficient or a normal diet during early-mid gestation. In addition, the difference in the responsiveness of the piglets to folate deficiency during early-mid pregnancy between reciprocal cross groups was investigated. Longissimus dorsi (LD) muscle samples were collected from newborn piglets and a 4 × 44K Agilent porcine oligo microarray was used for transcriptome analysis of porcine LD muscle. The results showed that folate deficiency during early-mid pregnancy affected piglet body weight, LD muscle fiber number and content of intramuscular triglyceride. The microarray results indicated that 3154 genes were differentially expressed between folate deficient and normal piglets from the LR♂ × LW♀ cross, and 3885 differentially expressed genes (DEGs) in the ones from the LW♂ × LR♀ cross. From functional analyses, sow folate deficiency affected almost all biological processes in the progeny. Lipid metabolism-related genes and associated metabolic pathways were regulated extensively by folate deficiency, especially in LR♂ × LW♀ cross piglets. Most of the genes that are regulated by folate deficiency in the LD muscle of piglets were different between LR♂ × LW♀ and LW♂ × LR♀ crosses, suggesting some epigenetic effects of FD exist in genes underlying myogenesis and intramuscular fat deposition in piglets.