Integrative network analysis revealed the molecular function of folic acid on immunological enhancement in a sheep model.

We previously observed the beneficial role of folic acid supplemented from maternal or offspring diet on lamb growth performance and immunity. Twenty-four Hu lambs from four groups (mother received folic acid or not, offspring received folic acid or not) were used in the current study, which was conducted consecutively to elucidate the molecular regulatory mechanisms of folic acid in lambs by analyzing blood metabolome, liver transcriptome, and muscle transcriptome. Serum metabolomics analysis showed that L-homocitrulline, hyodeoxycholic acid, 9-Hpode, palmitaldehyde, N-oleoyl glycine, hexadecanedioic acid, xylose, 1,7-dimethylxanthine, nicotinamide, acetyl-N-formyl-5-methoxykynurenamine, N6-succinyl adenosine, 11-cis-retinol, 18-hydroxycorticosterone, and 2-acetylfuran were down-regulated and methylisobutyrate was up-regulated by the feeding of folic acid from maternal and/or offspring diets. Meanwhile, folic acid increased the abundances of S100A12 and IRF6 but decreased TMEM25 in the liver. In the muscle, RBBP9, CALCR, PPP1R3D, UCP3, FBXL4, CMBL, and MTFR2 were up-regulated, CYP26B1 and MYH9 were down-regulated by the feeding of folic acid. The pathways of bile secretion, biosynthesis of unsaturated fatty acids, linoleic acid metabolism, and herpes simplex virus 1 infection were changed by folic acid in blood, liver, or muscle. Further integrated analysis revealed potential interactions among the liver, blood, and muscle, and the circulating metabolites, hub gene, and pathways, which might be the predominant acting targets of folic acid in animals. These findings provide fundamental information on the beneficial function of folic acid no matter from maternal or offspring, in regulating animal lipid metabolism and immune enhancement, providing a theoretical basis for the use of folic acid from the view of animal health care.

Folic acid supplementation during pregnancy modulates hepatic methyl metabolism and genes expression profile of neonatal lambs of different litter sizes.

Maternal folic acid (FA) plays an important role in the fetus development, but it is unknown the response of hepatic metabolism in the offspring from different litter sizes to maternal FA supplementation. In the present study, this was done by feeding the ewes with 0, 16 and 32 mg/(kg·DM) FA supplemented diet during pregnancy and analysing the hepatic one-carbon metabolism-related indices and gene expression in the neonatal lambs of different litter sizes (twins, TW; triplets, TR). Regardless of litter sizes, the concentrations of folate, methionine, S-adenosylmethionine and DNA methyltransferase increased significantly, but homocysteine and S-adenosylhomocysteine decreased in the liver of newborn lambs from ewes whose diet was supplemented with FA. In TW, maternal FA status has little effect on hepatic genes expression profile of newborn lambs, and no significant enriched pathway was found. However, DEG involved in cell proliferation such as CCNA2, CCNB2, CCNE2, CDK1 and BUB1 were significantly enriched when the ewes were supplemented with FA in TR groups. In addition, nucleotide synthesis-related genes such as POLD1, POLD2, MCM4 and MCM5 were enriched markedly in DNA replication and pyrimidine metabolism pathways in triplets when a higher FA ingestion [32 mg/(kg·DM)] was implemented in ewes. This finding demonstrated that the hepatic methyl metabolism in TW and TR newborn lambs was regulated by maternal FA status. The hepatic cell proliferation and nucleotide metabolism related genes in TR were more susceptible to maternal dietary FA supplementation during pregnancy.

Vitamin E Can Ameliorate Oxidative Damage of Ovine Hepatocytes In Vitro by Regulating Genes Expression Associated with Apoptosis and Pyroptosis, but Not Ferroptosis.

(1) Background: the current research was conducted to investigate the potential non-antioxidant roles of vitamin E in the protection of hepatocysts from oxidative damage. (2) Methods: primary sheep hepatocytes were cultured and exposed to 200, 400, 600, or 800 µmol/L hydrogen peroxide, while their viability was assessed using a CCK-8 kit. Then, cells were treated with 400 µmol/L hydrogen peroxide following a pretreatment with 50, 100, 200, 400, and 800 µmol/L vitamin E and their intracellular ROS levels were determined by means of the DCF-DA assay. RNA-seq, verified by qRT-PCR, was conducted thereafter: non-treated control (C1); cells treated with 400 µmol/L hydrogen peroxide (C2); and C2 plus a pretreatment with 100 µmol/L vitamin E (T1). (3) Results: the 200-800 µmol/L hydrogen peroxide caused significant cell death, while 50, 100, and 200 µmol/L vitamin E pretreatment significantly improved the survival rate of hepatocytes. ROS content in the cells pretreated with vitamin E was significantly lower than that in the control group and hydrogen-peroxide-treated group, especially in those pretreated with 100 µmol/L vitamin E. The differentially expressed genes (DEGs) concerning cell death involved in apoptosis (RIPK1, TLR7, CASP8, and CASP8AP2), pyroptosis (NLRP3, IL-1ß, and IRAK2), and ferroptosis (TFRC and PTGS2). The abundances of IL-1ß, IRAK2, NLRP3, CASP8, CASP8AP2, RIPK1, and TLR7 were significantly increased in the C1 group and decreased in T1 group, while TFRC and PTGS2 were increased in T1 group. (4) Conclusions: oxidative stress induced by hydrogen peroxide caused cellular damage and death in sheep hepatocytes. Pretreatment with vitamin E effectively reduced intracellular ROS levels and protected the hepatocytes from cell death by regulating gene expression associated with apoptosis (RIPK1, TLR7, CASP8, and CASP8AP2) and pyroptosis (NLRP3, IL-1ß, and IRAK2), but not ferroptosis (TFRC and PTGS2).

Specific recognition of protein by deep eutectic solvent-based magnetic ß-cyclodextrin molecularly imprinted polymer.

A magnetic ß-cyclodextrin (MCD) surface molecularly imprinted polymer (MIP) based on deep eutectic solvents (DESs) as cross-linker and functional monomer (MCD@DES-MIP) was successfully synthesized for the specific recognition of bovine hemoglobin (BHb). The adsorption behavior of MCD@DES-MIP for BHb was investigated by adsorption thermodynamics, adsorption kinetics, and pH control experiments. The maximum adsorption capacity of MCD@DES-MIP for BHb under the optimized conditions was 195.94 mg g-1 and the imprinting factor was 4.68. In addition, the competitive adsorption experiments demonstrated that MCD@DES-MIP showed excellent selective extraction ability for BHb in the binary mixture of BHb and bovine serum albumin (BSA). The actual sample analysis manifested that MCD@DES-MIP effectively separated BHb from complex samples. The results of circular dichroism spectra proved that the secondary structure of BHb did not change during elution. The result indicated that MCD@DES-MIP can be used as a new imprinting material for the separation and purification of BHb.Graphical abstract Magnetic imprinted microspheres (MCD@DES-MIP) were prepared by free radical polymerization using magnetic ß-cyclodextrin (MCD) as carrier, deep eutectic solvents (DESs) as functional monomer and cross-linker. MCD@DES-MIP show high adsorption capacity and excellent selectivity for BHb.

A green deep eutectic solvent modified magnetic titanium dioxide nanoparticles for the solid-phase extraction of chymotrypsin.

Magnetic titanium dioxide nanoparticles modified with green deep eutectic solvent (DES) composed of choline chloride (ChCl) and xylitol (Xyl) (Fe3O4@TiO2@[ChCl][Xyl]) were synthesized and applied to the solid-phase extraction(MSPE) of chymotrypsin (Chy). The physicochemical properties and morphology of Fe3O4@TiO2@[ChCl][Xyl] was characterized by Fourier transform infrared spectroscopy (FT-IR), thermogravimetric analysis (TGA), Zeta potential, X-ray diffraction (XRD), vibrating sample magnetometer (VSM) and transmission electron microscope (TEM). The experiment parameters such as initial concentration of Chy, extraction time, pH value, ionic strength, extraction temperature and sample matrix were effectively optimized. Under the optimal experimental conditions, the extraction capacity of Fe3O4@TiO2@[ChCl][Xyl] obtained a significantly improvement after the modification of Fe3O4@TiO2 nanoparticles by [ChCl][Xyl], and reached up to 347.8 mg g-1. In the elution experiment, 10% sodium dodecyl sulfate-acetic acid (SDS-HAc) was used as eluent, achieving an elution rate of 85.9% for the Chy on Fe3O4@TiO2@[ChCl][Xyl]. And the Fe3O4@TiO2@[ChCl][Xyl] still maintained a good extraction capacity for Chy after six times of reuse. The application result in the extraction of Chy from porcine pancreas crude extract showed a good practical application ability for Chy extraction. All the results indicated that the synthesized Fe3O4@TiO2@[ChCl][Xyl] has good application potential in the extraction of biomolecular molecules such as protein.

A novel"turn-off"fluorescence assay based on acid-copper nanoclusters in deep eutectic solvent micelles for co-aggregation inducing fluorescence enhancement and its application.

As mixtures, deep eutectic solvent (DES) is designability. By adjusting the long alkyl chain hydrogen bond acceptors (HBAs) or hydrogen bond donors (HBDs), the DES displays surfactant characteristics and can form micelles. Hence, a novel, simple, facile and green natural organic acids capped copper nanoclusters (Aci-CuNCs) was synthesized and the spectrum behavior of Aci-CuNCs in DES micelles was researched. It was found that the surfactant-like DES can form micellar co-aggregation with Aci-CuNCs, resulting in the fluorescence (FL) intensive of Aci-CuNCs increase. Corresponding performance of spectral properties of Aci-CuNCs in DES medium were systematically studied by fourier transform infrared spectrometer, 3D FL spectroscopy, FL emission/excitation spectra, ultraviolet absorption spectroscopy. In the mechanism exploration part, on the one hand, the existence of micellar co-aggregation was confirmed by the conductivity, the mass effect of DES, dynamic light scattering and transmission electron microscopy. On the other hand, the influence of different kinds of DESs (types of HBAs/HBDs, molar ratio) and some possible factors (ionic strength and temperature) were discussed in detail to investigate the main driving forces for the formation of micellar co-aggregates. The results of mechanism exploration prove that the long alkyl chain of DES is amphiphilic which can form micellar co-aggregation with Aci-CuNCs through hydrogen bonding. The DES micelle provides Aci-CuNCs with a relatively stable and closed micro-environment which can effectively prevent collisions with water molecules and weakening of fluorescence intensity. On the basic of the above research, a "turn-off" fluorimetric method based on Aci-CuNCs in DES medium was applied for the determination of Fe3+. Under the optimum conditions, the assay worked in the Fe3+ concentration ranges from 1 to -20 µM and had a detection limit of 0.0374 µM. Method validation study illustrates the proposed system can provide a good accuracy, repeatability and stability conditions. Furthermore, the real sample analysis result demonstrates that no obvious matrix effect is found. As a consequence, the FL assays (Aci-CuNCs-based DES) composed of natural organic acid capped CuNCs and green solvent DES provides a simple, gentle and environmentally friendly method for the detection of iron ions.

Maternal Folic Acid Supplementation Differently Affects the Small Intestinal Phenotype and Gene Expression of Newborn Lambs from Differing Litter Sizes.

The purpose of this study was to investigate the effect of maternal dietary folic acid (FA) supplementation during gestation on small intestinal development of newborn lambs of different litter sizes, focusing on the intestinal morphology and development-, apoptosis- and digestion-related genes expression. One hundred and twenty Hu ewes (Ovis aries) were inseminated and randomly allotted to three groups. One group received a control diet [without FA supplementation, control (CON)] and the other two groups received control diets supplemented with different amount of FA [16 or 32 mg FA per kg dry matter (DM), i.e., F16 and F32] during pregnancy. After lambing, according to the dietary FA levels and litter size (twins, TW; triplets, TR), the neonatal lambs were divided into 6 (TW-CON, TW-F16, TW-F32, TR-CON, TR-F16, TR-F32) treatment groups. The results showed that the ratio of small intestinal weight to live body weight and the thickness of the intestinal muscle layer in the offspring was enhanced significantly with increasing maternal FA supplementation (p < 0.05). Meanwhile, the expression levels of insulin-like growth factor I (IGF-I), B-cell lymphoma-2 (BCL-2) and sodium/glucose co-transporter-1 (SGLT1) in the small intestines of the newborn lambs were increased, while the opposite was true for Bcl2-associated × (BAX) in response to FA supplementation (p < 0.05). Moreover, the small intestinal weights of twins were significantly higher than those of triplets (p < 0.01), and the expression levels of IGF-I (p < 0.05), sucrase-isomaltase (SI) (p < 0.05) and solute carrier family 2 member 5 (SLC2A5) (p < 0.01) were significantly lower than those in triplets. These findings suggest that maternal FA supplementation could improve the offspring's small intestinal phenotype and the expression of development-, apoptosis- and digestion-related genes, so it could promote the small intestinal development of newborn lambs. Furthermore, the small intestine phenotypic development of twins was generally better than that of triplets, while the expression levels of the above genes of twins were lower than those of triplets.

A deep eutectic solvent modified magnetic ß-cyclodextrin particle for solid-phase extraction of trypsin.

An environmentally friendly deep eutectic solvent (DES) composed of trifluoroacetamide and benzyltrimethylammonium chloride modified ß-cyclodextrin (CD) grafted magnetic beads (MB-NH2@CD@DES) were synthesized for the first time and applied to the solid-phase extraction of trypsin. Among the five trypsin extractants prepared in this work (MB, MB-NH2, MB-NH2@CD, MB-NH2@DES, MB-NH2@CD@DES), the extractant MB-NH2@CD@DES with higher extraction capacity for trypsin was selected as final extractant. The extraction capacity of MB-NH2@CD@DES for trypsin can reach up to 549.87 mg⋅g-1 under the optimized conditions. The Langmuir adsorption equilibrium was found fitted better with equilibrium relation between MB-NH2@CD@DES and trypsin than Freundlich adsorption equilibrium. And a superior extraction for trypsin was verified by comparing the extraction capacity of MB-NH2@CD@DES for trypsin and four other common proteins. Compared with some reported trypsin extractants, the MB-NH2@CD@DES had a shorter extraction process, higher extraction capacity, more convenient operation of separation, a safer and more environmentally friendly synthesis process. With the optimized eluent, a great elution rate (74.32%) of trypsin was achieved. The absolute recovery of trypsin in trypsin standard solution was calculated to be 16.8%. And the extraction capacity of MB-NH2@CD@DES toward trypsin still maintained well after ten times recycling and reuse. The detection limit (LOD) and quantitative limit (LOQ) were 0.072 mg⋅mL-1 and 0.240 mg⋅mL-1 respectively. By sodium dodecyl sulfate polyacrylamide gel electrophoresis experiment, the extraction ability of MB-NH2@CD@DES to trypsin from real sample was fully demonstrated. All above results showed the potential of fabricated MB-NH2@CD@DES as a superior extractant for trypsin from real complex samples.

A new magnetic molecularly imprinted polymer based on deep eutectic solvents as functional monomer and cross-linker for specific recognition of bovine hemoglobin.

A novel magnetic molecularly imprinted polymer only based on deep eutectic solvents (DESs-MMIP) was successfully synthesized. The DESs-MMIP was constructed by using 2-hydroxyethyl methacrylate/tetrabutylammonium chloride deep eutectic solvent (DES1) as functional monomer, arylamide/(3-acrylamidopropyl) trimethylammonium chloride deep eutectic solvent (DES2) as cross-linker and bovine hemoglobin (BHb) as template through surface imprinting technology. The obtained DESs-MMIP was characterized by transmission electron microscope, X-ray diffraction, fourier transform infrared spectrometry, thermal gravimetric analysis and vibrating sample magnetometer. Under the optimized conditions, the maximum adsorption capacity of DESs-MMIP on BHb was 229.54 mg g-1 and the imprinting factor reached up to 21.89. The selective adsorption experiments indicated that compared with seven references, DESs-MMIP showed significant selectivity for BHb. The new-type DESs-MMIP exhibited higher adsorption capacity and imprinting factor on BHb than molecularly imprinted polymers constructed with traditional functional monomer and cross-linker in reported methods. The recognition of BHb by DESs-MMIP in calf blood samples demonstrated the practicality of the particles. The DESs-MMIP only based on deep eutectic solvents with excellent selectivity is expected to become an ideal candidate for selective recognition of BHb in complicated samples.

Effect of Dietary Folic Acid Supplementation during Pregnancy on Blood Characteristics and Milk Composition of Ewes.

The objective of the present study was to investigate the dynamic change of serum parameters and milk composition by dietary FA supplementation with ewes with different litter size from mating to lambing. The ewes were divided into six treatments (TW-CON, TW-F16, TW-F32, TR-CON, TR-F16, TR-F32) according to dietary FA levels (control, CON; 16 or 32 mg·kg-1 rumen-protect-FA supplementation, F16 and F32) and litter size (twin born, TW; and triplet born, TR). In serum, the concentration of folate increased linearly with dietary FA supplementation (P < 0.05), regardless of the litter size, they showed a quadratic response to gestation progression (P < 0.05). With dietary FA addition, IGFI-I levels significant increased from late gestation to after lambing (P < 0.05), and linearly increased immunoglobulin during the perinatal period (P < 0.05). In colostrum and milk at d 15, the content of folate, lactoferrin, and IgG were affected positively by FA supplementation (P < 0.05). IgG was higher in the TW group than TR in colostrum (P < 0.05), and lactoferrin in TW was lower than TR in milk of d 15 (P < 0.05). FA supplementation increased protein content in colostrum (P < 0.05), while it had no effect on the fat, lactose, and BUN of colostrum and milk of d 15 (P > 0.05). These results suggest that FA supplementation during gestation could regulate maternal blood metabolism and contribute to milk immune composition.

Maternal folic acid supplementation modulates the growth performance, muscle development and immunity of Hu sheep offspring of different litter size.

It is generally accepted that the phenotype and gene expression pattern of the offspring can be altered by maternal folic acid (FA) supplementation during the gestation period. The aims of this study were to investigate the effects of maternal FA supplementation on the growth performance, muscle development and immunity of newborn lambs of different litter size. According to litter size (twins, TW; triplets, TR) and maternal dietary FA supplementation levels (control, C; 16 or 32 mg·kg-1 FA supplementation, F16 and F32), neonatal lambs were randomly divided into six groups (TW-C, TW-F16, TW-F32, TR-C, TR-F16 and TR-F32). After farrowing, the birth weight in TW was higher than that in the TR group, and increased with FA supplementation of their mothers (P<.05). Folate, IGF-I, IgM and IgA concentrations of newborn lambs showed a litter size and FA supplementation interaction (P<.05). FA supplementation also increased diameter, area, perimeter and DNA content of the longissimus dorsi muscle of the lambs (P<.05) regardless of the litter size. Transcriptome analysis of the longissimus dorsi muscle revealed differentially expressed genes with dietary FA supplementation enriched in immunity- and cell development-related genes. Furthermore, FA supplementation upregulated the expression of myogenesis-related genes, while downregulated those involved in the inhibition of muscle development. In addition, immunity-related genes in the neonatal lambs showed lower expression levels in response to maternal dietary FA supplementation. Overall, maternal FA supplementation during gestation could increase the offspring's birth weight and modulate its muscle development and immunity.