The landscape of allelic expression and DNA methylation at the bovine SGCE/PEG10 locus.

Genomic imprinting is an epigenetic regulation in mammals in which a small subset of genes is monoallelically expressed dependent on their parental origin. A large imprinted domain, SGCE/PEG10 locus, is located on human chromosome 7q21s and mouse proximal chromosome 6. However, genomic imprinting of bovine SGCE/PEG10 cluster has not been systematically studied. In this study, we investigated allele expression of 14 genes of the SGCE/PEG10 locus in bovine somatic tissues and term placenta using a single nucleotide polymorphism (SNP)-based sequencing method. In addition to SGCE and PEG10, two conserved paternally expressed genes in human and mice, five other genes (TFPI2, GNG11, ASB4, PON1, and PON3) were paternally expressed. Three genes, BET1, COL1A2, and CASD1, exhibited tissue-specific monoallelic expression. CALCR showed monoallelic expression in tissues but biallelic expression in the placenta. Three genes, GNGT1, PPP1R9A, and PON2, showed biallelic expression in cattle. Five differentially methylated regions (DMRs) were found to be associated with the allelic expression of TFPI2, COL1A2, SGCE/PEG10, PON3, and ASB4 genes, respectively. The SGCE/PEG10 DMR is a maternally hypermethylated germline DMR, but TFPI2, COL1A2, PON3, and ASB4 DMRs are secondary DMRs. In summary, we identified five novel bovine imprinted genes (GNG11, BET1, COL1A2, CASD1, and PON1) and four secondary DMRs at the SGCE/PEG10 locus.

Layer-by-layer nanoparticle encapsulating all-trans retinoic acid and CpG as a mucosal adjuvant targeting colorectal cancer.

Colorectal cancer (CRC) ranks among the most prevalent cancers globally, demanding innovative therapeutic strategies. Immunotherapy, a promising avenue, employs cancer vaccines to activate the immune system against tumors. However, conventional approaches fall short of eliciting robust responses within the gastrointestinal (GI) tract, where CRC originates. Harnessing the potential of all-trans retinoic acid (ATRA) and cytosine-phosphorothioate-guanine (CpG), we developed layered nanoparticles using a layer-by-layer assembly method to co-deliver these agents. ATRA, crucial for gut immunity, was efficiently encapsulated alongside CpG within these nanoparticles. Administering these ATRA@CpG-NPs, combined with ovalbumin peptide (OVA), effectively inhibited orthotopic CRC growth in mice. Our approach leveraged the inherent benefits of ATRA and CpG, demonstrating superior efficacy in activating dendritic cells, imprinting T cells with gut-homing receptors, and inhibiting tumor growth. This mucosal adjuvant presents a promising strategy for CRC immunotherapy, showcasing the potential for targeting gut-associated immune responses in combating colorectal malignancies.

A novel imprinted locus on bovine chromosome 18 homologous with human chromosome 16q24.1.

Genomic imprinting is an epigenetic regulation mechanism in mammals resulting in the parentally dependent monoallelic expression of genes. Imprinting disorders in humans are associated with several congenital syndromes and cancers and remain the focus of many medical studies. Cattle is a better model organism for investigating human embryo development than mice. Imprinted genes usually cluster on chromosomes and are regulated by different methylation regions (DMRs) located in imprinting control regions that control gene expression in cis. There is an imprinted locus on human chromosome 16q24.1 associated with congenital lethal developmental lung disease in newborns. However, genomic imprinting on bovine chromosome 18, which is homologous with human chromosome 16 has not been systematically studied. The aim of this study was to analyze the allelic expressions of eight genes (CDH13, ATP2C2, TLDC1, COTL1, CRISPLD2, ZDHHC7, KIAA0513, and GSE1) on bovine chromosome 18 and to search the DMRs associated gene allelic expression. Three transcript variants of the ZDHHC7 gene (X1, X2, and X5) showed maternal imprinting in bovine placentas. In addition, the monoallelic expression of X2 and X5 was tissue-specific. Five transcripts of the KIAA0513 gene showed tissue- and isoform-specific monoallelic expression. The CDH13, ATP2C2, and TLDC1 genes exhibited tissue-specific imprinting, however, COTL1, CRISLPLD2, and GSE1 escaped imprinting. Four DMRs, established after fertilization, were found in this region. Two DMRs were located between the ZDHHC7 and KIAA0513 genes, and two were in exon 1 of the CDH13 and ATP2C2 genes, respectively. The results from this study support future studies on the molecular mechanism to regulate the imprinting of candidate genes on bovine chromosome 18.

A Synergistic Lipid Nanoparticle Encapsulating mRNA Shingles Vaccine Induces Potent Immune Responses and Protects Guinea Pigs from Viral Challenges.

Shingles is caused by the reactivation of varicella zoster virus (VZV) and manifests as painful skin rashes. While the recombinant protein-based vaccine proves highly effective, it encounters supply chain challenges due to a shortage of the necessary adjuvant. Messenger RNA (mRNA)-based vaccines can be rapidly produced on a large scale, but their effectiveness relies on efficient delivery and sequence design. Here, an mRNA-based VZV vaccine using a synergistic lipid nanoparticle (Syn-LNP) containing two different ionizable lipids is developed. Syn-LNP shows superior mRNA expression compared to LNPs formulated with either type of ionizable lipid and to a commercialized LNP. After encapsulating VZV glycoprotein E (gE)-encoding mRNA, mgE@Syn-LNP induces robust humoral and cellular immune responses in two strains of mice. The magnitude of these responses is similar to that induced by adjuvanted recombinant gE proteins and significantly higher than that observed with live-attenuated VZV. mgE@Syn-LNP exhibits durable humoral responses for over 7 months without obvious adverse effects. In addition, mgE@Syn-LNP protects vaccinated guinea pigs against live VZV challenges. Preliminary studies on the mRNA antigen design reveal that the removal of glycosylation sites of gE greatly reduces its immune responses. Collectively, Syn-LNP encapsulating gE-encoded mRNA holds great promise as a shingles vaccine.

Photoluminescence-Electrochemiluminescence Dual-Mode Sensor Arrays for Histidine and Its Metabolite Discrimination and Disease Identification.

Histidine (His) and its metabolite analysis is significant due to their vital roles in the diagnosis of diseases. In practical applications, simple and effective detection and discrimination of these metabolic species are still a great challenge due to their highly similar structures. Herein, photoluminescence (PL)-electrochemiluminescence (ECL) dual-mode sensor arrays consisting of a series of sensing elements were proposed for simultaneous quantitation and accurate discrimination of His and its four key metabolites (including histamine, imidazole-4-acetic acid, N-acetylhistamine, and imidazole propionate). The sensing elements of these sensor arrays were constructed by employing two solvent iridium(III) complexes ([Ir(pbz)2(DMSO)Cl] and [Ir(ppy)2(DMSO)Cl], pbz = 3-(2-pyridyl)benzoic acid, ppy = 2-phenylpyridine) with excellent PL and ECL performances as cross-responsive sensing units. Based on diverse coordination abilities of the two complexes with the imidazole group of the five targets, PL and ECL responses of each sensing unit can be enhanced to various degrees, which generate unique fingerprint patterns for the corresponding targets. Through principal component analysis, the multifarious patterns (two-, three-, and four-element sensor arrays) can be transformed into simple visualization modes, from which His and its four key metabolites can be effectively discriminated against each other. Moreover, the quantitation of an individual metabolic species at different concentrations and the recognition of the mixtures with different ratios were also accurately achieved. Notably, His and its four key metabolites in urine can also be successfully discriminated by the as-fabricated sensor arrays, and the patients with kidney diseases can be identified clearly, providing a promising way for disease diagnosis.

A fluorinated ionizable lipid improves the mRNA delivery efficiency of lipid nanoparticles.

The efficacy of messenger RNA (mRNA)-based vaccines or therapies relies on delivery vehicles that can transport them into the cytosol of cells. Lipid nanoparticles (LNPs) are the most clinically advanced carrier for mRNA. The chemical structure of an ionizable lipid is critical for the delivery efficiency of the LNPs. Herein, we synthesize a new ionizable lipid containing fluorinated alkyl chains (F-L319) and evaluate its mRNA delivery efficiency compared to its hydrocarbon counterpart (L319). While LNPs formulated with F-L319 alone showed decreased mRNA encapsulation and delivery efficiencies in comparison to the L319-LNP, we found that combining the appropriate ratios of F-L319 and L319 as hybrid ionizable lipids in LNPs (hybrid-LNPs) greatly enhanced mRNA delivery efficiency both in vitro and in vivo. Upon intravenous injection, the hybrid-LNP showed targeted mRNA expression in the spleen. Mechanistic studies indicate that the enhanced mRNA delivery of the hybrid-LNP is attributed to both improved mRNA encapsulation and cellular uptake. Collectively, fluorination of ionizable lipids represents a promising strategy to improve the delivery efficiency of LNPs.

PMM2 and NARFL are paternally imprinted genes in bovines.

In mammals, imprinted genes are required for both fetal development and postnatal growth. A novel candidate imprinted locus was found on human chromosome 16, and maternal uniparental disomy of this locus can cause a lethal developmental lung disease in human newborns. The PMM2 and NARFL genes are located in this region and its homologous region in cattle is on chromosome 25. Currently, there is no report on the genomic imprinting of the PMM2 and NARFL genes. In this study, we demonstrated that PMM2 and NARFL are two paternally imprinted genes in bovines using an SNP-based method. In addition, two differentially methylated regions of paternal methylation were found in the promoter region and the third intron of the bovine NARFL gene, which may be involved in regulating its imprinted expression. However, we did not find differential methylation in the promoter region or the seventh intron of the bovine PMM2 gene.

IMPACT and OSBPL1A are two isoform-specific imprinted genes in bovines.

The epigenetic process of genomic imprinting results in the monoallelic expression of genes based on their parental origin. Comparative analysis of imprinted genes between species is useful for investigating the biological significance and regulatory mechanisms of genomic imprinting. Mouse Impact is an imprinted gene, but its human ortholog IMPACT escapes genomic imprinting. Hrh4 and Osbpl1a are the two nearest neighbors of the Impact located in distal and proximal regions, respectively. This study aims to assess the allelic expression of bovine IMPACT, OSBPL1A and HRH4 genes and examine the differentially methylated regions (DMRs) associated with these three genes. Based on an expressed single-nucleotide polymorphism (SNP) approach, we found that both the IMPACT and OSBPL1A genes exhibit isoform-specific monoallelic expression in bovine adult tissues. In the seven detected bovine IMPACT transcripts, only one transcript variant (X6) is monoallelically expressed in bovine adult tissues and paternally expressed in the placenta. However, no DMR was found in the promoter region of the IMPACT gene. We obtained five transcript variants (V1-V5) of the bovine OSBPL1A gene of different lengths that start transcription from distinct alternative promoters by RT-PCR. Only the longest variant V1 was found to be expressed monoallelically in bovine adult tissues and a DMR was identified in its promoter region using the bisulfite sequencing method. Thus, the DMR in OSBPL1A V1 promoter region may contribute to its isoform-specific monoallelic expression. The bovine HRH4 gene is expressed biallelically. Hypermethylation was observed in brains without HRH4 expression, while hypomethylation was found in the spleens with HRH4 expression, so and the level of DNA methylation in the promoter seemed to be related to its expression in tissues.

Genomic imprinting of the IGF2R/AIR locus is conserved between bovines and mice.

Genomic imprinting is an epigenetic phenomenon that leads to genes monoallelically expressed in a parent-of-origin-specific manner and plays an important role in the embryonic development and postnatal growth of mammals. Imprinted genes usually occur in clusters in a chromosomal region and are regulated by a cis-acting imprinting control region that involves differential DNA methylation modification. Igf2r, Slc22a2 and Slc22a3 are three maternally expressed genes on mouse chromosome 17. The paternally expressed long noncoding RNA (lncRNA) Air and the nonimprinted gene Slc22a1 are also located in the imprinted region. Comparative characterization of imprinted clusters between species is useful for us to understand the biological significance and epigenetic regulating mechanism of genomic imprinting. The aim of this study was to analyze the allelic expression pattern of AIR and SLC22A1-3 genes in cattle and to determine the role of DNA methylation in regulating gene expression. Allelic expression analysis was performed in bovine adult tissues and term placenta using an SNP-based approach. We found that IGF2R, AIR and SLC22A3 were monoallelically expressed in all detected bovine somatic tissues, including heart, liver, spleen, lung, kidney, muscle, fat and brain. In bovine placenta, IGF2R and SLC22A3 are maternally expressed; however, the AIR gene is paternally expressed. Tissue-specific monoallelic expression of SLC22A2 is detected in bovines, with monoallelic expression in the spleen and brain but biallelic expression in kidney tissues. SLC22A1 is only detected in bovine liver and kidney tissues and is biallelicly expressed, which is consistent with the imprint expression in mice. To determine the possible role of DNA methylation in regulating the monoallelic/imprinted expression of bovine IGF2R, AIR, SLC22A2, and SLC22A3 genes, we analyzed the DNA methylation status of CpG islands in the first exon of SLC22A2, the promoter region of SLC22A3 and region 2 in the second intron of the IGF2R gene by bisulfite sequencing. Two differentially methylated regions (DMRs) were detected in the first exon of bovine SLC22A3 and the common regions of IGF2R and AIR. This suggests that DNA methylation is involved in the regulation of monoallelic/imprinted expression of IGF2R, AIR and SLC22A3 genes in cattle.

Effects of Dietary Glucose Oxidase Supplementation on the Performance, Apparent Ileal Amino Acids Digestibility, and Ileal Microbiota of Broiler Chickens.

This study aimed to investigate the effects of glucose oxidase (GOD) supplementation on growth performance, apparent ileal digestibility (AID) of nutrients, intestinal morphology, and short-chain fatty acids (SCFAs) and microbiota in the ileum of broilers. Six hundred 1-day-old male broilers were randomly allotted to four groups of 10 replicates each with 15 birds per replicate cage. The four treatments included the basal diet without antibiotics (Control) and the basal diet supplemented with 250, 500, or 1000 U GOD/kg diet (E250, E500 or E1000). The samples of different intestinal segments, ileal mucosa, and ileal digesta were collected on d 42. Dietary GOD supplementation did not affect daily bodyweight gain (DBWG) and the ratio of feed consumption and bodyweight gain (FCR) during d 1-21 (p > 0.05); however, the E250 treatment increased DBWG (p = 0.03) during d 22-42 as compared to control. Dietary GOD supplementation increased the AIDs of arginine, isoleucine, lysine, methionine, threonine, cysteine, serine, and tyrosine (p < 0.05), while no significant difference was observed among the GOD added groups. The E250 treatment increased the villus height of the jejunum and ileum. The concentrations of secreted immunoglobulin A (sIgA) in ileal mucosa and the contents of acetic acid and butyric acid in ileal digesta were higher in the E250 group than in the control (p < 0.05), whereas no significant differences among E500, E1000, and control groups. The E250 treatment increased the richness of ileal microbiota, but E500 and E100 treatment did not significantly affect it. Dietary E250 treatment increased the relative abundance of Firmicutes phylum and Lactobacillus genus, while it decreased the relative abundance of genus Escherichina-Shigella (p < 0.05). Phylum Fusobacteria only colonized in the ileal digesta of E500 treated broilers and E500 and E1000 did not affect the relative abundance of Firmicutes phylum and Lactobacillus and Escherichina-Shigella genera as compared to control. These results suggested that dietary supplementation of 250 U GOD/kg diet improves the growth performance of broilers during d 22-42, which might be associated with the alteration of the intestinal morphology, SCFAs composition, and ileal microbiota composition.