AtacAnnoR: a reference-based annotation tool for single cell ATAC-seq data.

Here, we present AtacAnnoR, a two-round annotation method for scATAC-seq data using well-annotated scRNA-seq data as reference. We evaluate AtacAnnoR's performance against six competing methods on 11 benchmark datasets. Our results show that AtacAnnoR achieves the highest mean accuracy and the highest mean balanced accuracy and performs particularly well when unpaired scRNA-seq data are used as the reference. Furthermore, AtacAnnoR implements a 'Combine and Discard' strategy to further improve annotation accuracy when annotations of multiple references are available. AtacAnnoR has been implemented in an R package and can be directly integrated into currently popular scATAC-seq analysis pipelines.

Protective effects of melatonin on deoxynivalenol-induced oxidative stress and autophagy in IPEC-J2 cells.

This study explored protective effects of melatonin (MEL) on deoxynivalenol (DON)-induced toxicity in porcine jejunum epithelial cells (IPEC-J2). Cells were preexposed to MEL and then exposed to DON to detect cell viability, apoptosis, and oxidative stress indicators. Compared to DON treatment, pretreatment with MEL significantly increased cell proliferation. (P < 0.01), intracellular catalase (CAT) and superoxide dismutase (SOD) levels (P < 0.05), decreased apoptosis and oxidative stress, and significantly attenuated the inflammatory response. RNA-seq analysis revealed that MEL protects IPEC-J2 from the adverse effects of DON by affecting the expression of tight junction and autophagy pathway-related genes. Similarly, further experiments revealed that MEL partly prevented DON-induced disruption of intestinal barrier function and decreased autophagy induced by DON via activation of the AKT/mTOR pathway. In conclusion, these results demonstrated the preventive properties of MEL against DON-induced cell damage by activating the antioxidant system and Inhibition of autophagy.

Integrative ATAC-seq and RNA-seq analyses of IPEC-J2 cells reveals porcine transcription and chromatin accessibility changes associated with Escherichia coli F18ac inhibited by Lactobacillus reuteri.

Escherichia coli is the main cause of postweaning diarrhea in pigs, leading to economic loss. As a probiotic, Lactobacillus reuteri has been used to inhibit E. coli in clinical applications; however, its integrative interactions with hosts remain unclear, especially in pigs. Here, we found that L. reuteri effectively inhibited E. coli F18ac adhering to porcine IPEC-J2 cells, and explored the genome-wide transcription and chromatin accessibility landscapes of IPEC-J2 cells by RNA-seq and ATAC-seq. The results showed that some key signal transduction pathways, such as PI3K-AKT and MAPK signaling pathways, were enriched in the differentially expressed genes (DEGs) between E. coli F18ac treatment with and without L. reuteri groups. However, we found less overlap between RNA-seq and ATAC-seq datasets; we speculated that this might be caused by histones modification through ChIP-qPCR detection. Furthermore, we identified the regulation of the actin cytoskeleton pathway and a number of candidate genes (ARHGEF12, EGFR, and DIAPH3) that might be associated with the inhibition of E. coli F18ac adherence to IPEC-J2 cells by L. reuteri. In conclusion, we provide a valuable dataset that can be used to seek potential porcine molecular markers of E. coli F18ac pathogenesis and L. reuteri antibacterial activity, and to guide the antibacterial application of L. reuteri.

LncRNA446 Regulates Tight Junctions by Inhibiting the Ubiquitinated Degradation of Alix after Porcine Epidemic Diarrhea Virus Infection.

Porcine epidemic diarrhea (PED) is a highly contagious disease, caused by porcine epidemic diarrhea virus (PEDV), which causes huge economic losses. Tight junction-associated proteins play an important role during virus infection; therefore, maintaining their integrity may be a new strategy for the prevention and treatment of PEDV. Long noncoding RNAs (lncRNAs) participate in numerous cellular functional activities, yet whether and how they regulate the intestinal barrier against viral infection remains to be elucidated. Here, we established a standard system for evaluating intestinal barrier integrity and then determined the differentially expressed lncRNAs between PEDV-infected and healthy piglets by lncRNA-seq. A total of 111 differentially expressed lncRNAs were screened, and lncRNA446 was identified due to significantly higher expression after PEDV infection. Using IPEC-J2 cells and intestinal organoids as in vitro models, we demonstrated that knockdown of lncRNA446 resulted in increased replication of PEDV, with further damage to intestinal permeability and tight junctions. Mechanistically, RNA pulldown and an RNA immunoprecipitation (RIP) assay showed that lncRNA446 directly binds to ALG-2-interacting protein X (Alix), and lncRNA446 inhibits ubiquitinated degradation of Alix mediated by TRIM25. Furthermore, Alix could bind to ZO1 and occludin and restore the expression level of the PEDV M gene and TJ proteins after lncRNA446 knockdown. Additionally, Alix knockdown and overexpression affects PEDV infection in IPEC-J2 cells. Collectively, our findings indicate that lncRNA446, by inhibiting the ubiquitinated degradation of Alix after PEDV infection, is involved in tight junction regulation. This study provides new insights into the mechanisms of intestinal barrier resistance and damage repair triggered by coronavirus. IMPORTANCE Porcine epidemic diarrhea is an acute, highly contagious enteric viral disease severely affecting the pig industry, for which current vaccines are inefficient due to the high variability of PEDV. Because PEDV infection can lead to severe injury of the intestinal epithelial barrier, which is the first line of defense, a better understanding of the related mechanisms may facilitate the development of new strategies for the prevention and treatment of PED. Here, we demonstrate that the lncRNA446 directly binds one core component of the actomyosin-tight junction complex named Alix and inhibits its ubiquitinated degradation. Functionally, the lncRNA446/Alix axis can regulate the integrity of tight junctions and potentially repair intestinal barrier injury after PEDV infection.

Water-in-water emulsion stabilized by cellulose nanocrystals and their high enrichment effect on probiotic bacteria.

HYPOTHESIS: The effect of the molecular weight and polymer concentration on the partition behavior of aqueous two-phase systems (ATPs) is significant for constructing water-in-water (W/W) emulsions. Hence, a long-term stable W/W emulsion system might be obtained through selecting the appropriate stabilizer and component phases, which could be a possible carrier for probiotics. EXPERIMENTS: Compared with the reported molecular weight difference between polyethylene oxide (PEO) and dextran (DEX) systems, PEO and dextran with lower molecular weight had been used for constructing the water in water (W/W) emulsion system. The W/W emulsions were stabilized using cellulose nanocrystals (CNCs), and the potential application of the W/W emulsion for the encapsulation of Lactobacillus was explored. FINDINGS: Emulsion stability exhibited a "dose-effect" relationship with the CNCs concentration and was decreased with the increase of the DEX concentration. The emulsion phase separation rate was increased with increasing ionic strength and temperature. Both Lactobacillus Plantarum and Lactobacillus helveticus were highly inclined to the DEX phase, and the emulsion droplets were deformed and aggregated when the encapsulation amount was increased. This long-term stability would provide a promising approach for designing high-density culture and fermentation of probiotics.

Depolymerization of chicken egg yolk granules induced by high-intensity ultrasound.

The effects of high-intensity ultrasound (HIU) treatment-induced depolymerization of chicken egg yolk granules were investigated. The results showed that the yolk granules were depolymerized after HIU treatment, and the average particle size was significantly reduced from 289.4 nm (untreated) to 181.4 nm (270-W HIU treatment). All contents of dry matter, protein, calcium and phosphorus in the supernatant of the HIU-treated yolk granule solution increased, which suggests that HIU treatment increases the dissolution of yolk granule components. Spectroscopic analysis showed that HIU treatment increased the polarity of the microenvironment and enhanced the hydrogen bond force of yolk granules. These changes induced by HIU treatment collectively enhanced the zeta potential, decreased the free sulfhydryl group content, and slightly improved the emulsifying activity index of yolk granules. The present study reveals the depolymerization effect of HIU treatment on egg yolk granules and can inspire new potential applications of egg yolk granules.

Molecular aggregation and property changes of egg yolk low-density lipoprotein induced by ethanol and high-density ultrasound.

Solvent and physical treatment are widely used in egg yolk processing, but the detailed changes in the molecular structure of egg yolk proteins during processing are unclear. The aim of this study was to investigate the effects of ethanol and ultrasonic treatments on chicken egg yolk low-density lipoprotein (LDL). The solubility, emulsifying activity and emulsifying stability decreased by 74.75%, 46.91%, and 81.58% after ethanol treatment, respectively. The average particle size of ethanol-treated LDL increased 13.3-fold to 937.85 nm. These results suggested that ethanol treatment induced wide-ranging aggregation of LDL. In contrast to ethanol treatment, ultrasonic treatment promoted the solubility and emulsifying stability of LDL and enhanced its zeta-potential (119.56%) and surface hydrophobicity (10.81%). Based on particle size analysis and transmission electron microscopy, approximately 34.65% of LDL had undergone aggregation and the molecular interface became more flexible after ultrasonic treatment. These results revealed the detailed changes in egg yolk LDL structure and properties during solvent (ethanol) and physical (ultrasound) processing.

Effects of high-intensity ultrasonic (HIU) treatment on the functional properties and assemblage structure of egg yolk.

The effects of high-intensity ultrasonic (HIU) treatment on the functional properties of egg yolk were studied in the present work. After HIU treatment, the emulsifying, foaming and gel properties of the egg yolk solution significantly increased, but the foam stability decreased. SDS-PAGE results showed that there was no obvious change in the protein bands of egg yolk, indicating that the yolk proteins did not undergo covalent crosslinking or degradation. HIU treatment enhanced the zeta potential of egg yolk components in solution and increased the free sulfhydryl content of egg yolk proteins. Moreover, the particle size distribution of egg yolk components in solution changed markedly, and these changes demonstrated that HIU treatment caused the aggregation of yolk low-density lipoprotein and the partial dissociation of yolk granules. These results revealed that HIU treatment could change the aggregation of yolk components, which in turn could influence the solution characteristics of egg yolk, finally resulting in changes to the functional properties of egg yolk.

Large-scale purification of ovalbumin using polyethylene glycol precipitation and isoelectric precipitation.

In addition to small amounts of minerals and carbohydrates, most of the dry matter of chicken egg white is protein, making egg white an ideal resource for obtaining food proteins. Ovalbumin (OVA), which accounts for more than 50% of the total egg white protein, is one of the most widely studied food proteins due to its multiple functional properties, and it has also been used as a model protein molecule in many research fields. The objective of this study was to develop a simple and rapid method for the purification of OVA from egg whites on large scale. First, OVA was separated from ovomucin, ovotransferrin, and ovomucoid by polyethylene glycol (PEG) concentration, using the following optimal parameters: the PEG concentration was 15%, the pH was 6.5, the salt concentration was 100 mmol/L, and the operating temperature was 10°C. The OVA-rich supernatant obtained from PEG precipitation was further purified by isoelectric precipitation at a pI of 4.5 and a temperature of 4°C, and the purified OVA was obtained with at a purity of 95.1% by HPLC, with a yield of 46.4%. After the extraction of OVA, the PEG solution was vacuum dried and then utilized cyclically in the PEG precipitation steps. The whole purification process could be finished within 2 to 3 h at a scale of several kilograms of egg white. This method has the advantages of rapidity, simplicity, low cost, and ease of scalability.

N-Glycoproteomic Analysis of Chicken Egg Yolk.

Posttranslational N-glycosylation of food proteins plays a critical role in their structure and function. However, the N-glycoproteome of chicken egg yolk (CEY) has not been studied yet. Glycopeptides hydrolyzed from CEY proteins were enriched, with deglycosylation occurring using PNGase F, and then were identified using a shotgun glycoproteomics strategy. A total of 217 N-glycosylation sites and 86 glycoproteins were identified in CEY, and these glycoproteins are mainly involved in the binding, biological regulation, catalytic activity, and metabolic processes. Among the identified CEY glycoproteins, 22 were recognized as proteases and protease inhibitors, suggesting that a proteinase/inhibitor regulation system exists in CEY; further, 15 were members of the complement and immune systems, which provide protection against potential threats during hatching. The study provides important structural information about CEY glycoproteins and aids in the understanding of the underlying mechanism of embryo development as well as changes in CEY functional characteristics during storage and processing.