Characterization of sexual maturity-associated N6-methyladenosine in boar testes.

BACKGROUND: The health and size of the testes are crucial for boar fertility. Testicular development is tightly regulated by epigenetics. N6-methyladenosine (m6A) modification is a prevalent internal modification on mRNA and plays an important role in development. The mRNA m6A methylation in boar testicular development still needs to be investigated. RESULTS: Using the MeRIP-seq technique, we identify and profile m6A modification in boar testes between piglets and adults. The results showed 7783 distinct m6A peaks in piglets and 6590 distinct m6A peaks in adults, with 2,471 peaks shared between the two groups. Enrichment of GO and KEGG analysis reveal dynamic m6A methylation in various biological processes and signalling pathways. Meanwhile, we conjointly analyzed differentially methylated and expressed genes in boar testes before and after sexual maturity, and reproductive related genes (TLE4, TSSK3, TSSK6, C11ORF94, PATZ1, PHLPP1 and PAQR7) were identified. Functional enrichment analysis showed that differential genes are associated with important biological functions, including regulation of growth and development, regulation of metabolic processes and protein catabolic processes. CONCLUSION: The results demonstrate that m6A methylation, differential expression and the related signalling pathways are crucial for boar testicular development. These results suggest a role for m6A modification in boar testicular development and provided a resource for future studies on m6A function in boar testicular development.

Copy number variants selected during pig domestication inferred from whole genome resequencing.

Over extended periods of natural and artificial selection, China has developed numerous exceptional pig breeds. Deciphering the germplasm characteristics of these breeds is crucial for their preservation and utilization. While many studies have employed single nucleotide polymorphism (SNP) analysis to investigate the local pig germplasm characteristics, copy number variation (CNV), another significant type of genetic variation, has been less explored in understanding pig resources. In this study, we examined the CNVs of 18 Wanbei pigs (WBP) using whole genome resequencing data with an average depth of 12.61. We identified a total of 8,783 CNVs (~30.07 Mb, 1.20% of the pig genome) in WBP, including 8,427 deletions and 356 duplications. Utilizing fixation index (Fst), we determined that 164 CNVs were within the top 1% of the Fst value and defined as under selection. Functional enrichment analyses of the genes associated with these selected CNVs revealed genes linked to reproduction (SPATA6, CFAP43, CFTR, BPTF), growth and development (NR6A1, SMYD3, VIPR2), and immunity (PARD3, FYB2). This study enhances our understanding of the genomic characteristics of the Wanbei pig and offers a theoretical foundation for the future breeding of this breed.

Integrated 4D Analysis of Intramuscular Fat Deposition: Quantitative Proteomic and Transcriptomic Studies in Wannanhua Pig Longissimus Dorsi Muscle.

Wannanhua (WH) is a pig breed indigenous to Anhui Province, China. This breed has a high intramuscular fat (IMF) content, making it an ideal model for investigating lipid deposition mechanisms in pigs. IMF content is one of the main indicators of meat quality in pigs and is regulated by multiple genes and metabolic pathways. Building upon our prior transcriptomic investigation, the present study focused on the longissimus dorsi muscle tissue of Wannanhua (WH) pigs in the rapid fat-deposition stages (120 and 240 days of age). Employing 4D label-free quantitative proteomic analysis, we identified 106 differentially expressed proteins (DEPs). Parallel reaction monitoring (PRM) technology was used to verify the DEPs, and the results showed that the 4D label-free results were reliable and valid. Functional enrichment and protein-protein interaction analyses showed that the DEPs were mainly involved in the skeletal-muscle-associated structural proteins, mitochondria, energy metabolism, and fatty acid metabolism. By integrating transcriptomic data, we identified seven candidate genes including ACADL, ACADM, ANKRD2, MYOZ2, TNNI1, UCHL1, and ART3 that play a regulatory role in fat deposition and muscle development. These findings establish a theoretical foundation for future analyses of lipid deposition traits, contributing to potential enhancements in pig meat quality during breeding and advancing the selection process for Chinese indigenous breeds.

AGIDB: a versatile database for genotype imputation and variant decoding across species.

The high cost of large-scale, high-coverage whole-genome sequencing has limited its application in genomics and genetics research. The common approach has been to impute whole-genome sequence variants obtained from a few individuals for a larger population of interest individually genotyped using SNP chip. An alternative involves low-coverage whole-genome sequencing (lcWGS) of all individuals in the larger population, followed by imputation to sequence resolution. To overcome limitations of processing lcWGS data and meeting specific genotype imputation requirements, we developed AGIDB (https://agidb.pro), a website comprising tools and database with an unprecedented sample size and comprehensive variant decoding for animals. AGIDB integrates whole-genome sequencing and chip data from 17 360 and 174 945 individuals, respectively, across 89 species to identify over one billion variants, totaling a massive 688.57 TB of processed data. AGIDB focuses on integrating multiple genotype imputation scenarios. It also provides user-friendly searching and data analysis modules that enable comprehensive annotation of genetic variants for specific populations. To meet a wide range of research requirements, AGIDB offers downloadable reference panels for each species in addition to its extensive dataset, variant decoding and utility tools. We hope that AGIDB will become a key foundational resource in genetics and breeding, providing robust support to researchers.

Rapid production of chimeric silkworm/spider silk with improved mechanical properties by infection of nonpermissive Bombyx mori with recombinant AcMNPV harboring native-size of spidroin genes.

Spider silks with excellent mechanical properties attract more attention from scientists worldwide, and the dragline silk that serves as the framework of the spider's web is considered one of the strongest fibers. However, it is unfeasible for large-scale production of spider silk due to its highly territorial, cannibalistic, predatory, and solitary behavior. Herein, to alleviate some of these problems and explore aneasy way to produce spider fibers, we constructed recombinant baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) simultaneously expressing Trichonephila clavipes native ampullate spidroin 2 (MaSp-G) and spidroin 1 (MaSp-C) driven by the promoters of silkworm fibroin genes, to infect the nonpermissive Bombyx mori larvae at the fifth instar. MaSp-G and MaSp-C were co-expressed in the posterior silk glands (PSGs) of infected silkworms and successfully secreted into the lumen of the silk gland for fibroin globule assembly. The integration of MaSp-G and MaSp-C into silkworm silk fibers significantly improved the mechanical properties of these chimeric silk fibers, especially the strength and extensibility, which may be caused by the increment of ß-sheet in the chimeric silkworm/spider silk fiber. These results demonstrated that silkworms could be developed as the nonpermissive heterologous host for the mass production of chimeric silkworm/spider silk fibers via the recombinant baculovirus AcMNPV.

Screening of Selenium/Glutathione-Enriched Candida utilis and Its Anti-inflammatory and Antioxidant Activities in Mice.

This study aimed to screen a mutant of Candida utilis SE-172 with high selenite tolerance and glutathione (GSH) biosynthesis capability via 60Co γ-radiation mutagenesis to prepare selenium (Se)-enriched yeast. The maximal intracellular contents of GSH and organic Se of 22.94 mg/g and 1308.1 µg/g were obtained, respectively, under a batch culture of SE-172. The physiological mechanism underlying increased GSH and organic Se contents in Se/GSH-enriched C. utilis SE-172 was revealed based on assaying activities of γ-glutamylcysteine synthase (γ-GCS) involved in GSH biosynthesis and selenophosphate synthase (SPS) related to organic Se bioconversion, and by determining intracellular ATP and NADH contents and ATP/ADP and NADH/NAD+ ratios associated with energy supply and regeneration. Moreover, the effect of this selenized yeast on anti-inflammatory and antioxidant activities in mice with colitis was investigated. The supplementation of Se/GSH-enriched yeast decreased the dextran sodium sulfate-induced damage to colon tissues, reduced the expression of pro-inflammatory factors [interleukin (IL)-6, IL-1ß, and tumor necrosis factor-α (TNF-α)] in serum, increased the antioxidant-related enzyme [superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px)] activities, and decreased the malondialdehyde content in colon. The Se/GSH-enriched C. utilis SE-172 showed potent anti-inflammatory and antioxidant activities in mice with colitis.

Genome-wide detection of CNV regions between Anqing six-end-white and Duroc pigs.

BACKGROUND: Anqing six-end-white pig is a native breed in Anhui Province. The pigs have the disadvantages of a slow growth rate, low proportion of lean meat, and thick back fat, but feature the advantages of strong stress resistance and excellent meat quality. Duroc pig is an introduced pig breed with a fast growth rate and high proportion of lean meat. With the latter breed featuring superior growth characteristics but inferior meat quality traits, the underlying molecular mechanism that causes these phenotypic differences between Chinese and foreign pigs is still unclear. RESULTS: In this study, copy number variation (CNV) detection was performed using the re-sequencing data of Anqing Six-end-white pigs and Duroc pigs, A total of 65,701 CNVs were obtained. After merging the CNVs with overlapping genomic positions, 881 CNV regions (CNVRs) were obtained. Based on the obtained CNVR information combined with their positions on the 18 chromosomes, a whole-genome map of the pig CNVs was drawn. GO analysis of the genes in the CNVRs showed that they were primarily involved in the cellular processes of proliferation, differentiation, and adhesion, and primarily involved in the biological processes of fat metabolism, reproductive traits, and immune processes. CONCLUSION: The difference analysis of the CNVs between the Chinese and foreign pig breeds showed that the CNV of the Anqing six-end-white pig genome was higher than that of the introduced pig breed Duroc. Six genes related to fat metabolism, reproductive performance, and stress resistance were found in genome-wide CNVRs (DPF3, LEPR, MAP2K6, PPARA, TRAF6, NLRP4).

Genome-scale CRISPR screen identifies TRIM2 and SLC35A1 associated with porcine epidemic diarrhoea virus infection.

Porcine epidemic diarrhoea (PED) caused by the porcine epidemic diarrhoea virus (PEDV) is the most devastating disease in the global pig industry due to its high mortality rate in piglets. The host factors critical for PEDV replication are poorly understood. Here, we designed a pooled African green monkey genome-scale CRISPR/Cas9 knockout (VeroCKO) library containing 75,608 single guide RNAs targeting 18,993 protein-coding genes. Subsequently, we use the VeroCKO library to identify key host factors facilitating PEDV infection in Vero E6 cells. Several previously unreported genes associated with PEDV infection are highly enriched post-PEDV selection. We discovered that knocking out the tripartite motif 2 (TRIM2) and the solute carrier family 35 member A1 (SLC35A1) inhibited PEDV replication. Virtual screening and molecular docking approaches showed that chem-80,048,685 (M2) s ignificantly inhibited PEDV attachment and late replication by impeding SLC35A1. Furthermore, we found that knocking out SLC35A1 in Vero E6 cells upregulated a disintegrin and metalloprotease protein-17 (ADAM17) by splicing porcine aminopeptidase N (pAPN) and angiotensin-converting enzyme 2 (ACE2) ectodomains to reduce PEDV-infection in a CMP-Sialic Acid (CMP-SA) cell entry-independent manner. These findings provide a new perspective for a better understanding of host-pathogen interactions and new therapeutic targets for PEDV infection.

Dietary administration with hydrolyzed silk sericin improves the intestinal health of diabetic rats.

Type II diabetes (T2D) is a global epidemic disease with an increased incidence and prevalence. Gut microbiota plays an important role in controlling T2D development. Dietary administration of prebiotics, probiotics, and drugs, including metformin, showed the regulatory impact on the change of gut microbiota, which is associated with the improvement of glucose tolerance. In this study, silk sericin was manufactured into hydrolyzed sericin peptide (HSP) powders as a dietary additive to investigate the effect on the gut microbiota of T2D model rats. The results indicated that the HSP-augmented dietary administration lowers the fast glucose level of diabetic rats, and HSP augmentation induces a change in the gut microbiota composition of T2D model rats toward the normal rats. Some key taxa, including Lactobacillus gasseri, were suggested to be involved in controlling T2D development. This finding provides new insight into developing sericin as functional food or therapeutic prebiotics against T2D in clinical practice.

Genome-Wide Detection and Analysis of Copy Number Variation in Anhui Indigenous and Western Commercial Pig Breeds Using Porcine 80K SNP BeadChip.

Copy number variation (CNV) is an important class of genetic variations widely associated with the porcine genome, but little is known about the characteristics of CNVs in foreign and indigenous pig breeds. We performed a genome-wide comparison of CNVs between Anhui indigenous pig (AHIP) and Western commercial pig (WECP) breeds based on data from the Porcine 80K SNP BeadChip. After analysis using the PennCNV software, we detected 3863 and 7546 CNVs in the AHIP and WECP populations, respectively. We obtained 225 (loss: 178, gain: 47) and 379 (loss: 293, gain: 86) copy number variation regions (CNVRs) randomly distributed across the autosomes of the AHIP and WECP populations, accounting for 10.90% and 22.57% of the porcine autosomal genome, respectively. Functional enrichment analysis of genes in the CNVRs identified genes related to immunity (FOXJ1, FOXK2, MBL2, TNFRSF4, SIRT1, NCF1) and meat quality (DGAT1, NT5E) in the WECP population; these genes were a loss event in the WECP population. This study provides important information on CNV differences between foreign and indigenous pig breeds, making it possible to provide a reference for future improvement of these breeds and their production performance.

Pangenome obtained by long-read sequencing of 11 genomes reveal hidden functional structural variants in pigs.

Long-read sequencing (LRS) facilitates both the genome assembly and the discovery of structural variants (SVs). Here, we built a graph-based pig pangenome by incorporating 11 LRS genomes with an average of 94.01% BUSCO completeness score, revealing 206-Mb novel sequences. We discovered 183,352 nonredundant SVs (63% novel), representing 12.12% of the reference genome. By genotyping SVs in an additional 196 short-read sequencing samples, we identified thousands of population stratified SVs. Particularly, we detected 7,568 Tibetan specific SVs, some of which demonstrate significant population differentiation between Tibetan and low-altitude pigs, which might be associated with the high-altitude hypoxia adaptation in Tibetan pigs. Further integrating functional genomic data, the most promising candidate genes within the SVs that might contribute to the high-altitude hypoxia adaptation were discovered. Overall, our study generates a benchmark pangenome resource for illustrating the important roles of SVs in adaptive evolution, domestication, and genetic improvement of agronomic traits in pigs.

Whole-crop biorefinery of corn biomass for pullulan production by Aureobasidium pullulans.

In the present study, corn starch, cob, and straw were biorefined and used as feedstocks for the production of pullulan. The titer and molecular weight (Mw) of pullulan significantly decreased when corn cob and straw hydrolysates were utilized by the parental strain Aureobasidium pullulans CCTCC M 2012259 (PS). Based on adaptive laboratory evolution of PS, an evolved strain A. pullulans EV6 with strong adaptability to the whole corn biomass hydrolysate and high capability of pullulan biosynthesis was screened. Batch pullulan fermentation results indicated that EV6 produced an increased titer of pullulan with a higher Mw than PS. The underlying reasons for these increases were revealed by assaying key enzymes activities and measuring intracellular uridine diphosphate glucose levels. Subsequently, whole-crop biorefinery of corn biomass was conducted, and the results confirmed that whole corn crop has immense potential for efficient pullulan production.

Circ-CREBBP inhibits sperm apoptosis via the PI3K-Akt signaling pathway by sponging miR-10384 and miR-143-3p.

Male reproductive diseases are becoming increasingly prominent, and sperm quality is an important indicator to reflect these diseases. Seminal plasma extracellular vesicles (SPEVs) are involved in sperm motility. However, their effects on sperm remain unclear. Here, we identified 222 differentially expressed circRNAs in SPEVs between boars with high or low sperm motility. We found that circ-CREBBP promoted sperm motility and inhibited sperm apoptosis by sponging miR-10384 and miR-143-3p. In addition, miR-10384 and miR-143-3p can regulate the expression of MCL1, CREB1 and CREBBP. Furthermore, we demonstrated that MCL1 interacted directly with BAX and that CREBBP interacted with CREB1 in sperm. We showed that inhibition of circ-CREBBP can reduce the expression of MCL1, CREB1 and CREBBP and increase the expression of BAX and CASP3, thus promoting sperm apoptosis. Our results suggest that circ-CREBBP may be a promising biomarker and therapeutic target for male reproductive diseases.

Genetic architecture and selection of Anhui autochthonous pig population revealed by whole genome resequencing.

The genetic resources among pigs in Anhui Province are diverse, but their value and potential have yet to be discovered. To illustrate the genetic diversity and population structure of the Anhui pigs population, we resequenced the genome of 150 pigs from six representative Anhui pigs populations and analyzed this data together with the sequencing data from 40 Asian wild boars and commercial pigs. Our results showed that Anhui pigs were divided into two distinct types based on ancestral descent: Wannan Spotted pig (WSP) and Wannan Black pig (WBP) origins from the same ancestor and the other four populations origins from another ancestor. We also identified several potential selective sweep regions associated with domestication characteristics among Anhui pigs, including reproduction-associated genes (CABS1, INSL6, MAP3K12, IGF1R, INSR, LIMK2, PATZ1, MAPK1), lipid- and meat-related genes (SNX19, MSTN, MC5R, PRKG1, CREBBP, ADCY9), and ear size genes (MSRB3 and SOX5). Therefore, these findings expand the catalogue and how these genetic differences among pigs and this newly generated data will be a valuable resource for future genetic studies and for improving genome-assisted breeding of pigs and other domesticated animals.

Population Structure and Selection Signatures Underlying Domestication Inferred from Genome-Wide Copy Number Variations in Chinese Indigenous Pigs.

Single nucleotide polymorphism was widely used to perform genetic and evolution research in pigs. However, little is known about the effect of copy number variation (CNV) on characteristics in pigs. This study performed a genome-wide comparison of CNVs between Wannan black pigs (WBP) and Asian wild boars (AWB), using whole genome resequencing data. By using Manta, we detected in total 28,720 CNVs that covered approximately 1.98% of the pig genome length. We identified 288 selected CNVs (top 1%) by performing Fst statistics. Functional enrichment analyses for genes located in selected CNVs were found to be muscle related (NDN, TMOD4, SFRP1, and SMYD3), reproduction related (GJA1, CYP26B1, WNT5A, SRD5A2, PTPN11, SPEF2, and CCNB1), residual feed intake (RFI) related (MAP3K5), and ear size related (WIF1). This study provides essential information on selected CNVs in Wannan black pigs for further research on the genetic basis of the complex phenotypic and provides essential information for direction in the protection and utilization of Wannan black pig.

MAL31, a sugar transporter involved in pullulan biosynthesis in Aureobasidium pullulans.

To investigate the role of the sugar transporter MAL31 on pullulan biosynthesis, the coding gene mal31 was respectively disrupted and overexpressed in the parental strain A. pullulans CCTCC M 2012259 to construct mutants of A. pullulans Δmal31 and A. pullulans Mal31. Batch pullulan production significantly decreased by 69.1 % in A. pullulans Δmal31 but increased by 15.9 % in A. pullulans Mal31, as compared to the parental strain. We performed kinetics analysis, assays of key enzymes, determination of intracellular UDPG, NADH, and ATP contents, and measurement of transcriptional levels of genes associated with pullulan biosynthesis and excretion. The results confirmed that the mal31 disruption decreased the glucose consumption rate, decreased the formation rate and titer of pullulan, but increased the intracellular UDPG supply for ß-glucan accumulation. In contrast, the mal31 overexpression increased the transcriptional levels of genes associated with pullulan biosynthesis, and accelerated the rates of glucose consumption and pullulan formation, thereby increased pullulan production. Our findings revealed that MAL31 is involved in the transport of precursors for pullulan biosynthesis. This study provides an accurate operating site for genetic modification of A. pullulans for improving pullulan production and also presents a feasible technique route for the overproduction of other polysaccharides.

miR-381-3p Inhibits Intramuscular Fat Deposition through Targeting FABP3 by ceRNA Regulatory Network.

Intramuscular fat (IMF) deposition is an important determinant of pork quality and a complex process facilitated by non-coding ceRNAs. In this study, 52 Berkshire × Anqing Sixwhite crossbred pigs were slaughtered to measure eight carcass and pork quality traits. Whole-transcriptome sequencing analysis was performed using longissimus dorsi samples of six low- and high-IMF samples; 34 ceRNA networks, based on 881, 394, 158 differentially expressed (DE) lncRNAs, miRNAs, and mRNAs, were constructed. Following weighted gene co-expression network analysis between the low and high IMF, only one ceRNA, lncRNA4789/miR-381-3p/FABP3, that showed similar DE trend in longissimus dorsi tissue was retained. Dual-luciferase reporter assays further indicated that FABP3 was a direct, functional target of miR-381-3p, where miR-381-3p overexpression inhibited the mRNA and protein expression of FABP3. In addition, overexpressed lncRNA4789 attenuated the effect of miR-381-3p on FABP3 by sponging miR-381-3p. Cell function verification experiment demonstrated that miR-381-3p suppressed IMF deposition by inhibiting preadipocyte cell differentiation and lipid droplet deposition via the suppression of FABP3 expression in the peroxisome proliferator-activated receptor signalling pathway, whereas lncRNA4789 rescued FABP3 expression by sponging miR-381-3p. Our study may aid in identifying novel molecular markers for its optimization in IMF which is of importance in breeding for improving pork quality.

CCR4-NOT Complex 2-A Cofactor in Host Cell for Porcine Epidemic Diarrhea Virus Infection.

The porcine epidemic diarrhea virus (PEDV) has catastrophic impacts on the global pig industry. However, there is no consensus on the primary receptor associated with the PEDV invasion of host cells. An increasing number of studies have reported that PEDV invading host cells may require collaboration between multiple receptors and to better understand the virus-host interaction during PEDV entry, surface plasmon resonance (SPR) assays are performed to investigate relevant host factors interacting with PEDV spike-1 protein (S1) in Vero and IPEC-J2 cell membranes. Subsequently, the rabbit anti-PEDV S1 polyclonal antibody is used as bait to recognize the complexes of IPEC-J2 membrane proteins with or without PEDV infection, followed by detection using liquid chromatography with tandem mass spectrometry (LC-MS-MS). Our results show that 13 and 10 proteins interacting between the S1 protein and plasma membrane protein of Vero or IPEC-J2 can be identified. More specifically, a total of 11 differentially expressed interacting proteins were identified in IPEC-J2 membrane proteins after PEDV infection, compared to the uninfected group. Furthermore, we found that the differentially interacting protein CCR4-NOT complex 2 (CNOT2), identified in PEDV S1 with plasma membrane proteins of Vero cells, is involved in viral infection. The results show that the knockout of CNOT2 significantly inhibits PEDV replication in vitro. These data provide novel insights into the entry mechanism of PEDV.

Reassessing acetyl-CoA supply and NADPH availability for mevalonate biosynthesis from glycerol in Escherichia coli.

Mevalonate is an important platform compound for the biosynthesis of isoprenoids. It can be synthesized from acetyl-CoA in the presence of nicotinamide adenine dinucleotide phosphate (NADPH) by the introduced mvaES operon in Escherichia coli. The influences of E. coli hosts, acetyl-CoA supply, and NADPH availability were assessed and engineered to improve the production titer and yield of mevalonate from glycerol. As a result, E. coli DH5α was found to be the best host with high specific capability and titer of mevalonate from glycerol. Through the engineering of phosphoketolase-phosphotransacetylase (xPK-PTA) bypass and NADPH availability, a final titer of 7.21 g/L with a specific capability of 1.36 g/g dry cell weight was gained in flask culture. Our work could offer new information to metabolically engineer the mevalonate pathway for the efficient production of isoprenoids.

Viral Circular RNAs and Their Possible Roles in Virus-Host Interaction.

Circular RNAs (circRNAs) as novel regulatory molecules have been recognized in diverse species, including viruses. The virus-derived circRNAs play various roles in the host biological process and the life cycle of the viruses. This review summarized the circRNAs from the DNA and RNA viruses and discussed the biogenesis of viral and host circRNAs, the potential roles of viral circRNAs, and their future perspective. This review will elaborate on new insights gained on viruses encoded circRNAs during virus infection.