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Intramuscular fat (IMF) content is an important factor in porcine meat quality. Previous studies have screened multiple candidate genes related to IMF deposition, but the lipids that affect IMF deposition and their lipid-protein network remain unknown. In this study, we performed proteomic and lipidomic analyses of the longissimus dorsi (LD) muscle from high-IMF (IMFH) and low-IMF (IMF-L) groups of Xidu black pigs. Eighty-eight proteins and 143 lipids were differentially abundant between the groups. The differentially abundant proteins were found to be involved in cholesterol metabolism, the PPAR signaling pathway, and ferroptosis. The triacylglycerols (TAGs) upregulated in the IMF-H group were mainly shown to be synthesized by saturated fatty acids (SFAs), while the downregulated TAGs were mainly synthesized by polyunsaturated fatty acids (PUFAs). All differentially abundant phosphatidylinositols (PIs) and phosphatidylserines (PSs) were found to be upregulated in the IMF-H group. A correlation analysis of the proteomic and lipidomic revealed candidate proteins (APOA4, VDAC3, PRNP, CTSB, GSPT1) related to TAG, PI, and PS lipids. These results revealed differences in proteins and lipids between the IMF-H and IMF-L groups, which represent new candidate proteins and lipids that should be investigated to determine the molecular mechanisms controlling IMF deposition in pigs. SIGNIFICANCE: Intramuscular fat (IMF) is a key factor affecting meat quality, and meat with a higher IMF content can have a better flavor. In this study, proteomic results show that the ferroptosis pathway, including the PRNP, VDAC3 and CP proteins, affects IMF deposition. Lipid composition is the key factor affecting IMF deposition, but there are few reports on this. In this study, through lipidomic analysis, we suggest that saturated fatty acid (SFA), phosphatidylinositol (PI), and phosphatidylserine (PS) may contribute to IMF deposition. A correlation analysis reveals the potential regulatory network between lipids and proteins. This study clarifies the difference in protein and lipid compositions in longissimus dorsi (LD) muscle with high and low IMF contents. This information suggests that it would be beneficial to increase the intramuscular fat content of pork not only from a genetic perspective but also from a nutritional perspective.

Fifty-four lactating multiparous Yorkshire sows were used to test the hypothesis that feeding a reduced protein diet with a near ideal AA (NIAA) profile increases the biological utilization efficiency of N and essential AA (EAA) for milk production in part as a result of reduced dietary Leu concentration. Sows were fed 1 of 3 isocaloric diets containing the following concentration of CP (% as-fed, analyzed): 18.74 (Control: CON), 13.78 (Optimal: OPT), and 14.25 (Optimal + Leu: OPTLEU). The OPT and OPTLEU diets contained the same concentration of crystalline AA (CAA) to meet requirements of the limiting AA. Crystalline Leu was added to OPTLEU to contain the same SID Leu concentration as that of CON. Sows were weighed on days 1 and 21 of lactation and piglets on days 1, 4, 8, 14, 18, and 21 of lactation. Nitrogen retention was measured for 48 or 72 h between days 4 and 8 (early) and days 14 and 18 (peak) of lactation. Sow BW change and ADFI did not differ between diets. Litter growth rate (LGR) during early lactation did not differ between diets. At peak lactation, LGR was higher in sows fed OPT compared with CON (P < 0.05) and lower in sows fed OPTLEU compared with OPT (P < 0.05). In early and peak lactation, total N retention, and milk N output efficiency were greater in OPT (P < 0.01) and OPTLEU (P < 0.05) than CON. Compared with CON, overall biological efficiency of N, Arg, His, Ile, Leu, Phe, and Trp were greater (P < 0.05), whereas those of Lys, Met, Thr, and Val did not differ in sows fed OPT and OPTLEU, except for Leu which did not differ between OPTLEU and CON. Compared with OPT, only Leu and Met efficiency were lower (P < 0.01) and tended to be lower (P = 0.10), respectively, in sows fed OPTLEU. Reducing CP with a NIAA profile to attain the minimum Leu requirement maintained overall lactation performance, improved utilization efficiency of N, Arg, His, Ile, Leu, Phe + Tyr, and Trp for milk production, and maximized efficiency of Ile, Leu, Lys, Met + Cys, Phe + Tyr, Thr, Trp, and Val. Addition of Leu did not reduce N and EAA utilization efficiency. This study provides revised and novel maximum biological efficiency value (MBEV) for Ile (65.4), Leu (75.1), Lys (63.2), Met + Cys (78.2), Phe + Tyr (69.5), Thr (71.0), Trp (70.1), and Val (57.0). These MBEV can be used to more accurately predict the requirement for those AA during lactation.

Porcine reproductive and respiratory syndrome (PRRS), which is caused by PRRS virus (PRRSV), is one of the most globally devastating swine diseases. It is essential to develop new strategy to control PRRS via an understanding of mechanisms that PRRSV utilizes to interfere with the host's innate immunity. In this study, we deeply sequenced and analyzed long noncoding RNA (lncRNA) and mRNA expression profiles of the porcine alveolar macrophages (PAMs) after PRRSV infection. 126 lncRNAs and 753 mRNAs were differentially expressed between PRRSV-infected and control PAMs. The co-expressed genes of down-regulated lncRNAs were significantly enriched within NF-kappa B and toll-like receptor signaling pathways. Co-expression network analysis indicated that part of the dysregulated lncRNAs associated with the interferon-induced genes. These dysregulated lncRNAs may play an important role in the host's innate immune responses to PRRSV infection. However, further research is required to characterize the function of these lncRNAs.

In our previous work, gene PPP1R11 (protein phosphatase 1 regulatory subunit 11) was significantly expressed in pigs after Streptococcus suis 2 (SS2) challenged. This study firstly confirmed that SS2 induced significant expression of PPP1R11 gene in porcine alveolar macrophage (PAM) cells, and apoptosis of PAM cells were observed. After that, the core promoter of porcine PPP1R11 was identified and its transcription factor AREB6 which significantly regulated PPP1R11. We also characterized that the PPP1R11 gene is a target of miR-34a. Further, we found that PPP1R11 helped to inhibit apoptosis of PAM cells under SS2 infecting, through transcription factor AREB6 was negatively correlated with apoptosis whereas miR-34a was positively correlated. Those findings provide a functional connection among the transcription factor AREB6, miR-34a, PPP1R11 gene and apoptosis of PAM cells in the pathogenesis of the SS2 infection.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes severe reproductive failure in sows, respiratory diseases, and high mortality in piglets, which results in serious economic losses to the swine industry worldwide. Previous studies have described that PRRSV could suppress the host immune system and had antiapoptotic activity in its initial phase of infection. Polyinosinic-polycytidylic acid (poly I:C), a synthesized analogue of viral double-strand RNA, activates innate immunity responses and induces apoptosis in cells. Therefore, we performed miRNA transcriptome analysis of poly I:C-stimulated and PRRSV-infected porcine alveolar macrophages (PAMs) using deep sequencing technology, to compare the different miRNA profiles between the statuses of innate immune activation and inactivation. After sequencing, 267 known mature miRNAs and 64 novel miRNAs were observed in PAMs, and a total of 197 miRNAs were significantly differently expressed in poly I:C-stimulated PAMs, compared with mock control cells. Thirty-three of them were also significantly alerted in PRRSV-infected PAMs. This indicated that PRRSV only slightly alerted the miRNA expression profile of host cells compared with poly I:C-stimulated PAMs, which confirmed that PRRSV could suppress host innate immune responses during the early stages of infection. Among the differentially expressed miRNAs, we found that ssc-miR-27b-3p could significantly inhibit PRRSV RNA and protein replication in MARC-145 cells and PAMs. Its antiviral mechanism needs further research in the future.

TCAP (also known as titin-cap or telethonin) is one of the titin interacting Z-disk proteins involved in the regulation and development of normal sarcomeric structure. In this study, we cloned the cDNA and promoter sequences of porcine TCAP gene, which contained a 504 bp full-length coding region. Quantitative real-time PCR (qRT-PCR) analyses showed that porcine TCAP was highly expressed in the skeletal muscle, heart, and kidney. During postnatal muscle development, TCAP expression was down-regulated from 30 days to 120 days in Large White and Meishan pigs. One single nucleotide polymorphism c.334 G>A in exon 2 of the TCAP gene was identified and detected by allele-specific primer-polymerase chain reaction (ASP-PCR). Association analysis revealed that the polymorphism had significant associations (P

The retina communicates with the brain using ≥30 parallel channels, each carried by axons of distinct types of retinal ganglion cells. In every mammalian retina one finds so-called "alpha" ganglion cells (αRGCs), identified by their large cell bodies, stout axons, wide and mono-stratified dendritic fields, and high levels of neurofilament protein. In the mouse, three αRGC types have been described based on responses to light steps: On-sustained, Off-sustained, and Off-transient. Here we employed a transgenic mouse line that labels αRGCs in the live retina, allowing systematic targeted recordings. We characterize the three known types and identify a fourth, with On-transient responses. All four αRGC types share basic aspects of visual signaling, including a large receptive field center, a weak antagonistic surround, and absence of any direction selectivity. They also share a distinctive waveform of the action potential, faster than that of other RGC types. Morphologically, they differ in the level of dendritic stratification within the IPL, which accounts for their response properties. Molecularly, each type has a distinct signature. A comparison across mammals suggests a common theme, in which four large-bodied ganglion cell types split the visual signal into four channels arranged symmetrically with respect to polarity and kinetics.

Imprinted genes play an essential role in the regulation of fetal growth, development and function of the placenta, however only a limited number of imprinted genes have been studied in swine. In this study, we cloned and characterized porcine MAGEL2 (melanoma antigen-like gene 2), and also identified its imprinting status during porcine fetal development. The complete open reading frame (ORF) encoding 1,193 amino acids was isolated and two single nucleotide polymorphisms (SNPs) (g.2592A>C and g.3277T>C) in the coding region were identified. The reciprocal Yorkshire × Meishan F1 hybrid model and the RT-PCR/RFLP method were used to detect the imprinting status of porcine MAGEL2 gene at two developmental stages of day 30 and 65 of gestation. Imprinting analysis showed that porcine MAGEL2 was paternally expressed in day 65 fetal tissues, including heart, liver, spleen, lung, kidney, stomach, small intestine, skeletal muscle, brain and placenta. Interestingly, we observed an imprinting variance of MAGEL2 gene in 30 dpc fetuses produced by the cross of Yorkshire boar × Meishan sow, in which seven heterozygous fetuses were monoallelically expressed from the paternal allele but two were biallelically expressed from both the paternal and maternal alleles. Association analysis in a Yorkshire × Meishan F2 resource population showed that the mutation of g.2592A>C was significantly associated with dressed carcass percentage (P < 0.05) and buttock fat thickness (P < 0.05). Our results suggest that MAGEL2, as a novel imprinted gene in pig, might be a candidate gene affecting carcass traits and could provide important information for the functional study of imprinted genes during porcine development.

An efficient vector, designated as pCAGX, was designed for direct cloning and enhanced expression of PCR-amplified ORFs in mammalian cells. It relied on the well-known TA-cloning principle, and utilized the CMV enhancer/chicken beta-actin/rabbit beta-globin (CAG) hybrid promoter instead of the classical CMV promoter to drive more efficient transgene expression in wider host cells. The specially designed cassette under CAG hybrid promoter contained two tandemly arrayed XcmI sites which were spaced by an additional EcoRV site. For direct cloning and expressing PCR-amplified ORFs, the T-vector was prepared by further digesting the EcoRV-linearized pCAGX with XcmI to produce T tails on both 3'-ends, which could efficiently minimize the non-recombinant background of T-vector and eliminate the necessity of selective marker genes such as LacZ that allowed blue/white screening. Various PCR fragments in length were prepared to verify the cloning efficiency by ligation with this vector, and GFP gene expression under control of the CAG hybrid promoter in different host cells was assayed by flow cytometry. The results indicated that this vector was higher efficient, especially suitable for cloning and expressing a number of interesting ORFs in parallel, and higher-level transgene expression in different mammalian cells was obtained than the reported vectors using the CMV promoter.

Klebsiella pneumoniae is an urgent public health threat because of resistance to carbapenems, antibiotics of last resort against Gram-negative bacterial infections. Despite the fact that K. pneumoniae is a leading cause of pneumonia in hospitalized patients, the bacterial factors required to cause disease are poorly understood. Insertion site sequencing combines transposon mutagenesis with high-throughput sequencing to simultaneously screen thousands of insertion mutants for fitness defects during infection. Using the recently sequenced K. pneumoniae strain KPPR1 in a well-established mouse model of pneumonia, insertion site sequencing was performed on a pool of >25,000 transposon mutants. The relative fitness requirement of each gene was ranked based on the ratio of lung to inoculum read counts and concordance between insertions in the same gene. This analysis revealed over 300 mutants with at least a 2-fold fitness defect and 69 with defects ranging from 10- to >2,000-fold. Construction of 6 isogenic mutants for use in competitive infections with the wild type confirmed their requirement for lung fitness. Critical fitness genes included those for the synthesis of branched-chain and aromatic amino acids that are essential in mice and humans, the transcriptional elongation factor RfaH, and the copper efflux pump CopA. The majority of fitness genes were conserved among reference strains representative of diverse pathotypes. These results indicate that regulation of outer membrane components and synthesis of amino acids that are essential to its host are critical for K. pneumoniae fitness in the lung., IMPORTANCE Klebsiella pneumoniae is a bacterium that commonly causes pneumonia in patients after they are admitted to the hospital. K. pneumoniae is becoming resistant to all available antibiotics, and when these infections spread to the bloodstream, over half of patients die. Since currently available antibiotics are failing, we must discover new ways to treat these infections. In this study, we asked what genes the bacterium needs to cause an infection, since the proteins encoded by these genes could be targets for new antibiotics. We identified over 300 genes that K. pneumoniae requires to grow in a mouse model of pneumonia. Many of the genes that we identified are found in K. pneumoniae isolates from throughout the world, including antibiotic-resistant forms. If new antibiotics could be made against the proteins that these genes encode, they may be broadly effective against K. pneumoniae.

The upstream stimulatory factor 1 (USF1) is a transcription factor controlling expression of several genes involved in lipid and glucose homeostasis. In this study, two isoforms of the porcine USF1 gene were detected by reverse transcription polymerase chain reaction (RT-PCR), termed USF1 wild-type (wt) and USF1/CD, both of them contain a helix-loop-helix leucine zipper (HLH-LZ) conserved domain. Tissue distribution analysis showed that the two transcripts of porcine USF1 gene were ubiquitously expressed in all tested tissues, except for heart. Moreover, we found that a single nucleotide polymorphism (SNP, C/T) in intron 10 was significantly associated with ratio of lean to fat (P < 0.05), dress percentage (P < 0.05), average backfat thickness (P < 0.05), loin eye width (P < 0.05), lean meat percentage (P < 0.01), loin eye height (P < 0.01), and loin eye area (P < 0.01). This result suggests that porcine USF1 gene may be a candidate gene of meat production trait.

Galanin-like peptide (GALP) gene, encoding a member of the galanin family of neuropeptides involved in reproduction, was differentially expressed in PMSG-hCG stimulated pre-ovulatory ovarian follicles of Chinese Taihu and Large White sows in our previous study. In the present study, promoter region and genetic mutations of the porcine GALP gene were determined. A 1,322 bp contig in 5'-flanking region was predicted to contain 5 potential transcription promoters by Neural Network Promoter Prediction version 2.2. 5'-deletion expression in both CHO and hela cells showed that there were a negative regulatory element at -852 to -803 bp and a positive regulatory element at -1,318 to -1,269 bp. Comparative sequence analyses of Chinese Taihu and Large White GALP gene sequence revealed the c.*27C>G mutation in the 3'-UTR and the c.88-1225C>G mutation in intron 1, which can be detected by HhaI and AluI PCR-RFLP, respectively. The association analysis with litter size traits showed that at both loci CC and GG genotypes were different for NBA for all parities in DIV pigs (P < 0.05). However, two SNPs were not in significant linkage disequilibrium analyzed using SHEsis online software, and could be used in pig breeding individually.

Imprinted genes play important roles in mammalian growth, development and behavior. In this study, we obtained 1568 bp mRNA sequence of porcine DIO3 (deiodinase, iodothyronine, type III), and also identified its imprinting status during porcine fetal development. The complete open reading frame (ORF) encoding 278 amino acids. The porcine DIO3 mRNA was expressed predominantly in backfat, mildly in liver, uterus, kidney, heart, small intestine, muscle and stomach, and almost absent in spleen and lung. A single nucleotide polymorphism in exon (A/C (687)) was used to investigate the allele frequencies in different pig breeds and the imprinting status in porcine embryonic tissues. The results indicate that DIO3 was imprinted in all the tested tissues. Statistical analysis showed the DIO3 gene polymorphism was significantly associated with almost all the fat deposition and carcass traits, including lean meat percentage (LMP), fat meat percentage (FMP), ratio of lean to fat (RLF), shoulder fat thickness (SFT), sixth-seventh rib fat thickness (RFT), buttock fat thickness (BFT), loin eye area (LEA), and intramuscular fat (IMF).

The lecithin cholesterol acyltransferase gene (LCAT) plays an important role in lipoprotein metabolism, especially in the process termed ‘reverse cholesterol transport’. In this study, we obtained the 1,434 bp mRNA sequence of porcine LCAT including the full coding region and encoding a protein of 472 amino acids. The sequence was deposited into the GenBank under the accession no. EU717835. The genomic sequence of this gene which contains six exons and five introns, is 3,712 bp in length (GQ379050). Bioinformatic analysis of the 5′ regulatory region has revealed that some transcription factor Sp1, AP-1, AP-2 and NF-kappaB were represented in this region. Tissue expression analysis showed that the porcine LCAT gene is ubiquitously expressed in all examined tissues. Phylogenetic tree was constructed by aligning the amino acid sequences of different species. Moreover, we found a single nucleotide polymorphism (SNP, C/G266) in intron 1 of the LCAT gene and association analysis showed that it was significantly associated with ratio of lean to fat (P

Receptors for the Fc regions of immunoglobin G (IgG) play a critical role in immunoregulation and immune defenses against pathogens. In this study, we describe the cloning, eukaryotic expression and IgG subclass specificity of ovine Fc gamma receptor III (FcγRIII). The newly cloned ovine FcγRIII cDNA contains a 940 bp open-reading frame (ORF), and is predicted to encode a 250 amino acid transmembrane glycoprotein composed of two immunoglobulin-like extracellular domains, a transmembrane region and a short cytoplasmic tail. The overall identity of the ovine FcγRIII amino acid sequence to its cattle, pig and human counterparts was 83.2%, 62.0%, 60.7%, respectively. Overlapping PCR was performed with the extracellular domain of ovine FcγRIII and the transmembrane and intracellular region of ovine Fc gamma chain to construct a chimeric receptor. Rosetting analysis showed that transfected COS-7 cells required Fc receptor gamma chain for the expression of FcγRIII on the surface. COS-7 cells expressing FcγRIII were able to bind chicken erythrocytes sensitized with ovine IgG1, but not IgG2. Identification of ovine FcγRIII will further our understanding of the ovine immune system.