Antibacterial activity of the antimicrobial peptide PMAP-36 in combination with tetracycline against porcine extraintestinal pathogenic Escherichia coli in vitro and in vivo.

The increase in the emergence of antimicrobial resistance has led to great challenges in controlling porcine extraintestinal pathogenic Escherichia coli (ExPEC) infections. Combinations of antimicrobial peptides (AMPs) and antibiotics can synergistically improve antimicrobial efficacy and reduce bacterial resistance. In this study, we investigated the antibacterial activity of porcine myeloid antimicrobial peptide 36 (PMAP-36) in combination with tetracycline against porcine ExPEC PCN033 both in vitro and in vivo. The minimum bactericidal concentrations (MBCs) of AMPs (PMAP-36 and PR-39) against the ExPEC strains PCN033 and RS218 were 10 µM and 5 µM, respectively. Results of the checkerboard assay and the time-kill assay showed that PMAP-36 and antibiotics (tetracycline and gentamicin) had synergistic bactericidal effects against PCN033. PMAP-36 and tetracycline in combination led to PCN033 cell wall shrinkage, as was shown by scanning electron microscopy. Furthermore, PMAP-36 delayed the emergence of PCN033 resistance to tetracycline by inhibiting the expression of the tetracycline resistance gene tetB. In a mouse model of systemic infection of PCN033, treatment with PMAP-36 combined with tetracycline significantly increased the survival rate, reduced the bacterial load and dampened the inflammatory response in mice. In addition, detection of immune cells in the peritoneal lavage fluid using flow cytometry revealed that the combination of PMAP-36 and tetracycline promoted the migration of monocytes/macrophages to the infection site. Our results suggest that AMPs in combination with antibiotics may provide more therapeutic options against multidrug-resistant porcine ExPEC.

AIEgens-enhanced rapid sensitive immunofluorescent assay for SARS-CoV-2 with digital microfluidics.

BACKGROUND: Sensitive and rapid antigen detection is critical for the diagnosis and treatment of infectious diseases, but conventional ELISAs including chemiluminescence-based assays are limited in sensitivity and require many operation steps. Fluorescence immunoassays are fast and convenient but often show limited sensitivity and dynamic range. RESULTS: To address the need, an aggregation-induced emission fluorgens (AIEgens) enhanced immunofluorescent assay with beads-based quantification on the digital microfluidic (DMF) platform was developed. Portable DMF devices and chips with small electrodes were fabricated, capable of manipulating droplets within 100 nL and boosting the reaction efficiency. AIEgen nanoparticles (NPs) with high fluorescence and photostability were synthesized to enhance the test sensitivity and detection range. The integration of AIEgen probes, transparent DMF chip design, and the large magnetic beads (10 µm) as capture agents enabled rapid and direct image-taking and signal calculation of the test result. The performance of this platform was demonstrated by point-of-care quantification of SARS-CoV-2 nucleocapsid (N) protein. Within 25 min, a limit of detection of 5.08 pg mL-1 and a limit of quantification of 8.91 pg mL-1 can be achieved using <1 µL sample. The system showed high reproducibility across the wide dynamic range (10-105 pg mL-1), with the coefficient of variance ranging from 2.6% to 9.8%. SIGNIFICANCE: This rapid, sensitive AIEgens-enhanced immunofluorescent assay on the DMF platform showed simplified reaction steps and improved performance, providing insight into the small-volume point-of-care testing of different biomarkers in research and clinical applications.

Whole-genome selective sweep analyses identifies the region and candidate gene associated with white earlobe color in Mediterranean chickens.

We compared the genomes of multiple domestic chicken breeds with red and white earlobes to identify the differentiated regions between groups of breeds differing in earlobe color. This was done using a selective sweep mapping approach based on whole-genome sequence data. The most significant selective sweep was identified on chromosome 11, where the white earlobe chicken breeds originated from Mediterranean share a common haplotype, and where multiple candidate genes are located. The most plausible functional candidate gene is the Melanocortin 1 Receptor (MC1R), a receptor known to regulate pigmentation in the skin and hair, and it is also the gene with the strongest positional support from the haplotype-based analyses. It, however, still needs to be explored experimentally to identify effects also on chicken earlobe color variation. Our study is the first exploration of the genetic basis of white earlobe color in Mediterranean chickens using a selective sweep mapping method based on whole-genome sequencing data and shows its value for identifying likely functional genes mediating the pigmentation in earlobe. It also indicates a potential novel role of MC1R in birds and exemplifies how selection on fancy traits has influenced the genome during formation of the modern chicken breeds.

Mapping and Functional Dissection of the Rumpless Trait in Piao Chicken Identifies a Causal Loss of Function Mutation in the Novel Gene Rum.

Rumpless chickens exhibit an abnormality in their tail development. The genetics and biology of this trait has been studied for decades to illustrate a broad variation in both the types of inheritance and the severity in the developmental defects of the tail. In this study, we created a backcross pedigree by intercrossing Piao (rumpless) with Xianju (normal) to investigate the genetic mechanisms and molecular basis of the rumpless trait in Piao chicken. Through genome-wide association and linkage analyses, the candidate region was fine-mapped to 798.5 kb (chromosome 2: 86.9 to 87.7 Mb). Whole-genome sequencing analyses identified a single variant, a 4.2 kb deletion, which was completely associated with the rumpless phenotype. Explorations of the expression data identified a novel causative gene, Rum, that produced a long, intronless transcript across the deletion. The expression of Rum is embryo-specific, and it regulates the expression of MSGN1, a key factor in regulating T-box transcription factors required for mesoderm formation and differentiation. These results provide genetic and molecular experimental evidence for a novel mechanism regulating tail development in chicken and report the likely causal mutation for the tail abnormity in the Piao chicken. The novel regulatory gene, Rum, will, due to its role in fundamental embryo development, be of interest for further explorations of a potential role in tail and skeletal development also in other vertebrates.

Genomic prediction based on selective linkage disequilibrium pruning of low-coverage whole-genome sequence variants in a pure Duroc population.

BACKGROUND: Although the accumulation of whole-genome sequencing (WGS) data has accelerated the identification of mutations underlying complex traits, its impact on the accuracy of genomic predictions is limited. Reliable genotyping data and pre-selected beneficial loci can be used to improve prediction accuracy. Previously, we reported a low-coverage sequencing genotyping method that yielded 11.3 million highly accurate single-nucleotide polymorphisms (SNPs) in pigs. Here, we introduce a method termed selective linkage disequilibrium pruning (SLDP), which refines the set of SNPs that show a large gain during prediction of complex traits using whole-genome SNP data. RESULTS: We used the SLDP method to identify and select markers among millions of SNPs based on genome-wide association study (GWAS) prior information. We evaluated the performance of SLDP with respect to three real traits and six simulated traits with varying genetic architectures using two representative models (genomic best linear unbiased prediction and BayesR) on samples from 3579 Duroc boars. SLDP was determined by testing 180 combinations of two core parameters (GWAS P-value thresholds and linkage disequilibrium r2). The parameters for each trait were optimized in the training population by five fold cross-validation and then tested in the validation population. Similar to previous GWAS prior-based methods, the performance of SLDP was mainly affected by the genetic architecture of the traits analyzed. Specifically, SLDP performed better for traits controlled by major quantitative trait loci (QTL) or a small number of quantitative trait nucleotides (QTN). Compared with two commercial SNP chips, genotyping-by-sequencing data, and an unselected whole-genome SNP panel, the SLDP strategy led to significant improvements in prediction accuracy, which ranged from 0.84 to 3.22% for real traits controlled by major or moderate QTL and from 1.23 to 11.47% for simulated traits controlled by a small number of QTN. CONCLUSIONS: The SLDP marker selection method can be incorporated into mainstream prediction models to yield accuracy improvements for traits with a relatively simple genetic architecture, however, it has no significant advantage for traits not controlled by major QTL. The main factors that affect its performance are the genetic architecture of traits and the reliability of GWAS prior information. Our findings can facilitate the application of WGS-based genomic selection.

Development of a novel magnetic metal-organic framework for the immobilization of short-chain dehydrogenase for the asymmetric reduction of pro-chiral ketone.

Short-chain dehydrogenase/reductase (SDR) acts as a biocatalyst in the synthesis of chiral alcohols with high optical purity. Herein, we achieved immobilization via crosslinking on novel magnetic metal-organic framework nanoparticles with a three-layer shell structure (Fe3O4@PDA@Cu (PABA)). The results of scanning electron microscopy, Fourier-transform infrared spectroscopy, X-ray diffraction, X-ray photoelectron spectroscopy, and energy dispersive X-ray spectroscopy confirmed the morphology and cross-linking property of immobilized SDR, which was more durable, stable, and reusable and exhibited better kinetic performance than free enzyme. The SDR and glucose dehydrogenase (GDH) were co-immobilized and then used for the asymmetric reduction of COBE and ethyl 2-oxo-4-phenylbutanoate (OPBE). These finding suggest that enzymes immobilized on novel MOF nanoparticles can serve as promising biocatalysts for asymmetric reduction prochiral ketones into chiral alcohols.

Tropinone reductase: A comprehensive review on its role as the key enzyme in tropane alkaloids biosynthesis.

TAs, including hyoscyamine and scopolamine, were used to treat neuromuscular disorders ranging from nerve agent poisoning to Parkinson's disease. Tropinone reductase I (TR-I; EC 1.1.1.206) catalyzed the conversion of tropinone into tropine in the biosynthesis of TAs, directing the metabolic flow towards hyoscyamine and scopolamine. Tropinone reductase II (TR-II; EC 1.1.1.236) was responsible for the conversion of tropinone into pseudotropine, diverting the metabolic flux towards calystegine A3. The regulation of metabolite flow through both branches of the TAs pathway seemed to be influenced by the enzymatic activity of both enzymes and their accessibility to the precursor tropinone. The significant interest in the utilization of metabolic engineering for the efficient production of TAs has highlighted the importance of TRs as crucial enzymes that govern both the direction of metabolic flow and the yield of products. This review discussed recent advances for the TRs sources, properties, protein structure and biocatalytic mechanisms, and a detailed overview of its crucial role in the metabolism and synthesis of TAs was summarized. Furthermore, we conducted a detailed investigation into the evolutionary origins of these two TRs. A prospective analysis of potential challenges and applications of TRs was presented.

Modelling porcine NAFLD by deletion of leptin and defining the role of AMPK in hepatic fibrosis.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is the most prevalent cause of chronic hepatic disease and results in non-alcoholic steatohepatitis (NASH), which progresses to fibrosis and cirrhosis. Although the Leptin deficient rodent models are widely used in study of metabolic syndrome and obesity, they fail to develop liver injuries as in patients. METHODS: Due to the high similarity with humans, we generated Leptin-deficient (Leptin-/-) pigs to investigate the mechanisms and clinical trials of obesity and NAFLD caused by Leptin. RESULTS: The Leptin-/- pigs showed increased body fat and significant insulin resistance at the age of 12 months. Moreover, Leptin-/- pig developed fatty liver, non-alcoholic steatohepatitis and hepatic fibrosis with age. Absence of Leptin in pig reduced the phosphorylation of JAK2-STAT3 and AMPK. The inactivation of JAK2-STAT3 and AMPK enhanced fatty acid ß-oxidation and leaded to mitochondrial autophagy respectively, and both contributed to increased oxidative stress in liver cells. In contrast with Leptin-/- pig, although Leptin deletion in rat liver inhibited JAK2-STAT3 phosphorylation, the activation of AMPK pathway might prevent liver injury. Therefore, ß-oxidation, mitochondrial autophagy and hepatic fibrosis did not occurred in Leptin-/- rat livers. CONCLUSIONS: The Leptin-deficient pigs presents an ideal model to illustrate the full spectrum of human NAFLD. The activity of AMPK signaling pathway suggests a potential target to develop new strategy for the diagnosis and treatment of NAFLD.

Insights into the Unusual Activity of a Novel Homospermidine Synthase with a Promising Application to Produce Spermidine.

Spermidine is a naturally occurring polyamine with multiple biological activities and potential food and agricultural applications. However, sustainable and scalable spermidine production has not yet been attained. In this study, a homospermidine synthase (HSS) from Pseudomonas frederiksbergensis (PfHSS) capable of catalyzing the synthesis of spermidine from 1,3-diaminopropane and putrescine was identified based on multiple sequence alignment using Blastochloris viridis HSS (BvHSS) as a template. The optimal reaction pH and temperature for purified PfHSS were determined to be 8.5 and 45 °C, respectively, and K+ was able to promote the enzyme activity. Further analysis of the structural and functional relationships through molecular docking and molecular dynamics simulation indicates that glutamic acid at position 359 is the essential residue for the enzyme-catalyzed synthesis of spermidine. The whole-cell catalytic reaction yielded 1321.4 mg/L spermidine and 678.2 mg/L of homospermidine. This study presents a novel, promising, and sustainable biological method for producing spermidine.

Serum metabolic profile and metabolome genome-wide association study in chicken.

BACKGROUND: Chickens provide globally important livestock products. Understanding the genetic and molecular mechanisms underpinning chicken economic traits is crucial for improving their selective breeding. Influenced by a combination of genetic and environmental factors, metabolites are the ultimate expression of physiological processes and can provide key insights into livestock economic traits. However, the serum metabolite profile and genetic architecture of the metabolome in chickens have not been well studied. RESULTS: Here, comprehensive metabolome detection was performed using non-targeted LC-MS/MS on serum from a chicken advanced intercross line (AIL). In total, 7,191 metabolites were used to construct a chicken serum metabolomics dataset and to comprehensively characterize the serum metabolism of the chicken AIL population. Regulatory loci affecting metabolites were identified in a metabolome genome-wide association study (mGWAS). There were 10,061 significant SNPs associated with 253 metabolites that were widely distributed across the entire chicken genome. Many functional genes affect metabolite synthesis, metabolism, and regulation. We highlight the key roles of TDH and AASS in amino acids, and ABCB1 and CD36 in lipids. CONCLUSIONS: We constructed a chicken serum metabolite dataset containing 7,191 metabolites to provide a reference for future chicken metabolome characterization work. Meanwhile, we used mGWAS to analyze the genetic basis of chicken metabolic traits and metabolites and to improve chicken breeding.

An atlas of regulatory elements in chicken: A resource for chicken genetics and genomics.

A comprehensive characterization of regulatory elements in the chicken genome across tissues will have substantial impacts on both fundamental and applied research. Here, we systematically identified and characterized regulatory elements in the chicken genome by integrating 377 genome-wide sequencing datasets from 23 adult tissues. In total, we annotated 1.57 million regulatory elements, representing 15 distinct chromatin states, and predicted about 1.2 million enhancer-gene pairs and 7662 super-enhancers. This functional annotation of the chicken genome should have wide utility on identifying regulatory elements accounting for gene regulation underlying domestication, selection, and complex trait regulation, which we explored. In short, this comprehensive atlas of regulatory elements provides the scientific community with a valuable resource for chicken genetics and genomics.

Review of the advances in lipid anchors-based biosensors for the isolation and detection of exosomes.

Exosomes are nanoparticles with a bilayer lipid structure that carry cargo from their cells of origin. These vesicles are vital to disease diagnosis and therapeutics; however, conventional isolation and detection techniques are generally complicated, time-consuming, and costly, thus hampering the clinical applications of exosomes. Meanwhile, sandwich-structured immunoassays for exosome isolation and detection rely on the specific binding of membrane surface biomarkers, which may be limited by the type and amount of target protein present. Recently, lipid anchors inserted into the membranes of vesicles through hydrophobic interactions have been adopted as a new strategy for extracellular vesicle manipulation. By combining nonspecific and specific binding, the performance of biosensors can be improved variously. This review presents the reaction mechanisms and properties of lipid anchors/probes, as well as advances in the development of biosensors. The combination of signal amplification methods with lipid anchors is discussed in detail to provide insights into the design of convenient and sensitive detection techniques. Finally, the advantages, challenges, and future directions of lipid anchor-based exosome isolation and detection methods are highlighted from the perspectives of research, clinical use, and commercialization.

Loss of MuRF1 in Duroc pigs promotes skeletal muscle hypertrophy.

Muscle mass development depends on increased protein synthesis and reduced muscle protein degradation. Muscle ring-finger protein-1 (MuRF1) plays a key role in controlling muscle atrophy. Its E3 ubiquitin ligase activity recognizes and degrades skeletal muscle proteins through the ubiquitin-proteasome system. The loss of Murf1, which encodes MuRF1, in mice leads to the accumulation of skeletal muscle proteins and alleviation of muscle atrophy. However, the function of Murf1 in agricultural animals remains unclear. Herein, we bred F1 generation Murf1+/- and F2 generation Murf1-/- Duroc pigs from F0 Murf1-/- pigs to investigate the effect of Murf1 knockout on skeletal muscle development. We found that the Murf1+/- pigs retained normal levels of muscle growth and reproduction, and their percentage of lean meat increased by 6% compared to that of the wild type (WT) pigs. Furthermore, the meat color, pH, water-holding capacity, and tenderness of the Murf1+/- pigs were similar to those of the WT pigs. The drip loss rate and intramuscular fat decreased slightly in the Murf1+/- pigs. However, the cross-sectional area of the myofibers in the longissimus dorsi increased in the adult Murf1+/- pigs. The skeletal muscle proteins MYBPC3 and actin, which are targeted by MuRF1, accumulated in the Murf1+/- and Murf1-/- pigs. Our findings show that inhibiting muscle protein degradation in MuRF1-deficient Duroc pigs increases the size of their myofibers and their percentage of lean meat without influencing their growth or pork quality. Our study demonstrates that Murf1 is a target gene for promoting skeletal muscle hypertrophy in pig breeding.

Transcription-wide impact by RESCUE-induced off-target single-nucleotide variants in mammalian cells.

RNA base editing is a promising tool in precise molecular therapy. Currently, there are two widely used RNA base editors, REPAIR and RESCUE. REPAIR only facilitates A-to-I conversions, while RESCUE performs both A-to-I and C-to-U conversions. Thus, RESCUE can generate twice the number of mutations compared to REPAIR. However, transcription-wide impact due to RESCUE-induced off-target single-nucleotide variants (SNVs) is not fully appreciated. Therefore, to determine the off-target effects of RESCUE-mediated editing, we employed transcription-wide sequencing on cells edited by RESCUE. The SNVs showed different off-target effects on mRNA, circRNA, lncRNA, and miRNA expression patterns and their interacting networks. Our results illustrate the transcription-wide impact of RESCUE-induced off-target SNVs and highlight the need for careful characterization of the off-target impact by this editing platform.

Chicken chromatin accessibility atlas accelerates epigenetic annotation of birds and gene fine-mapping associated with growth traits.

The development of epigenetic maps, such as the ENCODE project in humans, provides resources for gene regulation studies and a reference for research of disease-related regulatory elements. However, epigenetic information, such as a bird-specific chromatin accessibility atlas, is currently lacking for the thousands of bird species currently described. The major genomic difference between birds and mammals is their shorter introns and intergenic distances, which seriously hinders the use of humans and mice as a reference for studying the function of important regulatory regions in birds. In this study, using chicken as a model bird species, we systematically compiled a chicken chromatin accessibility atlas using 53 Assay of Transposase Accessible Chromatin sequencing (ATAC-seq) samples across 11 tissues. An average of 50 796 open chromatin regions were identified per sample, cumulatively accounting for 20.36% of the chicken genome. Tissue specificity was largely reflected by differences in intergenic and intronic peaks, with specific functional regulation achieved by two mechanisms: recruitment of several sequence-specific transcription factors and direct regulation of adjacent functional genes. By integrating data from genome-wide association studies, our results suggest that chicken body weight is driven by different regulatory variants active in growth-relevant tissues. We propose CAB39L (active in the duodenum), RCBTB1 (muscle and liver), and novel long non-coding RNA ENSGALG00000053256 (bone) as candidate genes regulating chicken body weight. Overall, this study demonstrates the value of epigenetic data in fine-mapping functional variants and provides a compendium of resources for further research on the epigenetics and evolution of birds and mammals.

PKD1 deficiency induces Bronchiectasis in a porcine ADPKD model.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is a prevalent genetic disorder, mainly characterized by the development of renal cysts, as well as various extrarenal manifestations. Previous studies have shown that ADPKD is related to bronchiectasis, while its pathogenic mechanism is unclear. In previous studies, we have generated the PKD1+/- pigs to simulate the progression of cyst formation and physiological alterations similar to those seen in ADPKD patients. METHODS: Phenotypic changes to airway epithelial cell and mesenchymal cell in PKD1+/- pigs were assessed by histological analysis. The molecular mechanisms driving these processes were investigated by using PKD1+/- pig lungs, human mesenchymal cells, and generating PKD1 deficient human epithelial cells. RESULTS: We identified bronchiectasis in PKD1+/- pigs, which is consistent with the clinical symptoms in ADPKD patients. The deficiency of PKD1 suppressed E-cadherin expression in the airway epithelial barrier, which aggravated invasion and leaded to a perpetuated inflammatory response. During this process, extracellular matrix (ECM) components were altered, which contributed to airway smooth muscle cell phenotype switch from a contractile phenotype to a proliferative phenotype. The effects on smooth muscle cells resulted in airway remodeling and establishment of bronchiectasis. CONCLUSION: To our knowledge, the PKD1+/- pig provides the first model recapitulating the pathogenesis of bronchiectasis in ADPKD. The role of PKD1 in airway epithelial suggests a potential target for development of new strategies for the diagnosis and treatment of bronchiectasis.

Plateau Adaptation Gene Analyses Reveal Transcriptomic, Proteomic, and Dual Omics Expression in the Lung Tissues of Tibetan and Yorkshire Pigs.

Elevated environments such as plateaus are often classified as low oxygen environments. The hypoxic adaptation mechanisms utilized by organisms in these conditions are not well understood. To address this, the differentially expressed genes (DEGs) involved in hypoxia adaptation were assessed using two pig breeds (Tibetan pig [TP] and Yorkshire sow [YY]). Genes related to lung tissue responses to hypoxia were assessed using transcriptomic (using RNA-seq) and proteomic (using iTRAQ) analysis. A total of 1021 DEGs were screened out. In the iTRAQ omics data, a total of 22,100 peptides were obtained and 4518 proteins were found after filtering. A total of 271 differentially expressed proteins [DEPs] were screened using the conditions of p < 0.05; FC ≤ 0.833; and FC ≥ 1.2. A total of 14 DEGs at the mRNA and protein levels were identified and found to be associated with regulation of the inflammatory response; blood particles; and MAPK cascade response regulation. Among the DEGs, six were associated with hypoxia adaptation function (mitochondria and glycolysis) in pigs. The results of this study identify novel candidate genes involved in porcine hypoxia adaptation mechanisms.