GW3, encoding a member of the P450 subfamily, controls grain width by regulating the GA4 content in spikelets of rice (Oryza sativa L.).

KEY MESSAGE: A stable QTL, GW3, controlling grain width was identified in two populations. Its causal gene LOC_Os03g04680 was verified by gene-based haplotype analysis, expression analysis, gene knockout and complementation transgenic tests. Grain width (GW) is one of the key traits affecting grain size and determines grain yield and appearance quality in rice. Mining gene loci and elite alleles controlling GW is necessary. The GW phenotypes of the two populations were investigated in three environments, which showed abundant phenotypic variation. GW3, encoding a P450 subfamily protein, was identified and validated as a causal gene by gene-based haplotype analysis, expression analysis, gene knockout and complementation transgenic tests. The accessions with large GW values had high gene expression levels. In addition, the GW of the accessions with the GG allele was significantly greater than that of the accessions with the AA allele. The Hap 1 and Hap 3 were identified as elite haplotypes, which can increase GW. The expression levels of OsKO1, OsGA3ox1, OsGA20ox1 and OsGA20ox2 in the young panicle of A7444 were significantly greater than those in the young panicle of the mutants, indicating that GW3 may be involved in the gibberellins (GA) biosynthesis pathway to regulate GW. GA4 content detection and electron scanning analysis revealed that GA4 regulates GW by affecting glume cell size. These results provide new insights for studying the genetic mechanism of rice GW and provide a material basis for breeding high-yield rice varieties.

N6-methyladenosine-modified lncRNA in Staphylococcus aureus-injured bovine mammary epithelial cells.

Staphylococcus aureus-induced mastitis is a serious disease in dairy bovine, with no currently effective treatment. Antibiotics demonstrate certain therapeutic potency in dairy husbandry; they generate drug-resistant bacteria, thereby harming public health. LncRNAs and m6A have been verified as potential targets in infectious diseases and have powerful regulatory capabilities. However, the biological regulation of lncRNAs with m6A modification in mastitis needs further investigation. This study aims to determine the m6A-modified lncRNAs in bovine mammary epithelial cells and their diversity during S. aureus induction. Heat-inactivated S. aureus was used to develop the cell injury model, and we subsequently found low cell viability and different m6A modification levels. Our analysis of m6A-modified lncRNA profiles through MeRIP-seq revealed significant differences in 140 peaks within 130 lncRNAs when cells were injured by S. aureus. Furthermore, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses revealed that these differential m6A-modified lncRNAs were mainly enriched in the WNT pathway, and their functions were associated with amino acid metabolism, lipid translocation, and metalloproteinase activity. Here, we report for the first time lncRNAs with m6A modification in regulating S. aureus infection, revealing potential mechanisms and targets of infectious diseases, such as mastitis, from an epigenetics perspective.

QTL Detection and Candidate Gene Identification for Eating and Cooking Quality Traits in Rice (Oryza sativa L.) via a Genome-Wide Association Study.

The eating and cooking quality (ECQ) directly affects the taste of rice, being closely related to factors such as gelatinization temperature (GT), gel consistency (GC) and amylose content (AC). Mining the quantitative trait loci (QTLs), and gene loci controlling ECQ-related traits is vital. A genome-wide association study on ECQ-related traits was conducted, combining 1.2 million single nucleotide polymorphisms (SNPs) with the phenotypic data of 173 rice accessions. Two QTLs for GT, one for GC and five for AC were identified, of which two were found in previously reported genes, and six were newly found. There were 28 positional candidate genes in the region of qAC11. Based on a linkage disequilibrium (LD) analysis, three candidate genes were screened within the LD region associated with AC. There were significant differences between the haplotypes of LOC_Os11g10170, but no significant differences were found for the other two genes. The qRT-PCR results showed that the gene expression levels in the accessions with high ACs were significantly larger than those in the accessions with low ACs at 35d and 42d after flowering. Hap 2 and Hap 3 of LOC_Os11g10170 reduced the AC by 13.09% and 10.77%, respectively. These results provide a theoretical and material basis for improving the ECQ of rice.

Gut microbiome variations in Rhinopithecus roxellanae caused by changes in the environment.

BACKGROUND: The snub-nosed monkey (Rhinopithecus roxellanae) is an endangered animal species mainly distributed in China and needs to be protected. Gut microbiome is an important determinant of animal health and population survival as it affects the adaptation of the animals to different foods and environments under kinetic changes of intrinsic and extrinsic factors. Therefore, this study aimed to elucidate gut fecal microbiome profiles of snub-nosed monkeys affected by several extrinsic and intrinsic factors, including raising patterns (captive vs. wild), age, sex, and diarrheal status to provide a reference for making protection strategies. RESULTS: The 16S rRNA gene sequencing was firstly used to pre-check clustering of 38 fecal samples from the monkeys including 30 wild and 8 captive (5 healthy and 3 diarrheal) from three Regions of Shennongjia Nature Reserve, Hubei Province, China. Then the 24 samples with high-quality DNA from 18 wild and 6 captive (4 healthy and 2 diarrheal) monkeys were subjected to shotgun metagenomic sequencing to characterize bacterial gut microbial communities. We discovered that the raising pattern (captive and wild) rather than age and sex was the predominant factor attributed to gut microbiome structure and proportionality. Wild monkeys had significantly higher bacterial diversity and lower Bacteroidetes/Firmicutes ratios than captive animals. Moreover, the gut microbiomes in wild healthy monkeys were enriched for the genes involved in fatty acid production, while in captive animals, genes were enriched for vitamin biosynthesis and metabolism and amino acid biosynthesis from carbohydrate intermediates. Additionally, a total of 37 antibiotic resistant genes (ARG) types were detected. Unlike the microbiome diversity, the captive monkeys have a higher diversity of ARG than the wild animals. CONCLUSION: Taken together, we highlight the importance of self-reprogramed metabolism in the snub-nosed monkey gut microbiome to help captive and wild monkeys adapt to different intrinsic and extrinsic environmental change.

Soluble expression and purification of recombinant bovine ferritin H-chain.

Ferritin is a potential medicine delivery vehicle and vaccine platform, and its efficient expression is a prerequisite for widespread application. This study introduces a soluble expression strategy for recombinant bovine ferritin heavy chain (rFTH) in a prokaryotic system and an improved protein purification method. The amplified rFTH gene was ligated into the prokaryotic expression vector pET30a. The recombinant vectors with the N-terminal His-tag(N-His) or C-terminal His-tag(C-His) were translated and expressed separately. The results showed that the solubility of rFTH with C-His was significantly higher than that with N-His. The expression of rFTH with C-His was attempted at 37 °C and 16 °C, respectively. The results showed that the proportion of soluble protein expressed at 37 °C was more than 90%, higher than that expressed at 16 °C. Then rFTH with C-His was purified successfully using anion exchange chromatography, modified PEG precipitation, and dialysis. The rFTH protein was characterized using SDS-PAGE, Native-PAGE, Western blot, transmission electron microscopy, and dynamic light scattering. The results demonstrated that the purified rFTH protein self-assembled into ferritin nanoparticles with a regular shape and uniform size. This study sheds new light on the soluble expression of ferritin and provides a foundation for the construction of bovine ferritin nanoparticle production platforms.

Identification of Differential Circular RNA Expression Profiles and Functional Networks in Human Macrophages Induced by Virulent and Avirulent Mycobacterium tuberculosis Strains.

Circular RNAs (circRNAs) are noncoding RNAs with diverse functions. However, most Mycobacterium tuberculosis (M.tb)-related circRNAs remain undiscovered. In this study, we infected THP-1 cells with virulent and avirulent M.tb strains and then sequenced the cellular circRNAs. Bioinformatic analysis predicted 58,009 circRNAs in all the cells. In total, 2035 differentially expressed circRNAs were identified between the M.tb-infected and uninfected THP-1 cells and 1258 circRNAs were identified in the virulent and avirulent M.tb strains. Further, the top 10 circRNAs were confirmed by Sanger sequencing, among which four circRNAs, namely circSOD2, circCHSY1, circTNFRSF21, and circDHTKD1, which were highly differentially expressed in infected cells compared with those in uninfected cells, were further confirmed by ring formation, specific primers, and RNase R digestion. Next, circRNA-miRNA-mRNA subnetworks were constructed, such as circDHTKD1/miR-660-3p/IL-12B axis. Some of the individual downstream genes, such as miR-660-3p and IL-12B, were previously reported to be associated with cellular defense against pathological processes induced by M.tb infection. Because macrophages are important immune cells and the major host cells of M.tb, these findings provide novel ideas for exploring the M.tb pathogenesis and host defense by focusing on the regulation of circRNAs during M.tb infection.

SARS-CoV-2 Infection Causes Hyperglycemia in Cats.

Isolated reports of new-onset diabetes in patients with coronavirus disease 2019 (COVID-19) have led researchers to hypothesize that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects human exocrine and endocrine pancreatic cells ex vivo and in vivo. However, existing research lacks experimental evidence indicating that SARS-CoV-2 can infect pancreatic tissue. Here, we found that cats infected with a high dose of SARS-CoV-2 exhibited hyperglycemia. We also detected SARS-CoV-2 RNA in pancreatic tissues of these cats, and immunohistochemical staining revealed the presence of SARS-CoV-2 nucleocapsid protein (NP) in islet cells. SARS-CoV-2 NP and spike proteins were primarily detected in glucagon-positive cells, and most glucagon-positive cells expressed ACE2. Additionally, immune protection experiments conducted on cats showed that blood glucose levels of immunized cats did not increase postchallenge. Our data indicate cat pancreas as a SARS-CoV-2 target and suggest that the infection of glucagon-positive cells could contribute to the metabolic dysregulation observed in SARS-CoV-2-infected cats.

An emerging role for cyclic dinucleotide phosphodiesterase and nanoRNase activities in Mycoplasma bovis: Securing survival in cell culture.

Mycoplasmas are host-restricted prokaryotes with a nearly minimal genome. To overcome their metabolic limitations, these wall-less bacteria establish intimate interactions with epithelial cells at mucosal surfaces. The alarming rate of antimicrobial resistance among pathogenic species is of particular concern in the medical and veterinary fields. Taking advantage of the reduced mycoplasma genome, random transposon mutagenesis was combined with high-throughput screening in order to identify key determinants of mycoplasma survival in the host-cell environment and potential targets for drug development. With the use of the ruminant pathogen Mycoplasma bovis as a model, three phosphodiesterases of the DHH superfamily were identified as essential for the proliferation of this species under cell culture conditions, while dispensable for axenic growth. Despite a similar domain architecture, recombinant Mbov_0327 and Mbov_0328 products displayed different substrate specificities. While rMbovP328 protein exhibited activity towards cyclic dinucleotides and nanoRNAs, rMbovP327 protein was only able to degrade nanoRNAs. The Mbov_0276 product was identified as a member of the membrane-associated GdpP family of phosphodiesterases that was found to participate in cyclic dinucleotide and nanoRNA degradation, an activity which might therefore be redundant in the genome-reduced M. bovis. Remarkably, all these enzymes were able to convert their substrates into mononucleotides, and medium supplementation with nucleoside monophosphates or nucleosides fully restored the capacity of a Mbov_0328/0327 knock-out mutant to grow under cell culture conditions. Since mycoplasmas are unable to synthesize DNA/RNA precursors de novo, cyclic dinucleotide and nanoRNA degradation are likely contributing to the survival of M. bovis by securing the recycling of purines and pyrimidines. These results point toward proteins of the DHH superfamily as promising targets for the development of new antimicrobials against multidrug-resistant pathogenic mycoplasma species.

Prevalence of bovine astroviruses and their genotypes in sampled Chinese calves with and without diarrhoea.

Bovine astrovirus (BoAstV) belongs to genus Mamastravirus (MAstV). It can be detected in the faeces of both diarrhoeal and healthy calves. However, its prevalence, genetic diversity, and association with cattle diarrhoea are poorly understood. In this study, faecal samples of 87 diarrhoeal and 77 asymptomatic calves from 20 farms in 12 provinces were collected, and BoAstV was detected with reverse transcription-polymerase chain reaction (RT-PCR). The overall prevalence rate of this virus in diarrhoeal and asymptomatic calves was 55.17 % (95 % CI: 44.13, 65.85 %) and 36.36 % (95 % CI: 25.70, 48.12 %), respectively, indicating a correlation between BoAstV infection and calf diarrhoea (OR=2.15, P=0.024). BoAstV existed mainly in the form of co-infection (85.53 %) with one to five of nine viruses, and there was a strong positive correlation between BoAstV co-infection and calf diarrhoea (OR=2.83, P=0.004). Binary logistic regression analysis confirmed this correlation between BoAstV co-infection and calf diarrhoea (OR=2.41, P=0.038). The co-infection of BoAstV and bovine rotavirus (BRV) with or without other viruses accounted for 70.77 % of all the co-infection cases. The diarrhoea risk for the calves co-infected with BoAstV and BRV was 8.14-fold higher than that for the calves co-infected with BoAstV and other viruses (OR=8.14, P=0.001). Further, the co-infection of BoAstV/BRV/bovine kobuvirus (BKoV) might increase the risk of calf diarrhoea by 14.82-fold, compared with that of BoAstV and other viruses (OR=14.82, P <0.001). Then, nearly complete genomic sequences of nine BoAstV strains were assembled by using next-generation sequencing (NGS) method. Sequence alignment against known astrovirus (AstV) strains at the levels of both amino acids and nucleotides showed a high genetic diversity. Four genotypes were identified, including two known genotypes MAstV-28 (n=3) and MAstV-33 (n=2) and two novel genotypes designated tentatively as MAstV-34 (n=1) and MAstV-35 (n=3). In addition, seven out of nine BoAstV strains showed possible inter-genotype recombination and cross-species recombination. Therefore, our results increase the knowledge about the prevalence and the genetic evolution of BoAstV and provide evidence for the association between BoAstV infection and calf diarrhoea.

Transcriptome Profiling of m6A mRNA Modification in Bovine Mammary Epithelial Cells Treated with Escherichia coli.

Mastitis is a common disease in dairy cows that is mostly caused by E. coli, and it brings massive losses to the dairy industry. N6-Methyladenosine (m6A), a methylation at the N6 position of RNA adenine, is a type of modification strongly associated with many diseases. However, the role of m6A in mastitis has not been investigated. In this study, we used MeRIP-seq to sequence the RNA of bovine mammary epithelial cells treated with inactivated E. coli for 24 h. In this in vitro infection model, there were 16,691 m6A peaks within 7066 mRNA transcripts in the Con group and 10,029 peaks within 4891 transcripts in the E. coli group. Compared with the Con group, 474 mRNAs were hypermethylated and 2101 mRNAs were hypomethylated in the E. coli group. Biological function analyses revealed differential m6A-modified genes mainly enriched in the MAPK, NF-κB, and TGF-ß signaling pathways. In order to explore the relationship between m6A and mRNA expression, combined MeRIP-seq and mRNA-seq analyses revealed 212 genes with concomitant changes in the mRNA expression and m6A modification. This study is the first to present a map of RNA m6A modification in mastitis treated with E. coli, providing a basis for future research.

Identification of Unique Key miRNAs, TFs, and mRNAs in Virulent MTB Infection Macrophages by Network Analysis.

Although Mycobacterium tuberculosis (MTB) has existed for thousands of years, its immune escape mechanism remains obscure. Increasing evidence signifies that microRNAs (miRNAs) play pivotal roles in the progression of tuberculosis (TB). RNA sequencing was used to sequence miRNAs in human acute monocytic leukemia cells (THP-1) infected by the virulent MTB-1458 strain and the avirulent vaccine strain Mycobacterium bovis Bacillus Calmette-Guérin (BCG). Sets of differentially expressed miRNAs (DE-miRNAs) between MTB-1458/BCG-infected groups and uninfected groups were identified, among which 18 were differentially expressed only in the MTB-1458-infected THP-1 group. Then, 13 transcription factors (TFs) and 81 target genes of these 18 DE-miRNAs were matched. Gene Ontology classification as well as Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that the candidate targets were predominantly involved in apoptotic-associated and interferon-γ-mediated signaling pathways. A TF-miRNA-mRNA interaction network was constructed to analyze the relationships among these 18 DE-miRNAs and their targets and TFs, as well as display the hub miRNAs, TFs, and target genes. Considering the degrees from network analysis and the reported functions, this study focused on the BHLHE40-miR-378d-BHLHE40 regulation axis and confirmed that BHLHE40 was a target of miR-378d. This cross-talk among DE-miRNAs, mRNAs, and TFs might be an important feature in TB, and the findings merited further study and provided new insights into immune defense and evasion underlying host-pathogen interactions.

Genome-Wide Analysis of LncRNA in Bovine Mammary Epithelial Cell Injuries Induced by Escherichia Coli and Staphylococcus Aureus.

Escherichia coli and Staphylococcus aureus are two common pathogenic microorganisms that cause mastitis in dairy cows. They can cause clinical mastitis and subclinical mastitis. In recent studies, lncRNAs have been found to play an important role in the immune responses triggered by microbial inducers. However, the actions of lncRNAs in bovine mastitis remain unclear. The purpose of this study was to investigate the effects of bovine mammary epithelial cell injuries induced by treatment with E. coli and S. aureus, and to explore the lncRNA profile on cell injuries. The lncRNA transcriptome analysis showed a total of 2597 lncRNAs. There were 2234 lncRNAs differentially expressed in the E. coli group and 2334 in the S. aureus group. Moreover, we found that the E. coli and S. aureus groups of maternal genes targeted signaling pathways with similar functions according to KEGG and GO analyses. Two lncRNA-miRNA-mRNA interaction networks were constructed in order to predict the potential molecular mechanisms of regulation in the cell injuries. We believe that this is the first report demonstrating the dysregulation of lncRNAs in cells upon E. coli and S. aureus infections, suggesting that they have the potential to become important diagnostic markers and to provide novel insights into controlling and preventing mastitis.

TRIM25 upregulation by Mycobacterium tuberculosis infection promotes intracellular survival of M.tb in RAW264.7 cells.

Tripartite motif 25 (TRIM25) is a TRIM family member which is involved in innate immunity. However, its role in the modulation of host defense against Mycobacterium tuberculosis (M.tb) infection has not been investigated. Therefore, this study aimed to demonstrate the significance of TRIM25 in the regulation of macrophage responses to M.tb infection. TRIM25 was found to be significantly overexpressed (3.476-fold) in peripheral blood mononuclear cells (PBMCs) of 67 patients with pulmonary tuberculosis compared with 48 healthy controls. TRIM25 expression was enhanced following M.tb infection of RAW264.7 cells, a macrophage cell line. Overexpression of TRIM25 in M.tb-infected RAW264.7 cells led to a significant increase in phosphorylated p38 levels; however, the production of IL-6, IL-1ß, and TNF-α were significantly reduced. Finally, M.tb intracellular survival increased by 90% at 12 h post-infection (PI) (p < 0.01). To validate the previous results, TRIM25 levels in M.tb-infected RAW264.7 macrophages were down-regulated using small interfering RNA (siRNA). Therefore, it was concluded that TRIM25 promotes intracellular survival of M.tb in RAW264.7 cells, likely by enhancing p38 pathways and thereby inhibiting the production of proinflammatory cytokines. These results contribute to the further understanding of the host defense against M.tb infection.

Indirubin Treatment of Lipopolysaccharide-Induced Mastitis in a Mouse Model and Activity in Mouse Mammary Epithelial Cells.

Indirubin is a Chinese medicine extracted from indigo and known to be effective for treating chronic myelogenous leukemia, neoplasia, and inflammatory disease. This study evaluated the in vivo anti-inflammatory activity of indirubin in a lipopolysaccharide- (LPS-) induced mouse mastitis model. The indirubin mechanism and targets were evaluated in vitro in mouse mammary epithelial cells. In the mouse model, indirubin significantly attenuated the severity of inflammatory lesions, edema, inflammatory hyperemia, milk stasis and local tissue necrosis, and neutrophil infiltration. Indirubin significantly decreased myeloperoxidase activity and downregulated the production of tumor necrosis factor-α, interleukin-1ß (IL-1ß), and IL-6 caused by LPS. In vitro, indirubin inhibited LPS-stimulated expression of proinflammatory cytokines in a dose-dependent manner. It also downregulated LPS-induced toll-like receptor 4 (TLR4) expression and inhibited phosphorylation of LPS-induced nuclear transcription factor-kappa B (NF-κB) P65 protein and inhibitor of kappa B. In addition to its effect on the NF-κB signaling pathway, indirubin suppressed the mitogen-activated protein kinase (MAPK) signaling by inhibiting phosphorylation of extracellular signal-regulated kinase (ERK), P38, and c-jun NH2-terminal kinase (JNK). Indirubin improved LPS-induced mouse mastitis by suppressing TLR4 and downstream NF-κB and MAPK pathway inflammatory signals and might be a potential treatment of mastitis and other inflammatory diseases.

Efficacy of Short Novel Antimicrobial Peptides in a Mouse Model of Staphylococcus pseudintermedius Skin Infection.

As the clinical application of antibiotics for bacterial skin infections in companion animals becomes increasingly prevalent, the issue of bacterial resistance has become more pronounced. Antimicrobial peptides, as a novel alternative to traditional antibiotics, have garnered widespread attention. In our study, synthetic peptides ADD-A and CBD3-ABU were tested against Staphylococcus pseudintermedius skin infections in KM mice. ADD-A was applied topically and through intraperitoneal injection, compared with control groups and treatments including CBD3-ABU, ampicillin sodium, and saline. Wound contraction, bacterial counts and histology were assessed on days 3 and 11 post-infection. ADD-A and ampicillin treatments significantly outperformed saline in wound healing (p < 0.0001 and p < 0.001, respectively). ADD-A also showed a markedly lower bacterial count than ampicillin (p < 0.0001). Histologically, ADD-A-applied wounds had better epidermal continuity and a thicker epidermis than normal, with restored follicles and sebaceous glands. ADD-A's effectiveness suggests it as a potential alternative to antibiotics for treating skin infections in animals.

MbovP0725, a secreted serine/threonine phosphatase, inhibits the host inflammatory response and affects metabolism in Mycoplasma bovis.

Mycoplasma species are able to produce and release secreted proteins, such as toxins, adhesins, and virulence-related enzymes, involved in bacteria adhesion, invasion, and immune evasion between the pathogen and host. Here, we investigated a novel secreted protein, MbovP0725, from Mycoplasma bovis encoding a putative haloacid dehalogenase (HAD) hydrolase function of a key serine/threonine phosphatase depending on Mg2+ for the dephosphorylation of its substrate pNPP, and it was most active at pH 8 to 9 and temperatures around 40°C. A transposon insertion mutant strain of M. bovis HB0801 that lacked the protein MbovP0725 induced a stronger inflammatory response but with a partial reduction of adhesion ability. Using transcriptome sequencing and quantitative reverse transcription polymerase chain reaction analysis, we found that the mutant was upregulated by the mRNA expression of genes from the glycolysis pathway, while downregulated by the genes enriched in ABC transporters and acetate kinase-phosphate acetyltransferase pathway. Untargeted metabolomics showed that the disruption of the Mbov_0725 gene caused the accumulation of 9-hydroxyoctadecadienoic acids and the consumption of cytidine 5'-monophosphate, uridine monophosphate, and adenosine monophosphate. Both the exogenous and endogenous MbvoP0725 protein created by purification and transfection inhibited lipopolysaccharide (LPS)-induced IL-1ß, IL-6, and TNF-α mRNA production and could also attenuate the activation of MAPK-associated pathways after LPS treatment. A pull-down assay identified MAPK p38 and ERK as potential substrates for MbovP0725. These findings define metabolism- and virulence-related roles for a HAD family phosphatase and reveal its ability to inhibit the host pro-inflammatory response. IMPORTANCE: Mycoplasma bovis (M. bovis) infection is characterized by chronic pneumonia, otitis, arthritis, and mastitis, among others, and tends to involve the suppression of the immune response via multiple strategies to avoid host cell immune clearance. This study found that MbovP0725, a haloacid dehalogenase (HAD) family phosphatase secreted by M. bovis, had the ability to inhibit the host pro-inflammatory response induced by lipopolysaccharide. Transcriptomic and metabolomic analyses were used to identify MbovP0725 as an important phosphatase involved in glycolysis and nucleotide metabolism. The M. bovis transposon mutant strain T8.66 lacking MbovP0725 induced a higher inflammatory response and exhibited weaker adhesion to host cells. Additionally, T8.66 attenuated the phosphorylation of MAPK P38 and ERK and interacted with the two targets. These results suggested that MbovP0725 had the virulence- and metabolism-related role of a HAD family phosphatase, performing an anti-inflammatory response during M. bovis infection.

A Novel Multiepitope Fusion Antigen as a Vaccine Candidate for the Prevention of Enterotoxigenic E. coli-Induced Calf Diarrhea.

Calf diarrhea caused by enterotoxigenic E. coli (ETEC) poses an enormous economic challenge in the cattle industry. Fimbriae and enterotoxin are crucial virulence factors and vaccine targets of ETEC. Since these proteins have complicated components with large molecular masses, the development of vaccines by directly expressing these potential targets is cumbersome Therefore, this study aimed to develop a multiepitope fusion antigen designated as MEFA by integrating major epitopes of FanC and Fim41a subunits and a toxoid epitope of STa into the F17G framework. The 3D modeling predicted that the MEFA protein displayed the epitopes from these four antigens on its surface, demonstrating the desired structural characteristics. Then, the MEFA protein was subsequently expressed and purified for mouse immunization. Following that, our homemade ELISA showed that the mouse antiserum had a consistent increase in polyclonal antibody levels with the highest titer of 1:217 to MEFA. Furthermore, the western blot assay demonstrated that this anti-MEFA serum could react with all four antigens. Further, this antiserum exhibited inhibition on ETEC adhesion to HCT-8 cells with inhibitory rates of 92.8%, 84.3%, and 87.9% against F17+, F5+, and F41+ ETEC strains, respectively. Additionally, the stimulatory effect of STa toxin on HCT-8 cells was decreased by approximately 75.3% by anti-MEFA serum. This study demonstrates that the MEFA protein would be an antigen candidate for novel subunit vaccines for preventing ETEC-induced diarrhea in cattle.

Identifying the Quantitative Trait Locus and Candidate Genes of Traits Related to Milling Quality in Rice via a Genome-Wide Association Study.

Milling quality directly affects production efficiency in rice, which is closely related to the brown rice recovery (BRR), the milled rice recovery (MRR) and the head milled rice recovery (HMRR). The present study investigated these three traits in 173 germplasms in two environments, finding abundant phenotypic variation. Three QTLs for BRR, two for MRR, and three for HMRR were identified in a genome-wide association study, five of these were identified in previously reported QTLs and three were newly identified. By combining the linkage disequilibrium (LD) analyses, the candidate gene LOC_Os05g08350 was identified. It had two haplotypes with significant differences and Hap 2 increased the BRR by 4.40%. The results of the qRT-PCR showed that the expression of LOC_Os05g08350 in small-BRR accessions was significantly higher than that in large-BRR accessions at Stages 4-5 of young panicle development, reaching the maximum value at Stage 5. The increase in thickness of the spikelet hulls of the accession carrying LOC_Os05g08350TT occurred due to an increase in the cell width and the cell numbers in cross-sections of spikelet hulls. These results help to further clarify the molecular genetic mechanism of milling-quality-related traits and provide genetic germplasm materials for high-quality breeding in rice.

The Establishment of a Novel γ-Interferon In Vitro Release Assay for the Differentiation of Mycobacterial Bovis-Infected and BCG-Vaccinated Cattle.

BCG vaccination is increasingly reconsidered in the effective prevention of bovine tuberculosis (bTB). However, the primary challenge in BCG vaccination for cattle is the lack of a technique for differentiating between infected and vaccinated animals (DIVA). This study aimed to establish a novel DIVA diagnostic test based on an interferon-gamma in vitro release assay (IGRA). The plasmid encoding three differential antigens (Rv3872, CFP-10, and ESAT-6) absent in BCG genes but present in virulent M. bovis was previously constructed. Thus, a recombinant protein called RCE (Rv3872, CFP-10, and ESAT-6) was expressed, and an RCE-based DIVA IGRA (RCE-IGRA) was established. The RCE concentration was optimized at 4 µg/mL by evaluating 97 cattle (74 of which were bTB-positive, and 23 were negative) using a commercial IGRA bTB diagnostic kit. Further, 84 cattle were tested in parallel with the RCE-IGRA and commercial PPD-based IGRA (PPD-IGRA), and the results showed a high correlation with a kappa value of 0.83. The study included BCG-vaccinated calves (n = 6), bTB-positive cattle (n = 6), and bTB-negative non-vaccinated calves (n = 6). After 3 months post-vaccination, PPD-IGRA generated positive results in both vaccinated and infected calves. However, RCE-IGRA developed positive results in infected calves but negative results in vaccinated calves. In conclusion, this DIVA method has broad prospects in differentiating BCG vaccination from natural infection to prevent bTB.

Genome-wide association mapping of quantitative trait loci for chalkiness-related traits in rice (Oryza sativa L.).

Grain chalkiness directly affects the commercial value of rice. Genes related to chalkiness reported thus far have been discovered in mutants, but it has not been identified whether these genes can be used to improve rice quality by breeding. Therefore, discovering more quantitative trait loci (QTLs) or genes related to chalkiness in the rice germplasm is necessary. This study entails a genome-wide association study on the degree of endosperm chalkiness (DEC) and percentage of grains with chalkiness (PGWC) by combining 1.2 million single-nucleotide polymorphisms (SNPs) with the phenotypic data of 173 rice accessions. Thirteen QTLs for DEC and nine for PGWC were identified, of which four were detected simultaneously for both DEC and PGWC; further, qDEC11/qPGWC11 was identified as the major QTL. By combining linkage disequilibrium analysis and SNP information, LOC_Os11g10170 was identified as the candidate gene for DEC. There were significant differences among the haplotypes of LOC_Os11g10170, and the Hap 1 of LOC_Os11g10170 was observed to reduce the DEC by 6.19%. The qRT-PCR results showed that the gene expression levels in accessions with high DEC values were significantly higher than those in accessions with low DEC values during days 21-42 after flowering, with a maximum at 28 days. These results provide molecular markers and germplasm resources for genetic improvement of the chalkiness-related traits in rice.