As an important economic livestock species, the reproductive efficiency of goats is greatly constrained by placental dysfunction during early pregnancy. The health of the goat placenta is crucial for fetal development and lambing rates. Establishing an in vitro placental model is necessary for elucidating its physiological and pathological characteristics. The purpose of this study is to establish goat placental trophoblast organoids and explore their application in placental diseases. Results indicate that goat placental trophoblast tissue (GPTT) from 80-90 d of gestation exhibits high proliferative activity, which is an ideal source for organoid construction. We successfully obtained goat placental trophoblast organoids in the optimized medium, which presented a typical cystic morphology and showed positive expression of trophoblast markers KRT23, CK7, E-cadherin, and the stemness factor SOX2. Importantly, these organoids also exhibit active metabolic and proliferative capabilities. Moreover, transcriptome analysis reveals that the gene expression profiles of the organoids are highly similar to those of in vivo tissues, and are mainly enriched in hormone response, cell adhesion, and pregnancy-related pathways. Notably, the expression of key placental trophoblast genes (WNT5A, VIM, VILL, INHBB) in the organoids is consistent with that in placental tissues. Finally, the disease model using goat placental organoids demonstrates that LPS stimulation induces time-dependent apoptosis in goat placental trophoblast organoids (GPTOs), which is analogous to placental damage caused by in vivo infection. This study firstly established a trophoblast organoid model in goats that has physiological and pathological responsibilities. Also providing an important platform for placental development research in ruminants, maternal-fetal interface regulation, and gestation-related diseases research.

The number of mammary epithelial cells in lactating dairy goats is important for milk yield and is influenced by both hormonal and nutritional factors. Previous studies have reported that the MOV10 is involved in the miRNA splicing process. Moreover, MOV10 has been confirmed to play an important regulatory role in the proliferation of goat epithelial cells; however, its specific molecular regulatory network remains unclear. Therefore, this study aims to further elucidate the mechanism of action of MOV10 in goat epithelial cells. This study demonstrates that MOV10 plays a critical regulatory role in the proliferation and apoptosis of mammary epithelial cells. Downregulating the expression of MOV10 inhibited proliferation and promoted apoptosis, whereas upregulating its expression produced the opposite effect. RNA immunoprecipitation (RIP) assays revealed that MOV10 binds to pre-miR-21 and promotes its processing into mature miR-21-5p. miR-21-5p subsequently activates the TGF-β/Smad signaling pathway by targeting and inhibiting Smad7, thereby promoting epithelial cell proliferation. This study provides a novel perspective on lactation in dairy goats, suggesting that targeting MOV10-mediated signaling may represent an important strategy for increasing the milk yield.

Subclinical mastitis causes substantial economic losses to the dairy industry, and the colonization of Staphylococcus aureus within mammary cells is a major reason for persistent difficulty in its treatment. The development of the Photorhabdus virulence cassette (PVC) delivery system has provided a potential solution for treating infections caused by intracellular bacteria. In this study, we first engineered a targeted delivery system for bacterial-infected cells by using homologous recombination to introduce the S. aureus SraP gene into the PVC13 tail protein and by loading the human β-defensin-3 (HBD3) antimicrobial peptide into PVC-regulated elements. The assembly yielded a PVC-SraP-targeted system capable of delivering HBD3. Mouse experiments demonstrated that the recombinant PVC-SraP system effectively delivered HBD3 to mammary tissue and cells, thereby eliminating S. aureus. Furthermore, infection and therapeutic experiments in mammary cells demonstrated that 2.5 mg/ml PVC can eliminate intracellular bacteria without distinct cytotoxicity. Transcriptomic and western blot analyses confirmed that HBD3 delivered via the PVC-targeted system inhibits NF-κB signaling, thereby reducing inflammation in mammary tissues. This study provides a technical foundation for the future treatment of intracellular and drug-resistant bacteria.

The development of biodegradable antimicrobial bioplastics for food packaging holds great promise for solving the pollution and safety problems caused by petrochemical plastics and spoiled food. Herein, a natural active-bioplastic synthesized from citrus peel biomass is presented for perishable fruit preservation. These plastics are characterized by the nanoscale entanglement and recombinant hydrogen bonding between the endogenous pectin, polyphenols and cellulose micro/nanofibrils. They have attractive flexibility, tensile strength, gas barrier properties and antimicrobial activities, and can effectively extend the shelf life of perishable fruits such as banana and mango when used as food packaging. Cytotoxicity, degradability tests and life-cycle assessment show that these plastics had excellent nontoxicity and can be safely degraded or easily recycled. This work demonstrates a sustainable strategy for converting peel waste into eco-friendly bioplastics, providing a unique and novel insight into radically reducing the pollution and life-health threats posed by petrochemical plastics and spoiled food.

This study aimed to obtain target cells with MRC1 gene-modified PCV2 and verify the inhibitory effect of the cells on PCV2 in vitro, which would provide a theoretical basis for the subsequent production of gene-edited pigs.In this study: 1) Firstly, a lentivirus-mediated high-efficiency homologous recombination vector and a CRISPR/Cas9 gene targeting system were established using genetic engineering technology; 2) The gene-edited MRC1 promoter in PK15 cells were screened by cell and molecular biology techniques; 3) Then, the anti-PCV2 effect was verified by PCV2 infection tests. The results showed that: 1) The pLV-LoxP-mCherry-puro-LoxP vector containing red fluorescent gene, puromycin gene, homologous LoxP sequence and multiple cloning sites was successfully constructed. The homologous arm was designed according to the differential sequences of MRC1 promoter among different pig breeds and the recombinant vector was constructed. Also the CRISPR/Cas9 mediated gene targeting vector were successfully constructed; 2) The PK15 cell line edited by MRC1 promoter gene was screened and verified by puromycin through virus packaging and co-transfection. The 14 bp upstream of the transcription start site of MRC1 gene was successfully knocked into PK15 cells; 3) The protein expression of MRC1 in PK15 cell line edited by MRC1 gene was significantly increased（P＜0.05）, and the replication of PCV2 was significantly reduced（P＜0.05）. The expression of MRC1 protein in PK15 cells edited by MRC1 promoter was significantly increased, which could be used for the preparation of anti-PCV2 pigs.

Mastitis results in great economic loss to the dairy goat industry. Many approaches have attempted to decrease the morbidity associated with this disease, and among these, transgenic strategy have been recognized as a potential approach. A previous mammalian study reports that interferon-gamma (IFN-γ) has potential anti-bacterial bioactivity against infection in vitro; however, its capacity in vivo is ambiguous. In this study, we initially constructed targeting and homologous recombination vectors (containing the IFN-γ gene) and then transferred the vectors into goat mammary gland epithelial cells (GMECs). Enzyme digestion and sequencing analysis indicated that the vectors used in this study were built correctly. Subsequently, monoclonal cells were selected using puromycin and the polymerase chain reaction (PCR) test indicated that IFN-γ was correctly inserted downstream of the casein promoter. Monoclonal cells were then assessed for reducible expression, and reverse transcriptase-PCR (RT-PCR) and Western blot tests confirmed that monoclonal cells could express IFN-γ. Finally, anti-bacterial capacity was evaluated using bacterial counts and flow cytometry analysis. Decreased bacterial counts and cell apoptosis rates in transgenic GMECs demonstrated that the secretion of IFN-γ could inhibit bacterial proliferation. Therefore, IFN-γ gene transfection in goat mammary epithelial cells could inhibit bacterial proliferation and reduce the risk of mammary gland infection in goats.

Previous studies demonstrated that progesterone (P4) can promote prostaglandin (PG) E2 production; however, how P4 mediates the synthesis of PGE2 remains unclear. In this study, cervical epithelial cells from mice during the follicular phase were cultured invitro and treated with different concentrations of P4 (5, 10, and 20nM). The results of the present study suggest that treatment of murine cervical epithelial cells with 10nM P4 for 24h contributed to: (1) significantly increased expression of protein kinase A (PKA), cytosolic phospholipase A2 (cPLA2) and PGE synthase (PGES)-1; (2) higher phosphorylated (p-) to total extracellular signal-regulated kinase (ERK) 1/2 and hormone-sensitive lipase (HSL) ratios; (3) a significant decrease in the number of lipid droplets (LDs) and fatty acid content within LDs in epithelial cells; and (4) enhanced arachidonic acid and PGE2 levels in cells compared with the control (0nM P4) group (P<0.01 for all findings). In contrast, the PKA inhibitor H89 contributed to significantly decreased cPLA2, PGES-1 and HSL expression, ERK1/2 phosphorylation and arachidonic acid and PGE2 levels, even in the presence of P4. These data show that P4 can act via the PKA/ERK1/2 pathway to stimulate lipolysis of triacylglycerol in the LD core and degradation of phospholipid in the LD membrane to promote PGE2 synthesis in murine cervical epithelial cells.

Since the first cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection were reported in late 2019, coronavirus disease 2019 (COVID-19) has claimed the lives of more than 4 million people worldwide.1 In the hepatology community, there has been recognition that patients with chronic liver disease, especially cirrhosis, may be among those at highest risk of a severe clinical course. In addition to the risk incurred by cirrhosis-related immune dysfunction,2 patients with cirrhosis often have comorbid conditions that predispose to severe COVID-19, such as diabetes, chronic kidney disease, and heart disease.

Lipid droplets (LDs) are reservoirs of arachidonoyl lipids for prostaglandin (PG) E2 synthesis, and progesterone can stimulate PGE2 synthesis; however, the relationship between progesterone and LD metabolism in the murine cervix remains unclear. In the present study we examined LD distribution and changes in the expression of proteins involved in lipolysis and autophagy in the murine cervix during pregnancy, and compared the findings with those in dioestrous mice. During mid-pregnancy, LDs were predominantly distributed in the cervical epithelium. Electron microscopy revealed the transfer of numerous LDs from the basal to apical region in the luminal epithelium, marked catabolism of LDs, an elevated number of LDs and autophagosomes and a higher LD:mitochondrion size ratio in murine cervical epithelial cells (P<0.05). In addition, immunohistochemical and western blotting analyses showed significantly higher cAMP-dependent protein kinase, adipose triglyceride lipase and hormone-sensitive lipase expression, and a higher light chain 3 (LC3) II:LC3I ratio in the stroma and smooth muscles and, particularly, in murine cervical epithelial cells, during mid-pregnancy than late dioestrus. In conclusion, these results suggest that the enhanced lipolysis of LDs and autophagy in murine cervical tissues were closely related to pregnancy and were possibly controlled by progesterone because LD catabolism may be necessary for energy provision and PGE2 synthesis to maintain a closed pregnant cervix.

Animal epidemic diseases caused by RNA viruses are the primary threat to the livestock industry, and understanding the mechanisms of RNA virus clearance from target cells is critical to establish an effective method to reduce economic losses. As an SF-1, ATP-dependent RNA helicase in the UPF1p family, MOV10 participates in the RNA degradation of multiple viruses mediated via miRNA pathways and therefore contributes to a decrease in the replication of RNA viruses. This review primarily focuses on the bioactivity of MOV10, the mechanism of RNA virus removal, and the potential roles of MOV10 in RNA virus clearance. In addition, clues are provided to reduce animal diseases caused by RNA viruses.

Staphylococcus aureus ( S. aureus ), a common mastitis pathogen widespread in the natural environment of dairy farms, is capable of invading mammary epithelial cells making treatment difficult. However, the mechanism of the response of bovine mammary epithelial cell to S. aureus invasion remains elusive. In this study, transcriptomic analysis and bioinformatics tools were applied to explore the differentially expressed RNAs in bovine mammary epithelial cells (bMECs) between the control and S. aureus -treated group. A total of 259 differentially expressed mRNAs (DEmRNAs), 27 differentially expressed microRNAs (DEmiRNAs), and 21 differentially expressed long non-coding RNAs (DElncRNAs) were found. These RNAs mainly enrich the inflammatory response, immune response, endocytosis, and cytokine-cytokine receptor interaction. qRT-PCR was used to analyze the quality of the RNA-seq results. In particular, to the defense mechanism of bovine mammary epithelial cells against intracellular S. aureus , the PPAR signaling pathway and the genes (ACOX2, CROT, and NUDT12) were found to be up-regulated to promote the production of peroxisomes and ROS, DRAM1 expression was also up-regulated to facilitate the activation of autophagy, indicating that the above mechanisms were involved in the elimination of intracellular S. aureus in bovine mammary epithelial cells.

Mammary epithelial cells are widely used as models in mastitis research and as tools for mammalian bioreactors; however, the short lifespan of these cells limits their utility. Several mammal epithelial cell line models have been established; however, the secretion capacity and the bacterial sensitivity of these lines have not been effectively evaluated. In this study, a stable immortalized goat mammary epithelial cell (GMEC) line was constructed by transfection with the SV40 gene. The monoclonal cells were then passaged through more than 50 generations after puromycin selection. The GMEC line was evaluated by reverse transcriptase polymerase chain reaction, the cell cycle, karyotype analysis, detection of apoptosis, Western blotting, and β-casein (CSN2) inducible assays. The GMEC line had a strong proliferation capacity relative to the primary GMECs. GMECs had the same karyotype as the primary cells. The GMEC lines maintained basic biological properties and had estrogen, prolactin, and progesterone receptors as same the primary cells. Additionally, the cells and the cell line could synthesize and secrete β-casein proteins. Finally, the rate of apoptosis of the transfected cells suggested that the cell line could provide a useful tool for signal research and mammary gland bioreactors.

Abstract Background: Mastitis results in great economic loss to the dairy goat industry. Many approaches have attempted to decrease the morbidity associated with this disease, and among these, transgenic strategies have been recognized as a crucial development. A previous mammalian study reports that interferon-gamma (IFN-γ) has potential anti-bacterial bioactivity against infection in vitro; however, its capacity in vivo is ambiguous. Results: In this study, we first constructed targeting and homologous recombination vectors (containing the IFN-γ gene) and then transferred the vectors into goat mammary gland epithelial cells (GMECs). Enzyme digestion and sequencing analysis indicated that the vectors used in this study were built correctly. Subsequently, monoclonal cells were selected using puromycin and the polymerase chain reaction (PCR) test indicated that IFN-γ was correctly inserted downstream of the casein promoter. Monoclonal cells were then assessed for reducible expression, and reverse transcriptase-PCR (RT-PCR) and Western blot tests confirmed that monoclonal cells could express IFN-γ. Finally, anti-bacterial capacity was evaluated using bacterial counts and flow cytometry analysis. Decreased bacterial counts and cell apoptosis rates in transgenic GMECs demonstrated that the secretion of IFN-γ could inhibit bacterial proliferation and reduce the risk of mammary infection in goats. Conclusions: Secretion of IFN-γ could inhibit bacterial proliferation and reduce the risk of mammary infection in goats.

PRRSV is an infectious illness causing lung injury and abortion in sows. Cells apoptosis in the interface between the endometrium and fetal placenta is a crucial factor causing abortion. Previous study confirmed PRRSV could cause apoptosis of macrophages but rarely produced an obvious change in porcine endometrial epithelial cells (PECs). Recently, PRRSV-induced abortion was attributed to fetal placental and endometrium epithelial cells (Sn+ and CD163+) apoptosis. However, the mechanism of abortion is still unrevealed because of the limit of porcine endometrium epithelial cells (PEC). The aim of this study was to establish a stable immortalized PECs lines and use it to reveal the abortion mechanism. In this study, highly purified primary PECs were harvested through differential digestion, and their characteristics were confirmed by CK18, ERɑ and PR staining. Cells were then immortalized by transfecting a lentiviral vector that expressed SV40 large T antigen. PECs lines were obtained after puromycin screening. Proliferation of cell line was evaluated by cell growth curve and cell cycle assays. Cell lines exhibited faster proliferation capacity than primary cells. Biological characteristics of cell line were assessed by Western blot, karyotype analysis and staining, which confirmed that the cell line retained the endometrium characteristics. Finally, PRRSV sensitivity was assessed; expression of Sn and CD163 indicated that primary PECs and cell lines were all potentially sensitive to PRRSV. PRRSV infection tests showed an obvious increase in apoptotic rate in the infected PEC cell line, which suggested its susceptibility. The newly constructed cell line is a useful tool for studying the mechanism of abortion caused by PRRSV.

Porcine reproductive and respiratory syndrome virus (PRRSV) can cause respiratory disease and reproductive failure in pregnant pigs. Previous transcriptome analyses in susceptive cells have mainly concentrated on pulmonary alveolar macrophages (PAM) and Marc-145 cells, and on the respiratory system. Some studies reported that apoptosis of placental cells and pig endometrial epithelial cells (PECs) is an obvious sign linked to reproductive failure in pregnant sows, but the mechanism is still unknown. In this study, Sn-positive PECs were isolated and apoptosis rates were assessed by flow cytometry. PRRSV-infected PECs exhibited apoptosis, indicative of their susceptibility to PRRSV. Subsequently, the whole transcriptome was compared between mock- and PRRSV-infected PECs and 54 differentially expressed microRNAs (DEmiRNAs), 104 differentially expressed genes (DEGs), 22 differentially expressed lncRNAs (DElncRNAs), and 109 isoforms were obtained, which were mainly enriched in apoptosis, necroptosis, and p53 signal pathways. Integration analysis of DEmiRNA and DEG profiles revealed two microRNAs (ssc-miR-339-5p and ssc-miR-181d-5p) and five genes (SLA-DQB1, THBS1, SLC3A1, ZFP37, and LOC100517161) participating in the apoptosis signal, of which THBS1 and SLC3A1 were mainly linked to the p53 pathway. Integration analysis of DEGs with DElncRNA profiles identified genes involved in apoptosis signal pathway are regulated by LTCONS_00010766 and LTCONS_00045988. Pathway enrichment revealed that the phagosome and p53 pathways are the two main signals causing apoptosis of PECs, and functional analysis revealed a role of miR-339-5p in regulating apoptosis of PECs after PRRSV inoculation.

Black swimming dots (BSDs) are extremely tiny dot-like contaminants found in dishes of cultured cells. They are harmful to cells and very hard to be removed. BSDs contamination has always been a worldwide unsolved conundrum. Now almost nothing is known about BSDs. It is generally accepted that BSDs come from serum, so speculations that BSDs are serum precipitates or some kind of unknown protozoa parasitizing in bovine serum are popular. And there are also some other proposed viewpoints to explain the identity of BSDs: cell debris, mycoplasma, Achromobacter, nanobacteria, nonliving calcified nanoparticles such as hydroxyapatite or CaCO3. Here we show the above speculations are incorrect or incomplete with firm empirical evidence. We demonstrate BSDs possess biotic-abiotic duality. Namely, BSDs per se are nonliving inorganic nanoparticles yet should derive from an unidentified airborne infectious organism. Therefore, we suggest that future investigations should focus on both the cells and the dots for the final identification of BSDs pathogen. We also present a new method of observing the perivitelline space of MII oocytes to screen out BSDs infected animals. This method can accurately tell whether the donor cattle for FBS production or the animals for primary cells isolation carry BSDs. Thus it is of great scientific and economic significance for the credibility and reproducibility of basic research and serum production enterprise. Moreover, we propose some criteria for judging whether tiny-black-dots are definitely BSDs or other BSDs-like-but-not-be contaminants when they are observed in cell or animal samples.

Bovine tuberculosis is a disease caused by Mycobacterium bovis (M. bovis) that leads to great economic losses in cattle production. The discovery of a reasonable bioagent to reduce M. bovis infection risk and environment contamination becomes significant and urgent. Previous study reported that human β-defensin-3 (HBD3) participated in Mycobacterial immunity and was recognized as a suitable candidate reagent. However, its minimal inhibitory concentration to M. bovis is not yet reported. In this study, we first purified HBD3 protein by recombinant-DNA technology and prokaryotic expression system. Subsequently, anti-bacterial tests were used to evaluate the basic bioactivity of the protein. Results revealed that recombinant HBD3 (rHBD3) protein inhibits Staphylococcus multiplication but not the host Escherichia coli. The growth curve of M. bovis showed that rHBD3 protein controls the proliferation of M. bovis in 20 μg/ml concentration. In addition, rHBD3 protein-incubated M. bovis exhibited reduced infectivity to alveolar epithelial cells and macrophages. In conclusion, the expression of rHBD3 protein is a potential ideal bio-regent for reducing M. bovis infection.

The Jining Gray goat is famous for its sexual precocity; however, the exact regulatory mechanism is still unknown. The hypothalamus is the key centrum in the process of animal reproduction, especially in signal transduction, and the initiation of puberty. The identification of potential genes and pathways in the hypothalamus of Jining Gray goat is critical to understanding the regulatory mechanism of sexual precocity in these goats. In this study, mRNA transcriptome analysis of the hypothalamus of juvenile and pubertal goats revealed eight genes (NTS, ADORA1, CRH, UCN3, E2F2, PDGFRB, GNRH1, and CACNA1C) and three pathways [neuroactive ligand-receptor interaction; gonadotropin-releasing hormone (GnRH) signal; melanoma] that are involved in this regulation. Subsequent methylation analysis on differentially methylated region (DMR) genes revealed the potential regulation network that influences pubertal onset. Correlation analysis verified the methylation level of some DMR genes correlates negatively with expression level. Integrated analysis between transcriptomes and methylomes identified 80 candidate genes involved in GnRH and neuroactive ligand signal pathways, of which CACNA1C and CRH were differentially expressed genes (DEGs) influenced by methylation level. The GnRH gene was the only DEG not affected by its methylation level. In summary, in this study, we identified eight genes and three pathways that are related to pubertal onset in Jining Gray goats, and the expression of CACNA1C and CRH genes of the GnRH and neuroactive ligand signal pathways were influenced by DNA methylation, while that of the GnRH gene was not affected.

Bovine tuberculosis results from infection with Mycobacterium bovis, a member of the Mycobacterium tuberculosis family. Worldwide, M. bovis infections result in economic losses in the livestock industry; cattle production is especially hard‐hit by this disease. Generating M. bovis‐resistant cattle may potentially mitigate the impact of this disease by reducing M. bovis infections. In this study, we used transgenic somatic cell nuclear transfer to generate cattle expressing the gene encoding human β‐defensin 3 (HBD3), which confers resistance to mycobacteria in vitro. We first generated alveolar epithelial cells expressing HBD3 under the control of the bovine MUC1 promoter, and confirmed that these cells secreted HBD3 and possessed anti‐mycobacterial capacity. We then generated and identified transgenic cattle by somatic cell nuclear transfer. The cleavage and blastocyst formation rates of genetically modified embryos provided evidence that monoclonal transgenic bovine fetal fibroblast cells have an integral reprogramming ability that is similar to that of normal cells. Five genetically modified cows were generated, and their anti‐mycobacterial capacities were evaluated. Alveolar epithelial cells and macrophages from these cattle expressed higher levels of HBD3 protein compared with non‐transgenic cells and possessed effective anti‐mycobacterial capacity. These results suggest that the overall risk of M. bovis infection in transgenic cattle is efficiently reduced, and support the development of genetically modified animals as an effective tool to reduce M. bovis infection.

The development of a bacteria-inducible expression system has several advantages compared with persistent expression of anti-bacterial proteins in milk to prevent and treat mastitis. The present study determined whether mastitis responsive promoters could regulate enhanced green fluorescent protein (EGFP) expression in goat mammary epithelial cells (GMECs) in response to challenges with Escherichia coli, Staphylococcus aureus or Streptococcus agalactiae. The level of expression of interleukin (IL)-1 alpha was significantly increased in GMECs challenged with E. coli, S. aureus or S. agalactiae compared with untreated GMECs. IL-1 beta was induced by E. coli and S. aureus, while Toll-like receptor 2 (TLR2) was induced by E. coli only.GMECs were transfected with IL-1 alpha, IL-1 beta and TLR2 promoter-EGFP reporter gene lentiviral expression vectors and the levels of expression of EGFP were measured by flow cytometry and Western blot analysis after bacterial challenge. EGFP expression driven by the IL-1 alpha and IL-1 beta promoters was higher in GMECs challenged with E. coli, S. aureus or S. agalactiae than in untreated GMECs. There were no differences in EGFP expression driven by the TLR2 promoter between GMECs challenged with S. aureus or S. agalactiae and untreated GMECs, but EGFP expression was significantly increased in GMECs challenged with E. coli. Overall, these results indicate that the promoters of some bacteria-inducible genes can regulate EGFP expression in GMECs in response to bacterial challenges. This bacteria-inducible expression strategy could be used for production of mastitis resistant animals by regulating the expression of anti-bacterial proteins in the mammary gland. (C) 2014 Elsevier Ltd. All rights reserved.