Multiplexed genome engineering for porcine fetal fibroblasts with gRNA-tRNA arrays based on CRISPR/Cas9.

Multiplex gene modifications are highly required for various fields of porcine research. In many species, the CRISPR/Cas9 system has been widely applied for genomic editing and provides a potential tool for introducing multiplex genome mutations simultaneously. Here, we present a CRISPR-Cas9 gRNA-tRNA array (GTR-CRISPR) for multiplexed engineering of porcine fetal fibroblasts (PFFs). We successfully produced multiple sgRNAs using only one Pol III promoter by taking advantage of the endogenous tRNA processing mechanism in porcine cells. Using an all-in-one construct carrying GTR and Cas9, we disrupted the IGFBP3, MSTN, MC4R, and SOCS2 genes in multiple codon regions in one PFF cell simultaneously. This technique allows the simultaneous disruption of four genes with 5.5% efficiency. As a result, this approach may effectively target multiple genes at the same time, making it a powerful tool for establishing multiple genes mutant cells in pigs.

Multiplexed Gene Engineering Based on dCas9 and gRNA-tRNA Array Encoded on Single Transcript.

Simultaneously, multiplexed genome engineering and targeting multiple genomic loci are valuable to elucidating gene interactions and characterizing genetic networks that affect phenotypes. Here, we developed a general CRISPR-based platform to perform four functions and target multiple genome loci encoded in a single transcript. To establish multiple functions for multiple loci targets, we fused four RNA hairpins, MS2, PP7, com and boxB, to stem-loops of gRNA (guide RNA) scaffolds, separately. The RNA-hairpin-binding domains MCP, PCP, Com and λN22 were fused with different functional effectors. These paired combinations of cognate-RNA hairpins and RNA-binding proteins generated the simultaneous, independent regulation of multiple target genes. To ensure that all proteins and RNAs are expressed in one transcript, multiple gRNAs were constructed in a tandemly arrayed tRNA (transfer RNA)-gRNA architecture, and the triplex sequence was cloned between the protein-coding sequences and the tRNA-gRNA array. By leveraging this system, we illustrate the transcriptional activation, transcriptional repression, DNA methylation and DNA demethylation of endogenous targets using up to 16 individual CRISPR gRNAs delivered on a single transcript. This system provides a powerful platform to investigate synthetic biology questions and engineer complex-phenotype medical applications.

Chemical characterization and multifunctional neuroprotective effects of sesquiterpenoid-enriched Inula britannica flowers extract.

Dried flowers of Inula britannica commercially serve as pharmaceutical/nutraceutical herbs in the manufacture of medicinal products and functional tea that has been reported to possess extensive biological property. However, the neuroprotective constituents in I. britannica flowers are not known. In the current study, phytochemicals of sesquiterpenoid-enriched I. britannica flowers extract and their potential multifunctional neuroprotective effects were investigated. Nineteen structurally diverse sesquiterpenoids, including two new sesquiterpenoid dimers, namely, inubritanolides A and B (1, 2), and four new sesquiterpenoid monomers (3-6), namely, 1-O-acetyl-6-O-chloracetylbritannilactone (3), 6-methoxybritannilactone (4), 1-hydroxy-10ß-methoxy-4αH-1,10-secoeudesma-5(6),11(13)-dien-12,8ß-olide (5) and 1-hydroxy-4αH-1,10-secoeudesma-5(6),10(14),11(13)-trien-12,8ß-olide (6), as well as 13 known congeners (7-19) were isolated from this source. The structures of compounds 1-6 were elucidated by 1D- and 2D- NMR and HR-ESI-MS data, and their absolute configurations were discerned by electronic circular dichroism (ECD) data analysis and single crystal X-ray diffraction. Interestingly, inubritannolide A (1) is a new type [4 + 2] Diels-Alder dimer featuring a hepta-membered cycloether skeleton. Most of the compounds showed potential multifunctional neuroprotective effects, including antioxidative, anti-neuroinflammatory, and microglial polarization properties. Specifically, 1 and 6 displayed slight strong neuroprotective potency against different types of neuronal cells mediated by various inducers including H2O2, 6-hydroxydopamine (6-OHDA), and lipopolysaccharide (LPS). Overall, this is the first report on multifunctional neuroprotective effects of sesquiterpenoid-enriched I. britannica flowers extract, which supports its potential pharmaceutical/nutraceutical application in neurodegenerative diseases.

[Study on clinical characteristics of Danshen Chuanxiongqin Injection in treatment of cerebral infarction in real world].

In this study, the clinical characteristics and drug combination rules of Danshen Chuanxiongqin Injection in the treatment of patients with cerebral infarction were analyzed. The inpatient information of 2 857 patients of cerebral infarction treated with Danshen Chuanxiongqin Injection in HIS database of 20 grade Ⅲ class A hospitals in China was collected, and a model was established by description analysis and Apriori algorithm, in order to explore the clinical characteristics and drug combination rules of Danshen Chuan-xiongqin Injection in the treatment of cerebral infarction. The results showed that among patients of cerebral infarction treated with Danshen Chuanxiongqin Injection, 1 727 patients were older than 65 years old, accounting for 69.61%, and 1 610 were males, accounting for 63.59%. Commonly used drugs included lipid-lowering agents, anticoagulant thrombolytic agents, antiplatelet agents, stimulants of brain metabolism, vasodilators and other Western drugs, as well as traditional Chinese medicines, such as blood-activating agents, heat-clearing agents and expectorant agents. The Western medicine with the highest use frequency in combination with Danshen Chuan-xiongqin Injection was aspirin enteric-coated tablets(1 528 cases, 53.48%). The traditional Chinese medicine with the highest use frequency in combination with Danshen Chuanxiongqin Injection was Xingnaojing Injection, with a total of 378 cases, accounting for 13.23%. Among them, the most commonly used Western drugs combined with Danshen Chuanxiongqin Injection were anticoagulant thrombolytic and antiplatelet drugs, with a usage rate as high as 83.48%. In order to further explore the drug combination rules of Danshen Chuanxiongqin Injection, the association analysis of drug combination in patients of cerebral infarction treated with Danshen Chuanxiongqin Injection was carried out. In clinical combination of two Western drugs, Atorvastatin Calcium Capsules+Cerebral Proteolytic Injection were the most common combination, with a support of 27.10%. In clinical combination with 3 Western drugs, Clopidogrel Bisulfate Tablets+Atorvastatin Calcium Capsules+Cerebral Proteolytic Injection were most commonly used, with a support of 15.90%. The results showed that the patients of cerebral infarction treated with Danshen Chuanxiongqin Injection were mainly elderly males, and often complicated with hypertension, coronary heart disease, diabetes and other basic diseases. The clinical application of Danshen Chuanxiongqin Injection was principally in line with the guidelines. In the treatment of cerebral infarction, it was often combined with Western medicine anticoagulant thrombolysis, antiplatelet drugs, traditional Chinese medicine blood-activating and stasis-dissolving prescription and other drugs with similar pharmacological effects, with an auxiliary therapeutic effect on patients of cerebral infarction complicated with other diseases, and can provide guidance for clinical medication.

Recombinant Newcastle disease virus expressing P53 demonstrates promising antitumor efficiency in hepatoma model.

BACKGROUND: Numerous studies have demonstrated that the NDV-mediated gene therapy is a promising new approach for treatment of cancers. P53 plays a vital role in tumor suppression and surveillance. Therefore, we hypothesize that a recombinant NDV expressing P53 would be an ideal agent for the hepatoma therapy. RESULTS: In the essay, the human P53 gene was incorporated into the genome of a lentogenic strain (named rNDV-P53), which did not affect viral replication kinetics and magnitude in HepG2 cells. Compared to the vehicle virus, rNDV-P53 increased cell growth suppressor ratio and early apoptosis by 2 folds, and decreased the mitochondrial membrane potential in HepG2 cells. In vivo studies, treatment with rNDV-P53 reduced tumor volume of tumor-bearing mice by more than 4 folds, tumor weight by more than 5 folds comparing with rNDV. The 120-day survival rate of rNDV-P53-treated mice was 75 %, survival rate of rNDV-treated mice was 12.5 %. TUNEL analysis showed a significant increase in the apoptosis rate in the tumor tissues of rNDV-P53-treated mice than that of rNDV-treated mice. Moreover, serum chemistries revealed an insignificant change of blood urea nitrogen (BUN), creatinine levels, alanine aminotransferase (ALT) and aspartate transaminase (AST) in rNDV-P53-treated group compared to normal mice, suggesting treatment with the recombinant virus was not toxic. CONCLUSION: rNDV-P53 is a potent candidate for carcinoma therapy especially for hepatocarcinoma.

Selection of affinity-improved neutralizing human scFv against HBV PreS1 from CDR3 VH/VL mutant library.

A CDR3 mutant library was constructed from a previously isolated anti-HBV neutralizing Homo sapiens scFv-31 template by random mutant primers PCR. Then the library was displayed on the inner membrane surface in Escherichia coli periplasmic space. Seven scFv clones were isolated from the mutant library through three rounds of screening by flow cytometry. Competition ELISA assay indicates that isolated scFv fragments show more efficient binding ability to HBV PreS1 compared with parental scFv-31. HBV neutralization assay indicated that two clones (scFv-3 and 59) show higher neutralizing activity by blocking the HBV infection to Chang liver cells. Our method provides a new strategy for rapid screening of mutant antibody library for affinity-enhanced scFv clones and the neutralizing scFvs obtained from this study provide a potential alternative of Hepatitis B immune globulin.

Competitive host-guest interaction between ß-cyclodextrin polymer and pyrene-labeled probes for fluorescence analyses.

We developed a novel homogeneous fluorescence analysis based on a novel competitive host-guest interaction (CHGI) mechanism between ß-cyclodextrin polymer (polyß CD) and pyrene-labeled probe for biochemical assay. Pyrene labeling with oligonucleotide strands can be recruited and reside in lipophilic cavities of polyß CD. This altered lipophilic microenvironment provides favored polarity for enhanced quantum efficiencies and extraordinarily increases the luminescence intensity of pyrene. However, with addition of complementary DNA, the pyrene-labeled probe formed double-strand DNA to hinder pyrene from entering the cavities of polyß CD. The release of pyrene from polyß CD, which are followed by fluorescence extinguishing, will provide the clear signal turn-off in the presence of target DNA. We also introduced Exodeoxyribonuclease I (Exo I) and Exodeoxyribonuclease III (Exo III) to improve the sensitivity of this system, and the following product of cleavage reaction, pyrene-nucleotide, could more easily host-guest interact with polyß CD and emit stronger fluorescence than pyrene-labeled probe. In addition, the successful detection of adenosine is also demonstrated by using the similar sensing scheme. Although this scheme might be easily interfered by some biomolecules in the real test sample, it holds promising potential for detecting a broad range of other types of aptamer-binding chemicals and biomolecules.

Multiple amplification detection of microRNA based on the host-guest interaction between ß-cyclodextrin polymer and pyrene.

MicroRNAs (miRNAs) participate in various biological processes during the course of life. The levels of miRNAs can be useful biomarkers for cellular events or cancer diagnosis, thus sensitive and accurate analysis of miRNA expression is crucial for better understanding its functions and the early diagnosis of human disease. Here, we developed a multiple amplification detection method for miRNA based on the host-guest interaction between ß-cyclodextrin polymer and pyrene, which takes advantage of the polymerase-aided strand displacement amplification and λ exonuclease-assisted cyclic enzymatic amplification. The proposed method allowed quantitative detection of miRNA-21 in a dynamic range of 1 pM to 5 nM with a detection limit of 0.3 pM and demonstrated good ability to discriminate the target sequence from the single-base mismatched miRNA sequence. Moreover, the assay was applied successfully in a complex biological matrix. We believe that this proposed sensitive and specific assay has great potential as a quantification method for miRNA detection in biomedical research and clinical diagnosis.

A multiple amplification strategy for nucleic acid detection based on host-guest interaction between the ß-cyclodextrin polymer and pyrene.

A multiple amplification strategy has been developed for nucleic acid detection based on host-guest interaction between the ß-cyclodextrin polymer (ß-CDP) and pyrene. Briefly, the detection system consists of three parts: the polymerase and nicking enzyme-assisted isothermal strand displacement amplification (SDA) activated by a target DNA or microRNA; the exonuclease III-aided cyclic enzymatic amplification (CEA); and the fluorescence enhancement effect based on host-guest interaction between ß-CDP and pyrene. This strategy showed a good positive linear correlation with target DNA concentrations in the range from 75 fM to 1 pM with a detection limit of 41 fM. Significantly, our amplification platform was further validated and evaluated successfully by assaying miRNA-21 in human serum. The proposed assay has great potential as a nucleic acid quantification method for use in biomedical research, clinical analysis and disease diagnostics.

[Research Advances in NLRP3 Inflammasome-related Regulatory Mechanisms].

Inflammasome is one of the pattern recognition receptors whose activation directly relates to the maturity and secretion of proinflammatory cytokines interleukin (IL)-1Β and IL-18. Thus, it plays an important role in the humoral immunity. A growing number of studies have found that inflammasome has a close relationship with the pathogenesis of various diseases including atherosclerosis,diabetes, and gout. However,the activation of the inflammasome and its specific regulatory mechanisms remain not clear. This article reviews the possible regulatory mechanisms of the inflammasome NLRP3 in terms of oxidative stress, endoplasmic reticulum stress,and autophagy reaction.

[Endoplasmic reticulum stress and vascular endothelial cell apoptosis].

Endoplasmic reticulum stress (ERS) is a new pathway of apoptosis following the discovery of death receptor signaling pathway and mitochondrial pathway. By activating the unfolded protein response (UPR), ERS can suspend protein synthesis, restore the endoplasmic reticulum homeostasis, and thus play a protective role for cells; however, if the inducing factors of ERS persist, ERS will continue to trigger C/EBP homologous protein, JNK, caspase, or other pathways to induce apoptosis. In addition, the injury and apoptosis of vascular endothelial cells are key links in various diseases and pathophysiologic processes, and research has also shown that vascular endothelial cell apoptosis is closely related with the ERS. Effective intervention of ERS may restrain apoptosis and protect the vascular endothelium. This article reviews the recent research advances in ERS and its role in vascular endothelial cell apoptosis.

CRISPR Ribonucleoprotein-Mediated Precise Editing of Multiple Genes in Porcine Fibroblasts.

The multi-gene editing porcine cell model can analyze the genetic mechanisms of multiple genes, which is beneficial for accelerating genetic breeding. However, there has been a lack of an effective strategy to simultaneously perform precise multi-gene editing in porcine cells. In this study, we aimed to improve the efficiency of CRISPR RNP-mediated precise gene editing in porcine cells. CRISPR RNP, including Cas9 protein, sgRNA, and ssODN, was used to generate precise nucleotide substitutions by homology-directed repair (HDR) in porcine fetal fibroblasts (PFFs). These components were introduced into PFFs via electroporation, followed by PCR for each target site. To enhance HDR efficacy, small-molecule M3814 and phosphorothioate-modified ssODN were employed. All target DNA samples were sequenced and analyzed, and the efficiencies of different combinations of the CRISPR RNP system in target sites were compared. The results showed that when 2 µM M3814, a small molecule which inhibits NHEJ-mediated repair by blocking DNA-PKs activity, was used, there was no toxicity to PFFs. The CRISPR RNP-mediated HDR efficiency increased 3.62-fold. The combination of CRISPR RNP with 2 µM M3814 and PS-ssODNs achieved an HDR-mediated precision gene modification efficiency of approximately 42.81% in mutated cells, a 6.38-fold increase compared to the control group. Then, we used the optimized CRISPR RNP system to perform simultaneous editing of two and three loci at the INS and RLN3 genes. The results showed that the CRISPR RNP system could simultaneously edit two and three loci. The efficiency of simultaneous editing of two loci was not significantly different from that of single-gene editing compared to the efficiency of single-locus editing. The efficiency of simultaneous precise editing of INS, RLN3 exon 1, and RLN3 exon 2 was 0.29%, 0.24%, and 1.05%, respectively. This study demonstrated that a 2 µM M3814 combination with PS-ssODNs improves the efficacy of CRISPR RNP-mediated precise gene editing and allows for precise editing of up to three genes simultaneously in porcine cells.

Evaluation of the in vitro and in vivo antimicrobial activity of alkaloids prepared from Chelidonium majus L. using MRSA- infected C. elegans as a model host.

Chelidonium majus L. contained alkaloids as its main component, exhibiting various biological activities, particularly antibacterial activity. This study aimed to extract alkaloids from C. majus L. (total alkaloids) and evaluate their antibacterial activity both in vitro and in vivo. Reflux extraction was carried out on C. majus L., and the extract was purified with HPD-600 macroporous resin and 732 cation exchange resin columns. Infection modeling of Caenorhabditis elegans (C. elegans) was established to investigate the impact of Methicillin-resistant Staphylococcus aureus (MRSA) and Methicillin-sensitive Staphylococcus aureus (MSSA) on the motility, longevity, and reactive oxygen species (ROS) levels of wild-type worms (N2 strain). The effects of total alkaloids on longevity and ROS were further evaluated in infected N2 worms. Additionally, the effect of total alkaloids on the stress resistance of C. elegans and the mechanism of action were investigated. By utilizing CB1370, DR26 and CF1038 transgenic strains of C. elegans to identify whether the antibacterial activity of total alkaloids was dependent on DAF-2/DAF-16 pathway. The results showed that total alkaloids exhibited a significant antibacterial activity against both MRSA and MSSA (MIC 31.25 µg/mL). Compared with MSSA, the MRSA exhibited a stronger inhibitory effect on the movement behavior and development of worms, along with faster pathogenicity and unique virulence factors. Total alkaloids also displayed the ability to extend the lifespan of C. elegans under oxidative stress and heat stress, and reduce the expression of ROS. The antibacterial activity of total alkaloids was primarily dependent on the DAF-2/DAF-16 pathway, and the presence of functional DAF-2 was deemed essential in total alkaloids mediated immune response against MRSA. Moreover, the antibacterial and anti-infection effects of total alkaloids were found to be associated with the daf-16 gene fragment.

Improving the Efficiency of CRISPR Ribonucleoprotein-Mediated Precise Gene Editing by Small Molecules in Porcine Fibroblasts.

The aim of this study was to verify whether small molecules can improve the efficiency of precision gene editing using clustered regularly interspaced short palindromic repeats (CRISPR) ribonucleoprotein (RNP) in porcine cells. CRISPR associated 9 (Cas9) protein, small guide RNA (sgRNA), phosphorothioate-modified single-stranded oligonucleotides (ssODN), and different small molecules were used to generate precise nucleotide substitutions at the insulin (INS) gene by homology-directed repair (HDR) in porcine fetal fibroblasts (PFFs). These components were introduced into PFFs via electroporation, followed by polymerase chain reaction (PCR) for the target site. All samples were sequenced and analyzed, and the efficiencies of different small molecules at the target site were compared. The results showed that the optimal concentrations of the small molecules, including L-189, NU7441, SCR7, L755507, RS-1, and Brefeldin A, for in vitro-cultured PFFs' viability were determined. Compared with the control group, the single small molecules including L-189, NU7441, SCR7, L755507, RS-1, and Brefeldin A increased the efficiency of HDR-mediated precise gene editing from 1.71-fold to 2.28-fold, respectively. There are no benefits in using the combination of two small molecules, since none of the combinations improved the precise gene editing efficiency compared to single small molecules. In conclusion, these results suggested that a single small molecule can increase the efficiency of CRISPR RNP-mediated precise gene editing in porcine cells.

OTUD1 promotes isoprenaline- and myocardial infarction-induced heart failure by targeting PDE5A in cardiomyocytes.

Heart failure represents a major cause of death worldwide. Recent research has emphasized the potential role of protein ubiquitination/deubiquitination protein modification in cardiac pathology. Here, we investigate the role of the ovarian tumor deubiquitinase 1 (OTUD1) in isoprenaline (ISO)- and myocardial infarction (MI)-induced heart failure and its molecular mechanism. OTUD1 protein levels were raised markedly in murine cardiomyocytes after MI and ISO treatment. OTUD1 deficiency attenuated myocardial hypertrophy and cardiac dysfunction induced by ISO infusion or MI operation. In vitro, OTUD1 knockdown in neonatal rat ventricular myocytes (NRVMs) attenuated ISO-induced injuries, while OTUD1 overexpression aggravated the pathological changes. Mechanistically, LC-MS/MS and Co-IP studies showed that OTUD1 bound directly to the GAF1 and PDEase domains of PDE5A. OTUD1 was found to reverse K48 ubiquitin chain in PDE5A through cysteine at position 320 of OTUD1, preventing its proteasomal degradation. PDE5A could inactivates the cGMP-PKG-SERCA2a signaling axis which dysregulate the calcium handling in cardiomyocytes, and leading to the cardiomyocyte injuries. In conclusion, OTUD1 promotes heart failure by deubiquitinating and stabilizing PDE5A in cardiomyocytes. These findings have identified PDE5A as a new target of OTUD1 and emphasize the potential of OTUD1 as a target for treating heart failure.

Fibroblast growth factor 21 mitigates lupus nephritis progression via the FGF21/Irgm 1/NLRP3 inflammasome pathway.

As an endocrine cytokine, fibroblast growth factor 21 (FGF21) exhibits anti-inflammatory properties. With the development of lupus nephritis (LN), which is tightly related to pathogenic factors, including inflammation and immune cell dysregulation, we explored the impact of Fibroblast Growth Factor 21 (FGF21) as well as its underlying mechanism. We induced an in vivo LN model using pristane in both wild-type C57BL/6 and FGF21 knockout (FGF21-/-) mice. LN serum obtained from 32-week-old wild-type LN mice was used to stimulate RAW264.7 and human renal tubular epithelial (HK-2) cells to mimic an in vitro LN model. Moreover, our findings revealed that FGF21-/- mice showed more severe kidney injury compared to wild-type mice, as evidenced by increased levels of renal function markers, inflammatory factors, and fibrosis markers. Notably, exogenous administration of FGF21 to wild-type LN mice markedly mitigated these adverse effects. Additionally, we used tandem mass tag (TMT)-based quantitative proteomics to detect differentially expressed proteins following FGF21 treatment. Results indicated that 121 differentially expressed proteins influenced by FGF21 were involved in biological processes such as immune response and complement activation. Significantly upregulated protein Irgm 1, coupled with modulated inflammatory response, appeared to contribute to the beneficial effects of FGF21. Furthermore, Western blot analysis demonstrated that FGF21 upregulated Irgm 1 while inhibiting nucleotide-binding oligomerization domain-like receptors family pyrin domain including 3 (NLRP3) inflammasome expression. Silencing Irgm 1, in turn, reversed FGF21's inhibitory effect on NLRP3 inflammasome. In summary, FGF21 can potentially alleviate pristane-induced lupus nephritis in mice, possibly through the FGF21/Irgm 1/NLRP3 inflammasome pathway.

Oncolytic Newcastle disease virus carrying the IL24 gene exerts antitumor effects by inhibiting tumor growth and vascular sprouting.

The second-leading cause of death, cancer, poses a significant threat to human life. Innovations in cancer therapies are crucial due to limitations in traditional approaches. Newcastle disease virus (NDV), a nonpathogenic oncolytic virus, exhibits multifunctional anticancer properties by selectively infecting, replicating, and eliminating tumor cells. To enhance NDV's antitumor activity, four oncolytic NDV viruses were developed, incorporating IL24 and/or GM-CSF genes at different gene loci using reverse genetics. In vitro experiments revealed that oncolytic NDV virus augmented the antitumor efficacy of the parental virus rClone30, inhibiting tumor cell proliferation, inducing tumor cell fusion, and promoting apoptosis. Moreover, NDV carrying the IL24 gene inhibited microvessel formation in CAM experiments. Evaluation in a mouse model of liver cancer confirmed the therapeutic efficacy of oncolytic NDV viral therapy. Tumors in mice treated with oncolytic NDV virus significantly decreased in size, accompanied by tumor cell detachment and apoptosis evident in pathological sections. Furthermore, oncolytic NDV virus enhanced T cell and dendritic cell production and substantially improved the survival rate of mice with hepatocellular carcinoma, with rClone30-IL24(P/M) demonstrating significant therapeutic effects. This study establishes a basis for utilizing oncolytic NDV virus as an antitumor agent in clinical practice.

Evaluation of the immune responses of biological adjuvant bivalent vaccine with three different insertion modes for ND and IBD.

Infectious bursal disease (IBD) is a widespread problem in the poultry industry, and vaccination is the primary preventive method. However, moderately virulent vaccines may damage the bursa, necessitating the development of a safe and effective vaccine. The Newcastle disease virus (NDV) has been explored as a vector for vaccine development. In this study, reverse genetic technology was used to obtain three recombinant viruses, namely, rClone30-VP2L (P/M)-chGM-CSF (NP), rClone30-chGM-CSF (P/M)-VP2L (NP), and rClone30-VP2L-chGM-CSF (P/M). Animal experiments showed that the three biological adjuvant bivalent vaccines effectively increased anti-NDV and anti-infectious bursal disease virus (IBDV) titres, enhancing both humoral and cellular immune responses in chickens without leading to any harm. Amongst the three biological adjuvant bivalent vaccines, the rClone30-chGM-CSF (P/M)-VP2L (NP) group had higher levels of anti-NDV antibodies at 14 days after the first immunization and stimulated a greater humoral immune response in 7-10 days. While, the rClone30-VP2L (P/M)-chGM-CSF (NP) group was the most effective in producing a higher level of IBDV antibody response. In conclusion, these three vaccines can induce immune responses more rapidly and effectively, streamline production processes, be cost-effective, and provide a new avenue for the development of Newcastle disease (ND) and IBD bivalent vaccines.

Evaluation of prevention and treatment effects of fibroblast growth factor-21 in BLM-induced pulmonary fibrosis.

Pulmonary fibrosis is a progressive and fatal fibrotic lung disease and associated with a high mortality rate. In the study, the prevention and treatment effects of fibroblast growth factor-21 (FGF-21) in bleomycin (BLM)-induced pulmonary fibrosis were investigated in vivo and vitro. In the prevention of pulmonary fibrosis studies, the results showed that interdict of FGF-21 could reduce the related gene and protein expression levels of pulmonary fibrosis. In addition, FGF-21 significantly reduced both the aggregation of inflammatory cells and deposition of collagen in the lung by histopathology. In therapy of pulmonary fibrosis studies, the results indicated that treatment with FGF-21 resulted in an amelioration of the pulmonary fibrosis in mice with reductions of the pathological score, collagen deposition and transforming growth factor (TGF)-ß and α-smooth muscle actin (α-SMA) expressions in the lung tissues at fibrotic stage, and late administration was also able to reduce the degree of pulmonary fibrosis and even better than these in the prevention group. Furthermore, BLM-induced THP-1 macrophage model was verified using FGF-21; the result showed that FGF-21 decreased the related gene expression level of pulmonary fibrosis. FGF-21 may have preventive and therapeutic effects on BLM-induced pulmonary fibrosis via inhibiting myofibroblast differentiation and inflammatory. Thus, FGF-21 represents a potential drug for the prevention and treatment of pulmonary fibrosis.

Development and evaluation of Newcastle disease - avian influenza bivalent vector vaccines in commercial chickens.

Avian influenza (AI) and Newcastle disease (ND) are regarded as the leading viral infectious diseases affecting the global poultry industry. Vaccination is a successful therapeutic intervention to safeguard birds against both ND and AI infections. In this research, ND-AI bivalent vaccines were developed through the incorporation of HA and IRES-GMCSF gene fragments at varying locations of NDV rClone30 vectors. The two constructed vaccines were rClone30-HA-IRES-GMCSF(PM) and rClone30-HA(PM)-IRES-GMCSF(NP). Next, 27-day-old Luhua chickens (the maternal antibody level was reduced to 1.4 log2) were inoculated with the same dose of the vaccines, and humoral and cellular immune responses were assessed at multiple time points. Compared to the commercial vaccine, the levels of anti-NDV antibodies following the administration of the ND-AI vaccines were above the theoretical protection value of 4 log2. The levels of anti-AIV antibodies in the bivalent vaccine group were notably higher than those in the commercial vaccine group. Furthermore, the content of inflammatory factors and transcription levels were significantly increased in chickens administered ND-AI vaccines. The ND-AI vaccines induced stronger proliferative responses of B cells or CD3+, CD8+, and CD4 + T cells. Hematoxylin and eosin staining showed that the tissue damage induced by the two recombinant vaccines was similar to that of commercial vaccines. The outcomes of the study suggest that the two bivalent ND-AI vaccine candidates produced using the reverse genetics approach are both secure and effective. This approach not only enables the multiuse of one vaccine but also provides a new concept for the development of other vaccines against infectious viral diseases.