Assessing immunogenicity of CRISPR-NCas9 engineered strain against porcine epidemic diarrhea virus.

Porcine epidemic diarrhea (PED) caused by porcine epidemic diarrhea virus (PEDV), is an acute and highly infectious disease, resulting in substantial economic losses in the pig industry. Given that PEDV primarily infects the mucosal surfaces of the intestinal tract, it is crucial to improve the mucosal immunity to prevent viral invasion. Lactic acid bacteria (LAB) oral vaccines offer unique advantages and potential applications in combatting mucosal infectious diseases, making them an ideal approach for controlling PED outbreaks. However, traditional LAB oral vaccines use plasmids for exogenous protein expression and antibiotic genes as selection markers. Antibiotic genes can be diffused through transposition, transfer, or homologous recombination, resulting in the generation of drug-resistant strains. To overcome these issues, genome-editing technology has been developed to achieve gene expression in LAB genomes. In this study, we used the CRISPR-NCas9 system to integrate the PEDV S1 gene into the genome of alanine racemase-deficient Lactobacillus paracasei △Alr HLJ-27 (L. paracasei △Alr HLJ-27) at the thymidylate synthase (thyA) site, generating a strain, S1/△Alr HLJ-27. We conducted immunization assays in mice and piglets to evaluate the level of immune response and evaluated its protective effect against PEDV through challenge tests in piglets. Oral administration of the strain S1/△Alr HLJ-27 in mice and piglets elicited mucosal, humoral, and cellular immune responses. The strain also exhibited a certain level of resistance against PEDV infection in piglets. These results demonstrate the potential of S1/△Alr HLJ-27 as an oral vaccine candidate for PEDV control. KEY POINTS: • A strain S1/△Alr HLJ-27 was constructed as the candidate for an oral vaccine. • Immunogenicity response and challenge test was carried out to analyze the ability of the strain. • The strain S1/△Alr HLJ-27 could provide protection for piglets to a certain extent.

Role of oncogenic long noncoding RNA KCNQ1OT1 in colon cancer.

The role of lncRNA KCNQ1 opposite strand/antisense transcript 1 (KCNQ1OT1) in colon cancer involves various tumorigenic processes and has been studied widely. However, the mechanism by which it promotes colon cancer remains unclear. Retroviral vector pSEB61 was retrofitted in established HCT116-siKCN and SW480-siKCN cells to silence KCNQ1OT1. Cellular proliferation was measured using CCK8 assay, and flow cytometry (FCM) detected cell cycle changes. RNA sequencing (RNA-Seq) analysis showed differentially expressed genes (DEGs). Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were carried out to analyze enriched functions and signaling pathways. RT-qPCR, immunofluorescence, and western blotting were carried out to validate downstream gene expressions. The effects of tumorigenesis were evaluated in BALB/c nude mice by tumor xenografts. Our data revealed that the silencing of KCNQ1OT1 in HCT116 and SW480 cells slowed cell growth and decreased the number of cells in the G2/M phase. RNA-Seq analysis showed the data of DEGs enriched in various GO and KEGG pathways such as DNA replication and cell cycle. RT-qPCR, immunofluorescence, and western blotting confirmed downstream CCNE2 and PCNA gene expressions. HCT116-siKCN cells significantly suppressed tumorigenesis in BALB/c nude mice. Our study suggests that lncRNA KCNQ1OT1 may provide a promising therapeutic strategy for colon cancer.

First principles insights into the interaction mechanism of iron doped thermally activated kaolinite with Cd and Pb pollutants in organic solid waste incineration flue gas.

Incineration of organic solid wastes is accompanied by the heavy metal emission through flue gas. As an inexpensive and efficient heavy metal adsorbent, the improvement of kaolinite adsorption performance for heavy metals has drawn widespread interests. In this work, the interaction mechanisms between various kaolinite surfaces and Cd/Pb species are explored through first principles calculations. The results show that the combination of Fe doping and dehydroxylation enhances the activity of kaolinite surfaces, analysis of adsorption configurations reveal that both Cd and Pb species are immobilized through chemisorption on the -H + Fe surface. At the microscopic level, further electronic structure analysis shows that the composite modified kaolinite surface has more electron transfer and more pronounced orbital hybridization and overlap compared to the original kaolinite surface, demonstrating that the modification means of dehydroxylation and Fe doping indeed enhanced the activity of the kaolinite surface, especially the activity of the O atoms in the vicinity of the Fe atom and that the O atoms are more efficiently bonded as ionic connecting Cd/Pb species for the purpose of trapping Cd/Pb species. This study points out the research direction and provides basic theoretical support for the development of new kaolinite adsorbents in the future.

Auxotrophic Lactobacillus Expressing Porcine Rotavirus VP4 Constructed Using CRISPR-Cas9D10A System Induces Effective Immunity in Mice.

Porcine rotavirus (PoRV) mainly causes acute diarrhea in piglets under eight weeks of age and has potentially high morbidity and mortality rates. As vaccine carriers for oral immunization, lactic acid bacteria (LAB) are an ideal strategy for blocking PoRV infections. However, the difficulty in knocking out specific genes, inserting foreign genes, and the residues of antibiotic selection markers are major challenges for the oral vaccination of LAB. In this study, the target gene, alanine racemase (alr), in the genome of Lactobacillus casei strain W56 (L. casei W56) was knocked out to construct an auxotrophic L. casei strain (L. casei Δalr W56) using the CRISPR-Cas9D10A gene editing system. A recombinant strain (pPG-alr-VP4/Δalr W56) was constructed using an electrotransformed complementary plasmid. Expression of the alr-VP4 fusion protein from pPG-alr-VP4/Δalr W56 was detected using Western blotting. Mice orally immunized with pPG-alr-VP4/Δalr W56 exhibited high levels of serum IgG and mucosal secretory immunoglobulin A (SIgA), which exhibited neutralizing effects against PoRV. Cytokines levels in serum detected using ELISA, indicated that the recombinant strain induced an immune response dominated by Th2 cells. Our data suggest that pPG-alr-VP4/Δalr W56, an antibiotic-resistance-free LAB, provides a safer vaccine strategy against PoRV infection.

Evaluation of the Immunogenicity in Mice Orally Immunized with Recombinant Lactobacillus casei Expressing Porcine Epidemic Diarrhea Virus S1 Protein.

Porcine epidemic diarrhea (PED), characterized by diarrhea, vomiting, and dehydration, is an acute enteric infectious disease of pigs. The disease is caused by porcine epidemic diarrhea virus (PEDV), which infects the intestinal mucosal surface. Therefore, mucosal immunization through the oral route is an effective method of immunization. Lactic acid bacteria, which are acid resistant and bile-salt resistant and improve mucosal immunity, are ideal carriers for oral vaccines. The S1 glycoprotein of PEDV mediates binding of the virus with cell receptors and induces neutralizing antibodies against the virus. Therefore, we reversely screened the recombinant strain pPG-SD-S1/Δupp ATCC 393 expressing PEDV S1 glycoprotein by Lactobacillus casei deficient in upp genotype (Δupp ATCC 393). Mice were orally immunized three times with the recombinant bacteria that had been identified for expression, and the changes of anti-PEDV IgG and secreted immunoglobulin A levels were observed over 70 days. The results indicated that the antibody levels notably increased after oral administration of recombinant bacteria. The detection of extracellular cytokines on the 42nd day after immunization indicated high levels of humoral and cellular immune responses in mice. The above results demonstrate that pPG-SD-S1/Δupp ATCC 393 has great potential as an oral vaccine against PEDV.

Characterization of four novel H5N6 avian influenza viruses with the internal genes from H5N1 and H9N2 viruses and experimental challenge of chickens vaccinated with current commercially available H5 vaccines.

Since 2014, highly pathogenic avian influenza H5N6 viruses have been responsible for outbreaks in poultry. In this study, four H5N6 virus strains were isolated from faecal samples of sick white ducks and dead chickens in Shandong in 2019. These H5N6 viruses were triple-reassortant viruses that have not been previously characterized. Their HA genes were derived from the H5 viruses and were closely related to the vaccine strain Re-11. Their NA genes all fell into the N6-like lineage and the internal gene were derived from H5N1 and H9N2 viruses. They all showed high pathogenicity in mice and caused lethal infection with high rates of transmission in chickens. Moreover, the SPF chickens inoculated with the currently used H5 (Re-11 and Re-12 strains)/H7 (H7-Re-2 strain) trivalent inactivated vaccines in China were completely protected from these four H5N6 viruses. Our study indicated the necessity of continued surveillance for H5 influenza A viruses and the importance of timely update of vaccine strains in poultry industry.

Design of a metamaterial film with excellent conformability and adhesion for bandage substrates.

Developing films with excellent conformability and adhesion has become a research hotspot in many fields, such as medical bandages. The conventional method for enhancing films conformability and adhesion is to make the films thinner or modify the material of the films, which usually compromises the function of the films. In this paper, a novel metamaterial film was proposed to cover the skin area of a human elbow during the rotation of elbow. This structure is composed of unit cells with rectangular perforations, whose Poisson's ratio (PR) is determined by the length of the perforation. With finite element analysis (FEA), relations among the stretch strain, Poisson's ratio and length of the perforation of unit cell was obtained. Then, the proposed film was generated by mapping unit cells with different PR to the target skin surface. With the same deformation behavior as the elbow skin, conformability and adhesion of the generated film can be guaranteed during the entire rotation process of the elbow, which has been verified by both FEA and experimental tests. Theoretically, by changing the arrangement of different PR unit cells, the proposed method can be applied to design films for other complex surface on human body. It also provides a new way to introduce materials with better biocompatibility but poor mechanical properties as bandage substrates. As a possible application, a prototype of smart bandage was developed by installing a high-resolution temperature sensor on the proposed film, which can monitor the inflammation of the wounded skin in real time.

Design and Mechanical Properties Verification of Gradient Voronoi Scaffold for Bone Tissue Engineering.

In order to obtain scaffold that can meet the therapeutic effect, researchers have carried out research on irregular porous structures. However, there are deficiencies in the design method of accurately controlling the apparent elastic modulus of the structure at present. Natural bone has a gradient porous structure. However, there are few studies on the mechanical property advantages of gradient bionic bone scaffold. In this paper, an improved method based on Voronoi-tessellation is proposed. The method can get controllable gradient scaffolds to fit the modulus of natural bone, and accurately control the apparent elastic modulus of porous structure, which is conducive to improving the stress shielding. To verify the designed structure can be fabricated by additive manufacturing, several designed models are obtained by SLM and EBM. Through finite element analysis (FEA), it is verified that the irregular porous structure based on Voronoi-tessellation is more stable than the traditional regular porous structure of the same structure volume, the same pore number and the same material. Furthermore, it is verified that the gradient irregular structure has a better stability than the non-gradient structure. An experiment is conducted successfully to verify the stability performance got by FEA. In addition, a dynamic impact FEA is also performed to simulate impact resistance. The result shows that the impact resistance of the regular porous structure, the irregular porous structure and the gradient irregular porous structure becomes better in turn. The mechanical property verification provides a theoretical basis for the structural design of gradient irregular porous bone tissue engineering scaffolds.

Establishment of stable Vero cell lines expressing TMPRSS2 and MSPL: A useful tool for propagating porcine epidemic diarrhea virus in the absence of exogenous trypsin.

Porcine epidemic diarrhea virus (PEDV) is the causative agent of porcine epidemic diarrhea, causing substantial economic losses to the swine industry worldwide. However, the development of PEDV vaccine is hampered by its low propagation titer in vitro, due to difficulty in adapting to the cells and complex culture conditions, even in the presence of trypsin. Furthermore, the frequent variation, recombination, and evolution of PEDV resulted in reemergence and vaccination failure. In this study, we established the Vero/TMPRSS2 and Vero/MSPL cell lines, constitutively expressing type II transmembrane serine protease TMPRSS2 and MSPL, in order to increase the stability and titer of PEDV culture and isolation in vitro. Our study revealed that the Vero/TMPRSS2, especially Vero/MSPL cell lines, can effectively facilitate the titer and multicycle replication of cell-adapted PEDV in the absence of exogenous trypsin, by cleaving and activating PEDV S protein. Furthermore, our results also highlighted that Vero/TMPRSS2 and Vero/MSPL cells can significantly enhance the isolation of PEDV from the clinical tissue samples as well as promote viral infection and replication by cell-cell fusion. The successful construction of the Vero/TMPRSS2 and Vero/MSPL cell lines provides a useful approach for the isolation and propagation of PEDV, simplification of virus culture, and large-scale production of industrial vaccine, and the cell lines are also an important system to research PEDV S protein cleaved by host protease.

Catalyst- and solvent-free approach to 2-arylated quinolines via [5 + 1] annulation of 2-methylquinolines with diynones.

A novel route for the synthesis of 2-arylated quinolines through a [5 + 1] annulation directly from 2-methylquinolines and diynones under catalyst-free and solvent-free conditions was disclosed. This synthetic process was atom-economic, with good tolerance of a broad range of functional groups, and with great practical worth.

Catalyst-Free Synthesis of Pyrrolo[1,2-a]quinolines via Dehydration/[3 + 2] Cycloaddition Directly from 2-Methylquinolines, Aldehydes, and Alkynoates.

A simple and efficient catalyst-free synthesis of pyrrolo[1,2-a]quinoline derivatives from 2-methylquinolines, aldehydes, and alkynoates via dehydration/[3 + 2] cycloaddition has been developed. The reaction conditions are tolerant to air, and H2O is the only byproduct of this transformation, thus offering an environmentally benign process with a wide range of potential applications in organic synthesis and medicinal chemistry.

Novel Approach for Isolation and Identification of Porcine Epidemic Diarrhea Virus (PEDV) Strain NJ Using Porcine Intestinal Epithelial Cells.

Porcine epidemic diarrhea virus (PEDV), which is the causative agent of porcine epidemic diarrhea in China and other countries, is responsible for serious economic losses in the pork industry. Inactivated PEDV vaccine plays a key role in controlling the prevalence of PEDV. However, consistently low viral titers are obtained during the propagation of PEDV in vitro; this represents a challenge to molecular analyses of the virus and vaccine development. In this study, we successfully isolated a PEDV isolate (strain NJ) from clinical samples collected during a recent outbreak of diarrhea in piglets in China, using porcine intestinal epithelial cells (IEC). We found that the isolate was better adapted to growth in IECs than in Vero cells, and the titer of the IEC cultures was 104.5 TCID50/0.1 mL at passage 45. Mutations in the S protein increased with the viral passage and the mutations tended towards attenuation. Viral challenge showed that the survival of IEC-adapted cultures was higher at the 45th passage than at the 5th passage. The use of IECs to isolate and propagate PEDV provides an effective approach for laboratory-based diagnosis of PEDV, as well as studies of the epidemiological characteristics and molecular biology of this virus.

Nephroprotective effect of astaxanthin against trivalent inorganic arsenic-induced renal injury in wistar rats.

Inorganic arsenic (iAs) is a toxic metalloid found ubiquitously in the environment. In humans, exposure to iAs can result in toxicity and cause toxicological manifestations. Arsenic trioxide (As2O3) has been used in the treatment for acute promyelocytic leukemia. The kidney is the critical target organ of trivalent inorganic As (iAs(III)) toxicity. We examine if oral administration of astaxanthin (AST) has protective effects on nephrotoxicity and oxidative stress induced by As2O3 exposure (via intraperitoneal injection) in rats. Markers of renal function, histopathological changes, Na(+)-K(+) ATPase, sulfydryl, oxidative stress, and As accumulation in kidneys were evaluated as indicators of As2O3 exposure. AST showed a significant protective effect against As2O3-induced nephrotoxicity. These results suggest that the mechanisms of action, by which AST reduces nephrotoxicity, may include antioxidant protection against oxidative injury and reduction of As accumulation. These findings might be of therapeutic benefit in humans or animals suffering from exposure to iAs(III) from natural sources or cancer therapy.