A novel CpG ODN compound adjuvant enhances immune response to spike subunit vaccines of porcine epidemic diarrhea virus

CpG oligodeoxynucleotides (CpG ODNs) boost the humoral and cellular immune responses to antigens through interaction with Toll-like receptor 9 (TLR9). These CpG ODNs have been extensively utilized in human vaccines. In our study, we evaluated five B-type CpG ODNs that have stimulatory effects on pigs by measuring the proliferation of porcine peripheral blood mononuclear cells (PBMCs) and assessing interferon gamma (IFN-γ) secretion. Furthermore, this study examined the immunoenhancing effects of the MF59 and CpG ODNs compound adjuvant in mouse and piglet models of porcine epidemic diarrhea virus (PEDV) subunit vaccine administration. The in vitro screening revealed that the CpG ODN named CpG5 significantly stimulated the proliferation of porcine PBMCs and elevated IFN-γ secretion levels. In the mouse vaccination model, CpG5 compound adjuvant significantly bolstered the humoral and cellular immune responses to the PEDV subunit vaccines, leading to Th1 immune responses characterized by increased IFN-γ and IgG2a levels. In piglets, the neutralizing antibody titer was significantly enhanced with CpG5 compound adjuvant, alongside a considerable increase in CD8+ T lymphocytes proportion. The combination of MF59 adjuvant and CpG5 exhibits a synergistic effect, resulting in an earlier, more intense, and long-lasting immune response in subunit vaccines for PEDV. This combination holds significant promise as a robust candidate for the development of vaccine adjuvant

Construction of a camelid VHH yeast two-hybrid library and the selection of VHH against haemagglutinin-neuraminidase protein of the Newcastle disease virus

Humoral immune response after immunization. Sera from IIama was collected, two-fold diluted and tested by HI using LaSota as antigen. Figure S1 Amplification of VHH through a nested PCR. (A) First round PCR to separate VH from VHH. The upper 900 bp bands represent the VH-CH1-Hinge-CH2 of conventional Abs (lane 1–8). The lower 600 bp bands represent the VHH-Hinge-CH2 of HCAbs (lane 1–8). (B) VHH amplified through nested PCR using 600 bp fragment recovered from first round PCR as template (lane 1–4). M in A and B was the DL2000 DNA marker. C in A and B represent the negative control. Figure S2 PCR identification of inserted VHH. 47 clones were randomly picked to determine the library functional diversity by PCR using universal primers T7 and 3’AD (Table 1). Meanwhile, Sterile water was used as negative controls. 45 clones have amplified the 500 bp VHH fragments (lane 1–47), while negative templates control haven’t amplified any bands (lane C). M indicated the DL2000 DNA marker. Figure S3 Detection of library capacity and library titer. (A) 10-3 dilution plating of the transformed cells calculated a library capacity of 1.25 × 107 independent clones. (B) 10-5 dilution plating of the cultured library indicated a library titer of 3.45 × 108 cfu/mL. Figure S4 Deduced amino acid aligment of 10 random picked VHH. Deduced amino acid sequences were analyzed according to the Kabat numbering. Differences in the sequences are pinked, and the dash represent the missing sequences. Two hallmark Cys residues are labeled by the thick-line boxes. The four conservative hallmark residues of VHH in FR2 are labeled by the dotted line boxes. Figure S5 pGBKT7-HN bait plasmid construction. (A) PCR was carried out to amplify a truncate HN gene (without transmembrane region) from La Sota strain. M, 5000 DNA marker. 1, Truncate HN. C, Negative control. (B) A truncate HN was cloned into pGBKT7 through BamH I and Sal I. M, 5000 DNA marker. 1, Double restriction enzyme digestion of pGBKT7-HN. Figure S6 pHSIE-VHH plasmid construction. (A) 7 positive VHH fragment were amplified from recovered positive clones containing pGADT7-VHH by PCR. M, 5000 DNA marker. 1–7, VHH 1–7. C, Negative control. (B) Double restriction enzyme digestion of pHSIE-VHHs. M, 5000 DNA marker. 1–7, pHSIE-VHH 1–7. Figure S7 Western blot analysis of bait protein expression. 2 mL of Y2HGold(pGBKT7-HN) culture liquid was extracted using yeast protein extraction reagent (Takara). c-Myc tag monoclonal antibody (1:4000 dilution) was used as first antibody and HRP-labeled goat anti-mouse antibody (1:5000) was used as second antibody. The immunoreactive was visualized with cECL Plus Western blotting detection reagent (CWBIO). (DOC 1129 kb

Quantitative MRI evaluation of articular cartilage in patients with meniscus tear

PurposeThe aim of this study was to assess quantitatively articular cartilage volume, thickness, and T2 value alterations in meniscus tear patients.Materials and methodsThe study included 32 patients with meniscus tears (17 females, 15 males; mean age: 40.16 ± 11.85 years) and 24 healthy controls (12 females; 12 males; mean age: 36 ± 9.14 years). All subjects were examined by 3 T magnetic resonance imaging (MRI) with 3D dual-echo steady-state (DESS) and T2 mapping images. All patients underwent diagnostic arthroscopy and treatment. Cartilage thickness, cartilage volume and T2 values of 21 subregions of knee cartilage were measured using the prototype KneeCaP software (version 2.1; Siemens Healthcare, Erlangen, Germany). Mann-Whitney-U tests were utilized to determine if there were any significant differences among subregional articular cartilage volume, thickness and T2 value between patients with meniscus tear and the control group.ResultsThe articular cartilage T2 values in all subregions of the femur and tibia in the meniscus tear group were significantly higher (p< 0.05) than in the healthy control group. The cartilage thickness of the femoral condyle medial, femur trochlea, femur condyle lateral central, tibia plateau medial anterior and patella facet medial inferior in the meniscus tear group were slightly higher than in the control group (p< 0.05). In the femur trochlea medial, patella facet medial inferior, tibia plateau lateral posterior and tibia plateau lateral central, there were significant differences in relative cartilage volume percentage between the meniscus tear group and the healthy control group (p< 0.05). Nineteen patients had no cartilage abnormalities (Grade 0) in the meniscus tear group, as confirmed by arthroscopic surgery, and their T2 values in most subregions were significantly higher (p< 0.05) than those of the healthy control group.ConclusionThe difference in articular cartilage indexes between patients with meniscus tears and healthy people without such tears can be detected by using quantitative MRI. Quantitative T2 values enable early and sensitive detection of early cartilage lesions

Rapid Construction of an Infectious Clone of Fowl Adenovirus Serotype 4 Isolate

Adenovirus vectors possess a good safety profile, an extensive genome, a range of host cells, high viral yield, and the ability to elicit broad humoral and cellular immune responses. Adenovirus vectors are widely used in infectious disease research for future vaccine development and gene therapy. In this study, we obtained a fowl adenovirus serotype 4 (FAdV-4) isolate from sick chickens with hepatitis–hydropericardium syndrome (HHS) and conducted animal regression text to clarify biological pathology. We amplified the transfer vector and extracted viral genomic DNA from infected LMH cells, then recombined the mixtures via the Gibson assembly method in vitro and electroporated them into EZ10 competent cells to construct the FAdV-4 infectious clone. The infectious clones were successfully rescued in LMH cells within 15 days of transfection. The typical cytopathic effect (CPE) and propagation titer of FAdV-4 infectious clones were also similar to those for wild-type FAdV-4. To further construct the single-cycle adenovirus (SC-Ad) vector, we constructed SC-Ad vectors by deleting the gene for IIIa capsid cement protein. The FAdV4 infectious clone vector was introduced into the ccdB cm expression cassette to replace the IIIa gene using a λ-red homologous recombination technique, and then the ccdB cm expression cassette was excised by PmeI digestion and self-ligation to obtain the resulting plasmids as SC-Ad vectors

A Study of Neutron Leakage in Finite Objects

Citation: Wilson JW, Slaba TC, Badavi FF, Reddell BD, and Bahadori AA. 2015. A Study of Neutron Leakage in Finite Objects NASA/TP-2015-218692 NASA Langley Research Center: Hampton, VA http://ntrs.nasa.gov/search.jsp?R=20150002822A computationally efficient 3DHZETRN code capable of simulating High charge (Z) and Energy (HZE) and light ions (including neutrons) under space-like boundary conditions with enhanced neutron and light ion propagation was recently developed for simple shielded objects. Monte Carlo (MC) benchmarks were used to verify the 3DHZETRN methodology in slab and spherical geometry, and it was shown that 3DHZETRN agrees with MC codes to the degree that various MC codes agree among themselves. One limitation in the verification process is that all of the codes (3DHZETRN and three MC codes) utilize different nuclear models/databases. In the present report, the new algorithm, with well-defined convergence criteria, is used to quantify the neutron leakage from simple geometries to provide means of verifying 3D effects and to provide guidance for further code development

Melatonin Relieves Busulfan-Induced Spermatogonial Stem Cell Apoptosis of Mouse Testis by Inhibiting Endoplasmic Reticulum Stress

Background/Aims: Busulfan is commonly used for cancer chemotherapy. Although it has the advantage of increasing the survival rate of patients, it can cause male infertility via damaging the testes and reducing sperm counts. Therefore, the underlying mechanism should be explored, and new agents should be developed to protect the male reproductive system from busulfan-induced damage. Endoplasmic reticulum stress (ERS) is considered a key contributor to numerous pathologies. Despite several studies linking ERS to toxicants, studies have yet to determine whether ERS is a contributing factor to busulfan-induced testicular damage. Melatonin is a well-known broad-spectrum antioxidant, anti-inflammatory and antitumour agent, but the effects of melatonin on busulfan-induced ERS in mouse testes damage are less documented. Methods: The effects of melatonin were measured by immunofluorescence staining, Western blot, qRT-PCR analysis and flow cytometry assay. The underlying mechanism was investigated by measuring ERS. Results: We found that ERS was strongly activated in mouse testes (in vivo) and the C18-4 cell line (in vitro) after busulfan administration. ERS-related apoptosis proteins such as caspase-12, CHOP and caspase-3 were activated, and the expression of apoptotic proteins such as P53 and PUMA were upregulated. Furthermore, we investigated whether melatonin reduced the extent of damage to mouse testes and improved the survival rates of busulfan-treated mice. When exploring the underlying mechanisms, we found melatonin could counteract ERS by decreasing the expression levels of the ERS markers GRP78, ATF6, pIRE1 and XBP1 in mouse testes and mouse SSCs (C18-4 cells). Moreover, it blocked the activation of ERS-related apoptosis proteins caspase-12, CHOP and caspase-3 and suppressed P53 and PUMA expression stimulated by busulfan both in vivo and in vitro. Conclusion: Our results demonstrate that ERS is an important mediator for busulfan-induced apoptosis. The attenuation of ERS by melatonin can prevent busulfan-treated SSCs apoptosis and protect busulfan-treated testes from damage. Thus, this study suggests that melatonin may alleviate the side effects of busulfan for male patients during clinical treatment

Restriction enzyme analysis of the recombinants drivered from the modified SLiCE cloning.

<p>After lethal CcdB gene screening on kanamycin plates, 48 randomly selected clones for the recombinant PAdV-3 vectors expressing each reporter gene were identified by <i>Hind</i> III digestion analysis. Lanes 1–20 of each group are the <i>Hind</i> III digestion of 20 clones among 48 selected clones. Lane Con is the <i>Hind</i> III digestion of the pFPAV3-CcdB-Cm parent vector which was used as the negative control for recombinant PAdV-3 vectors identification. Marker, DL15000.</p

A Novel and Simple Method for Rapid Generation of Recombinant Porcine Adenoviral Vectors for Transgene Expression

<div><p>Many human (different serotypes) and nonhuman adenovirus vectors are being used for gene delivery. However, the current system for isolating recombinant adenoviral vectors is either time-consuming or expensive, especially for the generation of recombinant non-human adenoviral vectors. We herein report a new and simple cloning approach for the rapid generation of a porcine adenovirus (PAdV-3) vector which shows promise for gene transfer to human cells and evasion of human adenovirus type 5 (HAdV-5) immunity. Based on the final cloning plasmid, pFPAV3-CcdB-Cm, and our modified SLiCE strategy (SLiCE cloning and lethal CcdB screening), the process for generating recombinant PAdV-3 plasmids required only one step in 3 days, with a cloning efficiency as high as 620±49.56 clones/ng and zero background (100% accuracy). The recombinant PAdV-3 plasmids could be successfully rescued in porcine retinal pigment epithelium cells (VR1BL), which constitutively express the HAdV-5 E1 and PAdV-3 E1B 55k genes, and the foreign genes were highly expressed at 24 h after transduction into swine testicle (ST) cells. In conclusion, this strategy for generating recombinant PAdV-3 vectors based on our modified SLiCE cloning system was rapid and cost-efficient, which could be used as universal cloning method for modification the other regions of PAdV-3 genome as well as other adenoviral genomes.</p></div