Background: Cone-beam computed tomography is being utilized in more clinical contexts and determining bone density with this method is becoming more important. Dentists, particularly dentomaxillofacial radiologists, orthodontists, and oral surgeons, must have a solid understanding of gray value. The gray values acquired from conebeam computed tomography images are used to assess dental implant bone density, diagnose dental ankylosis, and diagnose and differentiate pathological lesions. Objective: To determine the difference in the gray value of the trabecular bone in the impacted and normal erupted maxillary canine teeth using cone computed tomography. Methods: We retrospectively evaluated the cone-beam computed tomography images of patients scheduled for orthodontic treatment at the Universitas Airlangga Dental and Oral Hospital. On cross-sectional cone-beam computed tomography images, the region of interest determination of 5 mm2 in the area was placed in the trabecular bone and the gray value measurements were collected using Digital Imaging and Communications in Medicine (OnDemand3D™) dental software. The images were categorized by type of impacted canine teeth after assessing the gray values of all the teeth. Using images on the mesial, distal, buccal, and palatal areas, gray values of impacted and non-impacted teeth were compared. We used the SPSS 24 software. Results: From a total of 13 patient radiographs, we found types I (6/13), II (6/13), and VII (1/13). The mean pixel values of impacted maxillary unilateral canine teeth were 1972.92 (mesial), 2016.55 (distal), 1990.66 (buccal), and 1904.39 (palatal). The mean pixel values of normal erupted maxillary canines were 1754.93 (mesial), 1710.53 (distal), 1852.94 (buccal), and 1674.49 (palatal). There were significant differences between impacted and normal erupted maxillary canines: mesial (P = 0.018), distal (P = 0.000), buccal (P = 0.003), and palatal (P = 0.036). Conclusion There were statistically significant differences between affected and unaffected gray values in the canines in FOV size 51 × 55 mm. However, no statistically significant differences were found in the gray values in trabecular bone of unilateral maxillary impacted canines and normal erupted canines on the mesial, distal, buccal, and palatal sides.
impacted ; canine ; trabecular bone ; maxillary ; cone-beam computed tomography

The purpose of the present study was to determine the role of inositol 1,4,5-trisphosphate receptor 3 (IP₃R3) in renal cyst development in autosomal dominant polycystic kidney disease (ADPKD). 2-aminoethoxy-diphenyl borate (2-APB) and shRNA were used to suppress the expression of IP₃R3. The effect of IP₃R3 on cyst growth was investigated in Madin-Darby canine kidney (MDCK) cyst model, embryonic kidney cyst model and kidney specific Pkd1 knockout (PKD) mouse model. The underlying mechanism of IP₃R3 in promoting renal cyst development was investigated by Western blot and immunofluorescence staining. The results showed that the expression level of IP₃R3 was significantly increased in the kidneys of PKD mice. Inhibiting IP₃R3 by 2-APB or shRNA significantly retarded cyst expansion in MDCK cyst model and embryonic kidney cyst model. Western blot and immunofluorescence staining results showed that hyperactivated cAMP-PKA signaling pathway in the growth process of ADPKD cyst promoted the expression of IP₃R3, which was accompanied by a subcellular redistribution process in which IP₃R3 was translocated from endoplasmic reticulum to intercellular junction. The abnormal expression and subcellular localization of IP₃R3 further promoted cyst epithelial cell proliferation by activating MAPK and mTOR signaling pathways and accelerating cell cycle. These results suggest that the expression and subcellular distribution of IP₃R3 are involved in promoting renal cyst development, which implies IP₃R3 as a potential therapeutic target of ADPKD.
Animals ; Dogs ; Mice ; Cysts/genetics* ; Inositol 1,4,5-Trisphosphate Receptors/pharmacology* ; Kidney/metabolism* ; Polycystic Kidney Diseases/metabolism* ; Polycystic Kidney, Autosomal Dominant/drug therapy* ; Madin Darby Canine Kidney Cells

Transglutaminase 2 (TGM2) is a ubiquitous multifunctional protein, which is related to the adhesion of different cells and tumor formation. Previous studies found that TGM2 is involved in the interaction between host cells and viruses, but the effect of TGM2 on the proliferation of influenza virus in cells has not been reported. To explore the effect of TGM2 during H1N1 subtype influenza virus infection, a stable MDCK cell line with TGM2 overexpression and a knockout cell line were constructed. The mRNA and protein expression levels of NP and NS1 as well as the virus titer were measured at 48 hours after pot-infection with H1N1 subtype influenza virus. The results showed that overexpression of TGM2 effectively inhibited the expression of NP and NS1 genes of H1N1 subtype influenza virus, while knockout of TGM2 up-regulated the expression of the NP and NS1 genes, and the expression of the NP at protein level was consistent with that at mRNA level. Virus proliferation curve showed that the titer of H1N1 subtype influenza virus decreased significantly upon TGM2 overexpression. On the contrary, the virus titer in TGM2 knockout cells reached the peak at 48 h, which further proved that TGM2 was involved in the inhibition of H1N1 subtype influenza virus proliferation in MDCK cells. By analyzing the expression of genes downstream of influenza virus response signaling pathway, we found that TGM2 may inhibit the proliferation of H1N1 subtype influenza virus by promoting the activation of JAK-STAT molecular pathway and inhibiting RIG-1 signaling pathway. The above findings are of great significance for revealing the mechanism underlying the interactions between host cells and virus and establishing a genetically engineering cell line for high-yield influenza vaccine production of influenza virus.
Animals ; Cell Proliferation ; Dogs ; Humans ; Influenza A Virus, H1N1 Subtype/genetics* ; Influenza, Human ; Madin Darby Canine Kidney Cells ; Protein Glutamine gamma Glutamyltransferase 2

In order to establish an infectious clone for CDV-3, a commercial vaccine strain of canine distemper virus for mink, to provide reference for the studies of pathogenesis and novel vaccine development of CDV. Thirteen pairs of primers were used to amplify the full-length genome of CDV-3 strain. Five long fragments were obtained based on single restriction site analysis of the whole genome of CDV-3 by RT-PCR. Five fragments were successively inserted into the multiple clone sites in the modified eukaryotic vector of pcDNA3.2 by restriction enzymes and splicing. Meanwhile, the hammerhead ribozyme and hepatitis delta virus ribozyme sequences were added to the beginning of F1 fragment and the ending of F5 fragment, respectively. Then, the full-length cDNA recombinant plasmid of CDV-3 was obtained and named as pcDNA3.2-CDV-3. In addition, three helper plasmids, expressing the N protein, P protein and L protein of the CDV-3 strain respectively, were constructed. The 293T cells were transfected with the full-length cDNA recombinant plasmid and three helper plasmids by Lipofectamine™ 2000. At 3 days post transfection, the supernatant was added to the monolayer of Vero cells to observe the typical syncytium of CDV. Indirect immunofluorescence and artificial label identification of recombinant virus rCDV-3 were conducted after the occurrence of lesions. Finally, the growth characteristics of wtCDV-3 and rCDV-3 were compared after passaging of rCDV-3. The identification of the full-length cDNA recombinant plasmid and three helper plasmids by restriction enzyme digestion and sequencing were consistent with expected. The Vero cells infected with the recombinant rCDV-3 showed typical syncytic. The identification of indirect immunofluorescence and labeled marker, and observation under electron microscope proved that the rCDV-3 was indeed rescued from the recombinant plasmid of pcDNA3.2-CDV-3. In comparison of the virus titers of wtCDV-3, rCDV-3 replicated massively and rapidly and reached the maximize virus titer of 10⁷·⁶⁶⁷ TCID₅₀/mL within 36 h post infection (p.i.) in Vero cells, while wtCDV-3 grew gradually to 10⁶·⁶⁶⁷ TCID₅₀/mL at 72 h p.i. in Vero cells. This reverse genetic system of CDV-3 strain has been established successfully, to provide reference for the studies of pathogenesis and novel vaccine development of CDV.
Animals ; Chlorocebus aethiops ; Clone Cells ; DNA, Complementary ; Distemper Virus, Canine/genetics* ; Plasmids/genetics* ; Vero Cells

canine adenovirus type 2 (CAV-2) vaccine candidate using the recently isolated Korean CAV-2 strain; we termed the vaccine APQA1701-40P and evaluated its safety and immunogenicity in dogs.MATERIALS AND METHODS: To generate the anti-CAV-2 vaccine, APQA1701 was passaged 40 times in MDCK cells growing in medium containing 5 mM urea and the virus was inactivated using 0.05% (volume per volume) formaldehyde. Two vaccines were prepared by blending inactivated APQA1701-40P with two different adjuvants; both were intramuscularly injected (twice) into guinea pigs. The safety and immunogenicity of the Cabopol-adjuvanted vaccine were evaluated in seronegative dogs. The humoral responses elicited were measured using an indirect enzyme-linked immunosorbent assay (I-ELISA), and via a virus neutralization assay (VNA).RESULTS: The new, inactivated CAV-2 vaccine strain, APQA1701-40P, lacked six amino acids of the E1b-19K protein. In guinea pigs, the Cabopol-adjuvanted vaccine afforded a slightly higher VNA titer and I-ELISA absorbance than an IMS gel-adjuvanted vaccine 4 weeks post-vaccination (p>0.05). Dogs inoculated with the former vaccine developed a significantly higher immune titer than non-vaccinated dogs.CONCLUSION: The Cabopol-adjuvanted, inactivated CAV-2 vaccine was safe and induced a high VNA titer in dogs.]]>
Adenoviruses, Canine ; Amino Acids ; Animals ; Dogs ; Enzyme-Linked Immunosorbent Assay ; Formaldehyde ; Guinea Pigs ; Madin Darby Canine Kidney Cells ; Urea ; Vaccines

Tripartite motif (TRIM) family proteins are important effectors of innate immunity against viral infections. Here we identified TRIM35 as a regulator of TRAF3 activation. Deficiency in or inhibition of TRIM35 suppressed the production of type I interferon (IFN) in response to viral infection. Trim35-deficient mice were more susceptible to influenza A virus (IAV) infection than were wild-type mice. TRIM35 promoted the RIG-I-mediated signaling by catalyzing Lys63-linked polyubiquitination of TRAF3 and the subsequent formation of a signaling complex with VISA and TBK1. IAV PB2 polymerase countered the innate antiviral immune response by impeding the Lys63-linked polyubiquitination and activation of TRAF3. TRIM35 mediated Lys48-linked polyubiquitination and proteasomal degradation of IAV PB2, thereby antagonizing its suppression of TRAF3 activation. Our in vitro and in vivo findings thus reveal novel roles of TRIM35, through catalyzing Lys63- or Lys48-linked polyubiquitination, in RIG-I antiviral immunity and mechanism of defense against IAV infection.
A549 Cells ; Animals ; Apoptosis Regulatory Proteins/immunology* ; DEAD Box Protein 58/immunology* ; Dogs ; HEK293 Cells ; Humans ; Influenza A Virus, H1N1 Subtype/immunology* ; Madin Darby Canine Kidney Cells ; Mice ; Mice, Knockout ; Orthomyxoviridae Infections/pathology* ; Proteolysis ; RAW 264.7 Cells ; Signal Transduction/immunology* ; THP-1 Cells ; TNF Receptor-Associated Factor 3/immunology* ; Ubiquitination/immunology* ; Viral Proteins/immunology*

Canine adenovirus type 1 (CAV-1) infection results in hepatitis in dogs. In this study, we investigated the biologic and genetic characteristics of the CAV-1 vaccine strain (CAV1V) to improve quality control about CAV vaccine. The identity of CAV1V as CAV-1 was confirmed based on its cytopathic effects and the results of hemagglutination (HA) and immunofluorescence assays, and electron microscopy. The CAV1V strain reached 10(7.5) TCID(50)/mL in MDCK cells at 4 days post-inoculation and exhibited hemmagglutination activity of 256 U using guinea pig erythrocytes. Intranuclear fluorescence in the infected cells was observed and typical adenoviruses were observed in electon microscope. CAV1V strain was identified as a CAV-1 strain by nucleotide sequence analysis. In a comparison of the nucleotide sequences of the fiber genes of several CAV strains, CAV1V showed the highest similarity (99.8%) with the GLAXO strain, which was isolated in Canada. Our biological characterization of CAV1V will facilitate quality control of the canine hepatitis vaccine.
Adenoviridae ; Adenoviruses, Canine ; Animals ; Base Sequence ; Canada ; Dogs ; Erythrocytes ; Fluorescence ; Fluorescent Antibody Technique ; Guinea Pigs ; Hemagglutination ; Hepatitis ; Madin Darby Canine Kidney Cells ; Microscopy, Electron ; Quality Control

Hesperetin, an abundant bioactive component of citrus fruits, is poorly water-soluble, resulting in low oral bioavailability. We developed new formulations to improve the water solubility, antioxidant activity, and oral absorption of hesperetin. Two nano-based formulations were developed, namely hesperetin-TPGS (D-α-tocopheryl polyethylene glycol 1000 succinate) micelles and hesperetin-phosphatidylcholine (PC) complexes. These two formulations were prepared by a simple technique called solvent dispersion, using US Food and Drug Administration (FDA)-approved excipients for drugs. Differential scanning calorimetry (DSC) and dynamic light scattering (DLS) were used to characterize the formulations' physical properties. Cytotoxicity analysis, cellular antioxidant activity assay, and a pharmacokinetic study were performed to evaluate the biological properties of these two formulations. The final weight ratios of both hesperetin to TPGS and hesperetin to PC were 1:12 based on their water solubility, which increased to 21.5- and 20.7-fold, respectively. The hesperetin-TPGS micelles had a small particle size of 26.19 nm, whereas the hesperetin-PC complexes exhibited a larger particle size of 219.15 nm. In addition, the cellular antioxidant activity assay indicated that both hesperetin-TPGS micelles and hesperetin-PC complexes increased the antioxidant activity of hesperetin to 4.2- and 3.9-fold, respectively. Importantly, the in vivo oral absorption study on rats indicated that the micelles and complexes significantly increased the peak plasma concentration (C_max) from 2.64 μg/mL to 20.67 and 33.09 μg/mL and also increased the area under the concentration-time curve of hesperetin after oral administration to 16.2- and 18.0-fold, respectively. The micelles and complexes increased the solubility and remarkably improved the in vitro antioxidant activity and in vivo oral absorption of hesperetin, indicating these formulations' potential applications in drugs and healthcare products.
Administration, Oral ; Animals ; Antioxidants/chemistry* ; Biological Availability ; Calorimetry, Differential Scanning ; Dogs ; Dose-Response Relationship, Drug ; Drug Carriers ; Female ; Hep G2 Cells ; Hesperidin/chemistry* ; Humans ; Light ; Madin Darby Canine Kidney Cells ; Micelles ; Phosphatidylcholines/chemistry* ; Polyethylene Glycols/chemistry* ; Rats ; Rats, Sprague-Dawley ; Scattering, Radiation ; Solubility ; Solvents ; Vitamin E/chemistry* ; Water/chemistry* ; alpha-Tocopherol/chemistry*

To study the effect and underlying mechanism of Mahuang Tang against influenza A virus ， the influenza virus-infected Madin-Darby canine kidney(MDCK) cells were used as the carrier in this study to detect the median tissue culture-infective dose(TCID₅₀) of influenza A virus strains(A/PR8/34) on MDCK cells with cytopathic effect(CPE) assay. Blocking influenza virus invading host cells and anti-influenza virus biosynthesis were used as two different administration methods， and then the methyl thiazolyl tetrazolium(MTT) assay was utilized to determine the antiviral effective rate(ER)， median efficacious concentration(EC₅₀) and therapeutic index(TI) of Mahuang Tang. The quantitative Real-time polymerase chain reaction(RT-PCR) was used to measure virus load and the mRNA expression levels of TLR4， TLR7， MyD88 and TRAF6 in MDCK cells at 24， 48 h after the treatment. The experiment results indicated that TCID₅₀ of A/PR8/34 for MDCK cells was 1×10-4.32/mL. The EC₅₀ values of two different treatment methods were 4.92，1.59 g·L⁻¹ respectively， the TI values were 12.53， 38.78 respectively， and when the concentration of Mahuang Tang was 5.00 g·L⁻¹， ER values were 50.21%， 98.41% respectively， showing that Mahuang Tang can block influenza virus into the host cells and significantly inhibit their biosynthesis. Meanwhile， as compared with the virus group， the virus load was significantly inhibited in Mahuang Tang groups， and Mahuang Tang high and middle doses had the significant effect on decreasing the mRNA expression of TLR4， TLR7，MyD88 and TRAF6 at 24， 48 h after the treatment. It can be demonstrated that the mechanisms of Mahuang Tang against influenza A virus are related to the inhibition of influenza virus replication and the mRNA expression of correlative genes in TLR4 and TLR7 signaling pathways.
Animals ; Antiviral Agents ; pharmacology ; Dogs ; Drugs, Chinese Herbal ; pharmacology ; Influenza A Virus, H1N1 Subtype ; drug effects ; physiology ; Madin Darby Canine Kidney Cells ; Orthomyxoviridae Infections ; Toll-Like Receptor 4 ; metabolism ; Toll-Like Receptor 7 ; metabolism ; Virus Replication ; drug effects

Canine coronavirus is a single-stranded RNA virus that causes enteritis in dogs of any age. Coronaviral enteritis is seldom definitively diagnosed, since it is usually much less severe than many other types of enteritis and is self-limiting. Conventional diagnostics for the canine coronaviral enteritis such as polymerase chain reaction (PCR), virus isolation, and electron microscopic examination are inappropriate for small animal clinics due to the complicated experimental processes involved. Therefore, a commercially available lateral flow test kit based on chromatographic immunoassay techniques was tested to evaluate its performance as a first-line diagnostic test kit that could be used in clinics. The coronavirus antigen test kit detected canine coronavirus-infected dogs with 93.1% sensitivity and 97.5% specificity. The detection limit of the test kit was between 1.97 × 10⁴/mL and 9.85 × 10³/mL for samples with a 2-fold serial dilution from 1.25 × 10⁶ TCID₅₀ (TCID₅₀, 50% tissue culture infectious dose). Additionally, the test kit had no cross-reactivity with canine parvovirus, distemper virus, or Escherichia coli. Overall, the commercially available test kit showed good diagnostic performance in a clinical setting, with results similar to those from PCR, confirming their potential for convenient and accurate use in small animal clinics.
Animals ; Coronavirus ; Coronavirus, Canine ; Diagnostic Tests, Routine ; Distemper ; Dogs ; Enteritis ; Escherichia coli ; Immunoassay ; Limit of Detection ; Parvovirus, Canine ; Polymerase Chain Reaction ; Reagent Kits, Diagnostic ; RNA Viruses ; Sensitivity and Specificity