N-Linked Glycosylation Plays an Important Role in Budding of Neuraminidase Protein and Virulence of Influenza Viruses.

Neuraminidase (NA) has multiple functions in the life cycle of influenza virus, especially in the late stage of virus replication. Both of hemagglutinin (HA) and NA are highly glycosylated proteins. N-linked glycosylation (NLG) of HA has been reported to contribute to immune escape and virulence of influenza viruses. However, the function of NLG of NA remains largely unclear. In this study, we found that NLG is critical for budding ability of NA. Tunicamycin treatment or NLG knockout significantly inhibited the budding of NA. Further studies showed that the NLG knockout caused attenuation of virus in vitro and in vivo Notably, the NLG at 219 position plays an important role in the budding, replication, and virulence of H1N1 influenza virus. To explore the underlying mechanism, the unfolded protein response (UPR) was determined in NLG knockout NA overexpressed cells, which showed that the mutant NA was mainly located in the endoplasmic reticulum (ER), the UPR markers BIP and p-eIF2α were upregulated, and XBP1 was downregulated. All the results indicated that NLG knockout NA was stacked in the ER and triggered UPR, which might shut down the budding process of NA. Overall, the study shed light on the function of NLG of NA in virus replication and budding.IMPORTANCE NA is a highly glycosylated protein. Nevertheless, how the NLG affects the function of NA protein remains largely unclear. In this study, we found that NLG plays important roles in budding and Neuraminidase activity of NA protein. Loss of NLG attenuated viral budding and replication. In particular, the 219 NLG site mutation significantly attenuated the replication and virulence of H1N1 influenza virus in vitro and in vivo, which suggested that NLG of NA protein is a novel virulence marker for influenza viruses.

Complete genome sequence of a novel reassortant H11N2 avian influenza virus isolated from a live poultry market in eastern China.

A/chicken/Nanjing/908/2009(H11N2) (CK908) was isolated from a live poultry market in Nanjing, China. Using PCR and sequencing analysis, we obtained the complete genome sequences of the CK908 virus. The sequence analysis demonstrated that this H11N2 virus was a novel reassortant AIV whose PB1, PB2, PA, HA, NP, NA, M, and NS genes originated from H9N2, H7N7, H5N2, H11N8, H3N6, H6N2, H1N1, and H5N1, respectively. Knowledge regarding the complete genome sequences of the CK908 virus will be useful for epidemiological surveillance.

Dual function of anti-biofilm and modulating biofilm equilibrium of orthodontic cement containing quaternary ammonium salt.

The objectives of this study were to incorporate dimethylaminohexadecyl methacrylate (DMAHDM) into resin-modified glass ionomer cement (RMGI) to develop a novel orthodontic cement which endowed RMGI with strong antibacterial ability and investigated its modulation biofilm equilibrium from cariogenic state to non-cariogenic state for the first time. Cariogenic Streptococcus mutans (S. mutans), and non-cariogenic Streptococcus sanguinis (S. sanguinis) and Streptococcus gordonii (S. gordonii) were selected to form a tri-species biofilm model. RMGI incorporated with different mass fraction of DMAHDM was examined: biofilm colony-forming units, metabolic activity, live/dead staining, lactic acid and exopolysaccharides productions. TaqMan real-time polymerase chain reaction was used to determine changes of biofilm species compositions. The results showed RMGI containing 3% DMAHDM achieved strong antibacterial ability and suppressed the cariogenic species in biofilm, modulating biofilm equilibrium from cariogenic state to non-cariogenic state tendency. The novel bioactive cement containing DMAHDM is promising in fixed orthodontic treatments and protecting tooth enamel.

RNA-Seq Analysis of Influenza A Virus-Induced Transcriptional Changes in Mice Lung and Its Possible Implications for the Virus Pathogenicity in Mice.

The influenza A virus (IAV) is an important cause of respiratory disease worldwide. It is well known that alveolar epithelial cells are the target cells for the IAV, but there is relatively limited knowledge regarding the role of macrophages during IAV infection. Here, we aimed to analyze transcriptome differences in mouse lungs and macrophage (RAW264.7) cell lines infected with either A/California/04/2009 H1N1 (CA09) or A/chicken/SD/56/2015 H9N2 (SD56) using deep sequencing. The uniquely differentially expressed genes (UDEGs) were analyzed with the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases; the results showed that the lungs infected with the two different viruses had different enrichments of pathways and terms. Interestingly, CA09 virus infection in mice was mostly involved with genes related to the extracellular matrix (ECM), while the most significant differences after SD56 infection in mice were in immune-related genes. Gene set enrichment analysis (GSEA) of RAW264.7 cells revealed that regulation of the cell cycle was of great significance after CA09 infection, whereas the regulation of the immune response was most enriched after SD56 infection, which was consistent with analysis results in the lung. Similar results were obtained from weighted gene co-expression network analysis (WGCNA), where cell cycle regulation was extensively activated in RAW264.7 macrophages infected with the CA09 virus. Disorder of the cell cycle is likely to affect their normal immune regulation, which may be an important factor leading to their different prognoses. These results provide insight into the mechanism of the CA09 virus that caused a pandemic and explain the different reactivities of monocytes/macrophages infected by H9N2 and H1N1 IAV subtypes.

microRNA-132 inhibits osteogenic differentiation of periodontal ligament stem cells via GDF5 and the NF-κB signaling pathway.

BACKGROUND: Periodontal ligament stem cells (PDLSCs) could differentiate into osteoblasts and have a great prospect in treating bone diseases. microRNAs (miRs) and nuclear factor kappa-B (NF-κB) signaling pathway have proved pivotal in regulating osteogenic differentiation. This study intended to discuss the mechanism of miR-132 and NF-κB in PDLSC osteogenesis. METHODS: PDLSCs were firstly cultured, induced, and identified by detecting the surface markers and observing cell morphology. Levels of osteogenic markers alkaline phosphatase (ALP), bone morphogenetic proteins 2 (BMP2), runt-related transcription factor 2 (Runx2) and osteocalcin (OCN), along with miR-132 expression were measured. The osteoblast activity and mineral deposition were detected by ALP and alizarin red S (ARS) stainings. The targeting relationship between miR-132 and growth differentiation factor 5 (GDF5) was verified. The gain-and loss-of-function was performed to discuss roles of miR-132 and GDF5 in osteogenic differentiation of PDLSCs. Besides, levels of NF-κB signaling pathway-related proteins were measured. RESULTS: In osteogenic differentiation of PDLSCs, levels of ALP, BMP2, Runx2 and OCN were upregulated while miR-132 was downregulated. Overexpressing miR-132 reduced levels of osteogenic markers, osteoblast activity, ALP and ARS intensity and the activation of NF-κB axis. GDF5 is a target of miR-132 and GDF5 overexpression reversed the inhibitory effects of overexpressed miR-132 on PDLSC osteogenesis. CONCLUSION: Together, miR-132 could inhibit PDLSC osteogenesis via targeting GDF5 and activating NF-κB axis. These data provide useful information for PDLSC application in periodontal therapy.

Limited adaptation of chimeric H9N2 viruses containing internal genes from bat influenza viruses in chickens.

Influenza virus-like sequences of H17N10 and H18N11 were identified in bats, despite there has been no live virus isolated. The genetic analysis indicated that they have distinct but relatively close evolutionary relationships to known influenza A viruses. However, the infectivity and adaptation of bat influenza viruses in avian species remain unclear. In this study, two modified bat influenza viruses cH9cN2/H17 and cH9cN2/H18 containing HA and NA coding regions replaced with those of H9N2 influenza A virus were generated in the background of the H17N10 or H18N11 viruses. These two modified viruses replicated less efficiently than wild type H9N2 virus in cultured chicken cells. The mini-genome assay showed that viral ribonucleoproteins (vRNPs) of H9N2 has significantly higher polymerase activity than that of bat influenza viruses in avian cells. In chicken study, compared with H9N2 virus, which replicated and transmitted efficiently in chickens, the cH9cN2/H17 and cH9cN2/H18 viruses only replicated in chicken tracheas with lower titers. Pathological examination showed that the H9N2 caused severer lesions in lung and trachea than the modified bat influenza viruses. Notably, the cH9cN2/H18 transmitted among chickens, but not cH9cN2/H17, and chicken IFN-ß antagonism results showed that H18N11 NS1 protein inhibited chicken IFN-ß response more efficiently than H17N10 NS1 protein in avian cells. Taken together, our data indicated that the internal genes of bat influenza viruses adapted poorly to chickens, while the internal genes of H18N11 seemed to adapt to chickens better than H17N10.

Phase transformation analysis of varied nickel-titanium orthodontic wires.

BACKGROUND: The shape memory effect of nickel-titanium (NiTi) archwires is largely determined by the phase transition temperature. It is associated with a reversible transformation from martensite to austenite. The aim of this study was to characterize austenite, martensite and R phase temperatures as well as transition temperature ranges of the commonly used clinical NiTi orthodontic arch wires selected from several manufacturers. METHODS: Differential scanning calorimetry (DSC) method was used to study the phase transformation temperatures and the phase transition processes of 9 commonly used clinical NiTi alloys (types: 0.40 mm (0.016 inch), 0.40 mm x 0.56 mm (0.016 inch x 0.022 inch)). RESULTS: The austenite finish temperatures (Af) of 0.40 mm Smart, Ormco and 3M NiTi wires were lower than the room temperature, and no phase transformation was detected during oral temperature. Therefore, we predicted that these types of NiTi did not possess shape memory property. For 0.40 mm and 0.40 mm x 0.56 mm Youyan I NiTi wires, no phase transformation was detected during the scanning temperature range, suggesting that these two types of wires did not possess shape memory either. The Af of 0.40 mm x 0.56 mm Smart, L&H, Youyan II Ni-Ti wires were close to the oral temperature and presented as martensitic-austenitic structures at room temperature, suggesting the NiTi wires listed above have good shape memory effect. Although the 0.40 mm x 0.56 mm Damon CuNiTi wire showed martensitic-austenitic structures at oral temperature, its Af was much higher than the oral temperature. It means that transformation from martensite to austenite for this type of NiTi only finishes when oral temperature is above normal. CONCLUSION: The phase transformation temperatures and transformation behavior varied among different commonly used NiTi orthodontic arch wires, leading to variability in shape memory effect.

Changes in force associated with the amount of aligner activation and lingual bodily movement of the maxillary central incisor.

OBJECTIVE: The purposes of this study were to measure the orthodontic forces generated by thermoplastic aligners and investigate the possible influences of different activations for lingual bodily movements on orthodontic forces, and their attenuation. METHODS: Thermoplastic material of 1.0-mm in thickness was used to manufacture aligners for 0.2, 0.3, 0.4, 0.5, and 0.6 mm activations for lingual bodily movements of the maxillary central incisor. The orthodontic force in the lingual direction delivered by the thermoplastic aligners was measured using a micro-stress sensor system for the invisible orthodontic technique, and was monitored for 2 weeks. RESULTS: Orthodontic force increased with the amount of activation of the aligner in the initial measurements. The attenuation speed in the 0.6 mm group was faster than that of the other groups (p < 0.05). All aligners demonstrated rapid relaxation in the first 8 hours, which then decreased slowly and plateaued on day 4 or 5. CONCLUSIONS: The amount of activation had a substantial influence on the orthodontic force imparted by the aligners. The results suggest that the activation of lingual bodily movement of the maxillary central incisor should not exceed 0.5 mm. The initial 4 or 5 days is important with respect to orthodontic treatment incorporating an aligner.

Development of a PCR-Based Reverse Genetics System for an Attenuated Duck Tembusu Virus Strain.

The infectious disease caused by the duck Tembusu virus (DTMUV) has resulted in massive economic losses to the Chinese duck industry in China since 2010. Research on the molecular basis of DTMUV pathogenicity has been hampered by the lack of a reliable reverse genetics system for this virus. Here we developed a PCR-based reverse genetics system with high fidelity for the attenuated DTMUV strain FX2010-180P. The rescued virus was characterized by using both indirect immunofluorescence assays (IFA) and whole genome sequencing. The rescued virus (rFX2010-180P) grew to similar titers as compared with the wild-type virus in DF-1 cells, and had similar replication and immunogenicity properties in ducks. To determine whether exogenous proteins could be expressed from DTMUV, both an internal ribosomal entry site (IRES) and the enhanced green fluorescent protein (eGFP) gene were introduced between the NS5 gene and the 3' non-coding sequence of FX2010-180P. A recombinant DTMUV expressing eGFP was rescued, but eGFP expression was unstable after 4 passages in DF-1 cells due to a deletion of 1,294 nucleotides. The establishment of a reliable reverse genetics system for FX2010-180P provides a foundation for future studies of DTMUV.

[Measurement of orthodontic forces exerted on the upper right central incisor with the increase of the distance of tooth movement and thickness of the aligner].

OBJECTIVE: To measure the orthodontic forces exerted on the upper right central incisor with the increase of the distance of tooth movement and the thickness of the aligner. METHODS: The labial movement of upper right central incisor at various distances (0.3, 0.6, 0.9, 1.2, 1.5, 1.8 mm) was designed and the stereolithography model (2 times bigger than the original model) was created with 3-D scanning and tomography output. These models were used to fabricate the aligners with different thicknesses (0.8, 1.0, 1.5 and 2.0 mm) of the thermoplastic materials (6 samples for various distances of tooth movement and thicknesses). Orthodontic forces exerted on the upper right central incisor were measured with the micro-stress sensor measurement system. RESULTS: The orthodontic forces increased with the increase of the thickness of the aligner at the same distance of tooth movement (P < 0.05). The orthodontic force was (1.237 ± 0.082), (1.543 ± 0.059), (3.602 ± 0.102), (6.734 ± 0.063) N when the labial movement of upper right central incisor was 0.3 mm with the aligner of 0.8, 1.0, 1.5, 2.0 mm. The orthodontic forces also increased with the increase of the distance of the tooth movement at the same thickness of the aligner(P < 0.05). The orthodontic force of the aligner of 0.8 mm were (1.354 ± 0.039), (1.288 ± 0.037), (1.479 ± 0.031), (1.799 ± 0.039) N when the upper right central tooth labial movement at 0.6, 0.9, 1.2, 1.5 mm. CONCLUSIONS: The orthodontic forces increased with the increase of the distance of tooth movement and the thickness of the aligner.

[Adult orthodontic technique: development and challenge].

Orthodontic treatments have increasingly become accepted by adults. However, the treatment therapies and philosophies for adults and adolescents have numerous differences. Orthodontic treatment for adults requires more careful planning, flexible management, interdisciplinary cooperation, and rational expectations. New techniques, such as mini-screw implants, invisalign, and self-ligating brackets, have recently been used to update treatments and widen the application of adult orthodontics by improving the treatment results. However, orthodontists still face a number of risks and challenges.

[Establishment of the micro-stress sensor measurement system for invisible aligner technique].

OBJECTIVE: To design and build the micro-stress sensor measurement system for invisible aligner technique. METHODS: A measurement system based on silicon-on-insulator piezoresistive stress sensor was developed. A four-point-bending based experimental apparatus was constructed to calibrate the piezoresistive coefficients of this stress sensor. A chemical-mechanical polishing process was developed for thinning the stress sensor dies. A packaging solution using flexible printed circuit to get signals out was designed. RESULTS: The developed silicon stress sensor chip was 7.0 mm × 6.0 mm × 0.1 mm in size, and 13 sensor rosettes and 4 calibration rosettes were fabricated in one sensor. And a main testing PCB and a Lab View program were designed to carry out the automation measurement of the stress sensor. The stress state during the process was obtained through this test system. And measuered the stress of the 13 sensor unit. CONCLUSIONS: A stress measurement system was established for measuring stress during orthodontic treatment with invisable aligner.

[Properties of NiTi wires with direct electric resistance heat treatment method in three-point bending tests].

OBJECTIVE: To investigate the mechanical properties of Ni-Ti wires with direct electric resistance heat treatment (DERHT) method in three-point bending tests. METHODS: Two superelastic Ni-Ti wires (wire A: Smart SE, wire B: SENTALLOY SE, 0.406 mm × 0.559 mm) and 2 heat-actived Ni-Ti wires (wire C: Smart SM, wire D: L&H TITAN, 0.406 mm × 0.559 mm) were selected. They were heat-treated using the DERHT method by a controlled electric current (6.36 A) applied for different period of time [0 (control), 1.0, 1.5, 2.0, 2.5 seconds). Then, a three-point bending test was performed under controlled temperature (37°C) to examine the relationships between the deflection and the load in the bending of wires. RESULTS: After DERHT treatment, the plateau in the force-deflection curve of superelastic Ni-Ti wires and heat-activated Ni-Ti wires were increased. When the wires were heated for 2.0 seconds and deflected to 1.5 mm, the loading force of A, B, C and D Ni-Ti wires increased from (3.85 ± 0.11), (3.62 ± 0.07), (3.28 ± 0.09), (2.91 ± 0.23) N to (4.33 ± 0.07), (4.07 ± 0.05), (4.52 ± 0.08), (3.27 ± 0.15) N respectively. CONCLUSIONS: DERHT method is very convenient for clinical use. It is possible to change the arch form and superelastic force of NiTi wires. The longer the heating time is, the more the superelastic characteristics of the wires are altered.

[Study on the thickness-change of different thickness thermoplastic materials after thermoforming and saliva immersion].

OBJECTIVE: To survey and compare the thickness-change of different thickness thermoplastic materials under different test condition and make sure the relationship between the thickness-change and the material initial thickness in order to provide a guide in selecting the suitable thickness thermoplastic in practice. METHODS: To choose Biolon, the thickness include 1.0 mm, 0.75 mm, 0.5 mm. Used Electron Vernier caliper to measure the thickness-change of different thickness thermoplastic materials under different processing mode. The data was analyzed by SPSS 10.0. RESULTS: After thermoforming the thickness of thermoplastic became thinner, the thickness of Biolon 0.75 mm decreased by 0.14 mm, Biolon 1.0 mm decreased by 0.22 mm and Biolon 0.5 mm decreased by 0.14 mm. After saliva immersion the thickness became thicker. The thickness of Biolon 0.75 mm increased by 0.02 mm, Biolon 1.0 mm increased by 0.03 mm and Biolon 0.5 mm increased by 0.02 mm. CONCLUSION: 1)The influence of different processing mode to the thickness-change had relation to the material initial thickness. 2)The Biolon 0.75 mm had certain superiority in thickness stability compared to the homogeneous brand through the above research.

[Differential scanning calorimetry analyses of phase transformations in different nickel-titanium orthodontic wires].

OBJECTIVE: To characterize austenite, martensite and R phase temperatures as well as transition temperature ranges of the commonly used nickel-titanium (NiTi) orthodontic arch wires selected from several manufacturers. METHODS: Differential scanning calorimetry (DSC) method was used to study the phase transformation temperatures and the phase transition processes of 9 commonly used NiTi alloys (types: 0.406 mm, 0.406 mm x 0.559 mm). RESULTS: The austenite finish temperatures of A, B, D NiTi wires were 22.4 CT, 21.9 degrees C, 22.5 degrees C, respectively. No phase transformation was detected during oral temperature. It indicated that these types of NiTi wires did not possess shape memory property. For C and H NiTi wires, no phase transformation was detected during the scanning temperature range, suggesting that these two types of wires did not possess shape memory either. The austenite finish temperatures of E, G and I NiTi wires were 34.3 degrees C, 36.6 degrees C, 38.5 degrees C, respectively, which were close to the oral temperature and presented as martensitic-austenitic structures at room temperature, suggesting that the NiTi wires listed above had good shape memory effect. Although F NiTi wire also showed martensitic-austenitic structures at room temperature, its austenite finish temperature (61.5 degrees C) was much higher than oral temperature. CONCLUSIONS: The transformation phase temperatures and transformation behavior were varied among different NiTi alloys, leading to variability in shape memory effect.