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To prepare an ophthalmic solution with a cell-permeable TAT peptide (TAT-N24) as the main cell-permeable peptide inhibitor of p55PIK signaling and observe its therapeutic effect on suture-induced corneal neovascularization (CNV) in rats. Sprague-Dawley rats were used to establish a corneal suture (CS) model of CNV. The vehicle and 0.9% TAT-N24 ophthalmic solution was topically administered. CNV induction was assessed on the basis of the clinical performance of each group. Hematoxylin-eosin staining was used to observe pathological changes, and immunohistochemical staining and confocal immunofluorescence were used to determine the localization of factors associated with corneal tissue. The mRNA expression levels of hypoxia-inducible factor (HIF-1α), vascular endothelial growth factor (VEGF-A), nuclear transcription factor κB (NF-κB p65), tumor necrosis factor (TNF-α), interleukin-1β (IL-1β), and interleukin (IL)-6 were determined using real-time quantitative polymerase chain reaction. Western blotting was performed to detect the protein expression levels of HIF-1α and NF-κB p65. TAT-N24 slowed CNV production and reduced the expression of HIF-1α and inflammatory factors in CS models. The mRNA levels of HIF-1α, VEGF-A, NF-kB, TNF-α, IL-1β, and IL-6 significantly decreased. Moreover, the protein levels of HIF-1α and NF-κB p65 were significantly decreased. TAT-N24 can treat CNV and ocular inflammation by inhibiting the HIF-1α/NF-κB signaling pathway in CS. In the early treatment of corneal foreign body trauma, topical application of TAT-N24 can not only reduce the inflammatory response but also inhibit corneal neovascularization.

Epstein-Barr virus (EBV) is an important human dsDNA virus, which has been shown to be associated with several malignancies including about 10% of gastric carcinomas. How EBV enters an epithelial cell has been an interesting project for investigation. “Cell-in-cell” infection was recently reported an efficient way for the entry of EBV into nasopharynx epithelial cells. The present approach was to explore the feasibility of this mode for EBV infection in gastric epithelial cells and the dynamic change of host inflammatory reaction. The EBV-positive lymphoblastic cells of Akata containing a GFP tag in the viral genome were co-cultured with the gastric epithelial cells (GES-1). The infection situation was observed under fluorescence and electron microscopies. Real-time quantitative PCR and Western-blotting assay were employed to detect the expression of a few specific cytokines and inflammatory factors. The results demonstrated that EBV could get into gastric epithelial cells by “cell-in-cell” infection but not fully successful due to the host fighting. IL-1β, IL-6 and IL-8 played prominent roles in the cellular response to the infection. The activation of NF-κB and HSP70 was also required for the host antiviral response. The results imply that the gastric epithelial cells could powerfully resist the virus invader via cell-in-cell at the early stage through inflammatory and innate immune responses. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12250-019-00097-1) contains supplementary material, which is available to authorized users.

Purpose Previously we developed TAT-N24 as a synthetic cell-permeable peptide inhibitor of p55PIK signaling and demonstrated its anti-inflammatory effects. This study aimed to evaluate the potential of TAT-N24 as a new agent for the treatment of ocular inflammatory diseases. Methods The endotoxin-induced uveitis (EIU) model was established by intravitreal injection of lipopolysaccharide (LPS) in BALB/c mice and experimental autoimmune uveitis (EAU) model was established by subcutaneous injection of a peptide spanning amino acid residues 161–180 of interphotoreceptor retinoid binding protein (IRBP161-180) with complete Freund's adjuvant (CFA) in B10.RIII mice. TAT-N24 was topically administered in EIU model and intraperitoneally administered in EAU model. The severity levels of uveitis were assessed by clinical and histopathological scores. The mRNA levels of inflammatory cytokines in iris-ciliary body (ICB) and retina were analyzed by reverse transcription quantitative polymerase chain reaction (RT-qPCR). The protein levels of inflammatory factors were determined by ELISA or Western blotting. Results The results showed that TAT-N24 alleviated clinical signs, decreased inflammatory cell infiltration and the expression of inflammatory cytokines in both EIU and EAU models. Furthermore, protein levels of tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) in aqueous humor and mRNA and protein levels of NF-κB p65 in the ICB significantly decreased in EIU model. In EAU model, TAT-N24 application induced a significant decrease of IFN-gamma (IFN-γ) and interleukin-17 (IL-17) in the retina, which were secreted by Th1 and Th17 cells, respectively. Conclusion In conclusion, TAT-N24 suppressed intraocular inflammation in both EIU and EAU models, and the anti-inflammatory effects were mediated by suppressing the expression of inflammatory cytokines by PI3K/NF-κB signaling pathway. TAT-N24 could be potential candidate for the treatment of ocular inflammatory diseases.

A tumor model that Epstein-Barr virus (EBV) latent infection facilitated the tumorigenicity was previously established using the Maxi-EBV system. In the present approach, EBV-lost cell clones demonstrated significantly decreased tumorigenesis. On the other hand, the LMP1 gene in Maxi-EBV genome was replaced by that of nasopharyngeal carcinoma origin. The resultant cell line, 293–1/NL showed much lower malignancy than the original 293-EBV. The result was opposite to our expectation. The change of 293 sublineage cells for EBV harboring also got similar result. To seek the underlying reason, the copy number of EBV genome in all the cell lines was detected. The result indicated that 293-EBV contained about 4.5-fold higher EBV copies than 293–1/NL did. Parallel EBV genomes led to relatively stable copies in different 293 sublineages, suggesting the viral genome structure is a factor for the sustainability of EBV's copy number. Moreover, the LMP1 transcription in high copy-containing cells showed abnormally high level. Furthermore, the main LMP1-driven pathway, transcription factor NF-κB, was highly activated in high-copy cells. Here we first manifest by experimental model that the copy number of EBV latent genome correlates with the viral pathogenesis, which depends on the activation level of LMP1 and NF-κB. Overall, both the presence and amount of EBV genome are crucial for the viral oncogenicity.

MicroRNAs (miRNAs) are a class of short non-coding RNAs that regulate gene expression at the post-transcriptional level. It has been demonstrated that miRNAs serve a crucial role in tissue development and the pathogenesis of numerous diseases. The aim of the current study was to investigate the alterations in miRNA expression in a cultured retinal ganglion cell line (RGC-5 cells) following ionizing radiation injury. Cultured RGC-5 cells were exposed to X-rays at doses of 2, 4, 6 and 8 Gy using a medical linear accelerator. Alterations in cellular morphology were observed under a phase contrast microscope and cell viability was measured using the MTT assay. Subsequent to exposure to X-ray radiation for 5 days, the viability of RGC-5 cells was significantly reduced in the 6 and 8 Gy groups, accompanied by morphological alterations. Total RNA was then extracted from RGC-5 cells and subjected to miRNA microarray analysis subsequent to exposure to 6 Gy X-ray radiation for 5 days. The results of the microarray analysis indicated that the expression levels of 12 miRNAs were significantly different between the 6 Gy and control groups, including 6 upregulated miRNAs and 6 downregulated miRNAs. To verify microarray results, a reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis was performed. The data obtained from RT-qPCR analysis was similar to that of the the microarray analysis for alterations in the expression of the 12 miRNAs. The results of the current study indicated that miRNA expression was sensitive to ionizing radiation, which may serve an important role in mechanisms of radiation injury in retinal ganglion cells.

Oxide-doped PbFe 1/4 Sc 1/4 Nb 1/2 O 3 (PFSN) ceramics were prepared by the conventional ceramic processing via a B-site oxide mixing route. The oxide-doped PFSN ceramics exhibit pure rhombohedral perovskite structure and high relative density. Due to the MnO 2 and Li 2 CO 3 doping, the synthesized ceramics exhibit improved dielectric property, and excellent ferroelectric and piezoelectric properties, which can be attributed to a charge compensation mechanism where donor defects are generated by different ionization paths. The values of remnant polarization P r , electromechanical coupling coefficient K p and piezoelectric constant d 33 of the MnO 2 - and Li 2 CO 3 -doped PFSN ceramics are 16.35 μC/cm 2 , 0.232 and 112.7 pC/N, and 29.22 μC/cm 2 , 0.241 and 112.5 pC/N, respectively. Therefore, oxide doping is an efficient method to improve dielectric, ferroelectric and piezoelectric properties of the PFSN-based ferroelectric ceramics.

The incidence of cervical cancer is expected to rise sharply in China. A reliable routine human papillomavirus (HPV) detection and genotyping test to be supplemented by the limited Papanicolaou cytology facilities is urgently needed to help identify the patients with cervical precancer for preventive interventions. To this end, we evaluated a nested polymerase chain reaction (PCR) protocol for detection of HPV L1 gene DNA in cervicovaginal cells. The PCR amplicons were genotyped by direct DNA sequencing. In parallel, split samples were subjected to a Digene HC2 HPV test which has been widely used for “cervical cancer risk” screen. Of the 1826 specimens, 1655 contained sufficient materials for analysis and 657 were truly negative. PCR/DNA sequencing showed 674 infected by a single high-risk HPV, 188 by a single low-risk HPV, and 136 by multiple HPV genotypes with up to five HPV genotypes in one specimen. In comparison, the HC2 test classified 713 specimens as infected by high-risk HPV, and 942 as negative for HPV infections. The high-risk HC2 test correctly detected 388 (57.6%) of the 674 high-risk HPV isolates in clinical specimens, mislabeled 88 (46.8%) of the 188 low-risk HPV isolates as high-risk genotypes, and classified 180 (27.4%) of the 657 “true-negative” samples as being infected by high-risk HPV. It was found to cross-react with 20 low-risk HPV genotypes. We conclude that nested PCR detection of HPV followed by short target DNA sequencing can be used for screening and genotyping to formulate a paradigm in clinical management of HPV-related disorders in a rapidly developing economy.

A dimeric rhodium(ii) complex catalyses the chemoselective dehydrogenative esterification of aldehydes and alcohols., Dehydrogenative cross-coupling of aldehydes with alcohols as well as dehydrogentive cross-coupling of primary alcohols to produce esters have been developed using a Rh-terpyridine catalyst. The catalyst demonstrates broad substrate scope and good functional group tolerance, affording esters highly selectively. The high chemoselectivity of the catalyst stems from its preference for dehydrogenation of benzylic alcohols over aliphatic ones. Preliminary mechanistic studies suggest that the active catalyst is a dimeric Rh(ii) species, operating via a mechanism involving metal–base–metal cooperativity.

To obtain recombinant Epstein-Barr virus (EBV) particles with the neomycin resistance from BAC-EBV with hygrymicin (hyg) gene, homologous recombination was employed for altering the original hyg gene to neo resistance gene in the BAC-EBV genome. The kanamycin marker gene (kan) containing the recombinant protein FLP recognition sites (FRTs) was connected to the neo gene in the vector of pGEM-T using conventional cloning techniques. Both ends of the connective are the arms with 20bp of homology sequences responding to the ends next to the hyg gene. PCR and cloning techniques were further performed to extend homology arms to make them reach 89bp. The linear fragment kan-neo with 89bp of arms was then electroporated into the bacteria containing BAC-EBV genome. Under the mediation of the redαβγ system, the hyg gene in BAC-EBV was replaced by neo gene. A series of methods including antibiotic selection, PCR amplification, sequencing and restriction enzyme digestion were used for the identification of the mutated plasmids. The results showed that BAC-EBV/neo, was constructed as expected. Through the transfection of the BAC-EBV/neo plasmid, G418 selection, and GFP observation, we established 293-EBV/neo cell line. This research lays a foundation for the establishment of broader cell models in the study of EBV-associated pathogenic mechanisms.

Epstein-Barr virus nuclear antigen 1 (EBNA1) is essential for EBV episome's replication and mitotic segregation. During this process, EBNA1 may interact with a lot of host factors. Bimolecular complementation (BiMC) assay is a useful tool for a detection of protein-protein interaction in living cells. In the present study, a plasmid for expressing a fusion protein CYFP-EBNA1 was constructed. As expected, it could express CYFP-EBNA1 and localize in the cellular nuclear. The result also demonstrated that the plasmid is allowed to be used in the BiMC system. KEYWORD: Bimolecular complementation; Epstein-Barr virus nuclear antigen 1; Protein-protein interaction

Hemodilution is a commonly used technique in cardiopulmonary bypass (CPB) and deep hypothermic circulation arrest (DHCA). Our previous study showed that lower hematocrit aggravated the brain injury after DCHA. Because the excitatory amino acids are critical pathways of ischemic neuronal damage, the purpose of the present study was to investigate the effects of different degrees of hemodilution on the excitatory amino acid content in different brain areas after DHCA Adult Sprague-Dawley rats were randomly divided into four groups: group I hematocrit (Hct) 10% (H1), group II Hct 20% (H2), group III Hct 30% (H3), and control group (C). All animals except those in the control group underwent DHCA at 18°C for 90 minutes. Different degrees of hemodilution were accomplished by changing the composition and volume of the priming solution used in CPB. High-performance liquid chromatography was used to determine the concentration of glutamate (Glu), aspartate (Asp), glycine (Gly), gamma-aminobutyric acid (GABA), and taurine (Tau) in the cerebral cortex, hippocampus, and thalamus. We found that the concentration of these five amino acids in the hippocampus and cortex were all increased after DHCA. Glu, Asp, and Gly in the hippocampus and cortex were significantly lower in the Hct 30% group than in the other two groups (p

Dehydrogenative cross-coupling of aldehydes with alcohols as well as dehydrogentive cross-coupling of primary alcohols to produce esters have been developed using a Rh-terpyridine catalyst. The catalyst demonstrates broad substrate scope and good functional group tolerance, affording esters highly selectively. The high chemoselectivity of the catalyst stems from its preference for dehydrogenation of benzylic alcohols over aliphatic ones. Preliminary mechanistic studies suggest that the active catalyst is a dimeric Rh(II) species, operating via a mechanism involving metal–base–metal cooperativity.

In this paper, we propose a new method that can acquire the complex hologram of a 3D object for optical scanning holography (OSH) by using polarized optical elements and a 90 degrees 2×4 optical hybrid. The 3D object is scanned by a wave front combined by a plane and spherical wave with orthogonal polarization. The transmitted/reflected signal light is divided into two beams by a polarized beamsplitter (PBS). Then after the polarization control, the two separated beams are heterodyne detected by a 90 degrees 2×4 optical hybrid with two balanced detectors. The 3D object can be reconstructed from the complex hologram acquired from the balanced detectors. Compared with the conventional OSH, the acousto-optic modulator and complex electronic system are not needed in this method. Another advantage of this method lies in its high efficiency since there is almost no energy loss in the generation of the scanning beam by using the polarized optical elements. This method should be used for 3D holographic microscopy and 3D holographic television.