Methyltransferase-Deficient Avian Flaviviruses Are Attenuated Due to Suppression of Viral RNA Translation and Induction of a Higher Innate Immunity.

The 5' end of the flavivirus genome contains a type 1 cap structure formed by sequential N-7 and 2'-O methylations by viral methyltransferase (MTase). Cap methylation of flavivirus genome is an essential structural modification to ensure the normal proliferation of the virus. Tembusu virus (TMUV) (genus Flavivirus) is a causative agent of duck egg drop syndrome and has zoonotic potential. Here, we identified the in vitro activity of TMUV MTase and determined the effect of K61-D146-K182-E218 enzymatic tetrad on N-7 and 2'-O methylation. The entire K61-D146-K182-E218 motif is essential for 2'-O MTase activity, whereas N-7 MTase activity requires only D146. To investigate its phenotype, the single point mutation (K61A, D146A, K182A or E218A) was introduced into TMUV replicon (pCMV-Rep-NanoLuc) and TMUV infectious cDNA clone (pACYC-TMUV). K-D-K-E mutations reduced the replication ability of replicon. K61A, K182A and E218A viruses were genetically stable, whereas D146A virus was unstable and reverted to WT virus. Mutant viruses were replication and virulence impaired, showing reduced growth and attenuated cytopathic effects and reduced mortality of duck embryos. Molecular mechanism studies showed that the translation efficiency of mutant viruses was inhibited and a higher host innate immunity was induced. Furthermore, we found that the translation inhibition of MTase-deficient viruses was caused by a defect in N-7 methylation, whereas the absence of 2'-O methylation did not affect viral translation. Taken together, our data validate the debilitating mechanism of MTase-deficient avian flavivirus and reveal an important role for cap-methylation in viral translation, proliferation, and escape from innate immunity.

Suppression of miR-4463 promotes phenotypic switching in VSMCs treated with Ox-LDL.

Vascular smooth muscle cell (VSMC) phenotypic switching is a hallmark of vascular remodeling that contributes to atherosclerotic diseases. MicroRNA 4463 (miR-4463) has been implicated in the development of arteriosclerosis obliterans, whereas the underlying mechanisms in VSMCs have not been fully addressed. In this study, we assessed whether miR-4463 is involved in the phenotypic switching process in VSMCs. Oxidized low-density lipoprotein (Ox-LDL, 50 mg/L) was used to simulate the oxidative stress condition, and miR-4463 expression in VSMCs was detected by a quantitative polymerase chain reaction. To determine the effect of Ox-LDL-mediated regulation of miR-4463 on the phenotypic switching of VSMCs, cell counting kit-8, cell migration assays, and cytoskeleton test were performed. After using specific antagonists of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK), the relationship between miR-4463 and its downstream signaling proteins was explored. Ox-LDL induced oxidative stress to promote VSMC transformation from contraction to secretion, which clearly decreased the level of miR-4463. Then, downregulated miR-4463 enhanced the migration and phenotypic transformation of VSMCs and activated the phosphorylation of JNK and ERK; these effects were increased after Ox-LDL induction. As expected, inhibiting the two signaling proteins blocked the effect of the miR-4463 inhibitor combined with Ox-LDL. In addition, inhibition of miR-4463 led to the upregulation of basic fibroblast growth factor (bFGF) expression. The results of this study demonstrate that miR-4463 is a novel regulator of VSMC function in hypoxic conditions and modulates VSMC phenotypic switching via the JNK and ERK signaling pathways; bFGF may be the target gene of miR-4463.

Dye-enhanced third upconversion Raman overtone in the ultraviolet region under intense excitation of a 532 nm laser.

We report, to our best knowledge, the first observation of enhanced third upconversion Raman overtone of water in the ultraviolet region 240-380 nm by Rhodamine 6G (R6G) under intense excitation of a 532 nm laser. Its spectral peak is mostly around 288 nm. This ultraviolet spectrum has also been obtained for purified water on the Raman spectrometer LRS-3 and fluorescence spectrometer RF-5301PC, which have photomultiplier tubes. The spectral profile is approximately a triangle in most cases. Moreover, an intense and broad background is also observed to appear in the spectrum. Additionally, the conventional visible luminescence spectrum of R6G is discovered to coexist with the ultraviolet one. In comparison, the visible spectrum is much more intense.

The Antibacterial Mechanism of Terpinen-4-ol Against Streptococcus agalactiae.

Streptococcus agalactiae, a highly contagious mastitis pathogen, caused huge economic losses; meanwhile, repeated use of antibiotics results in the emergence of serious antibiotic residues and drug resistance. Therefore, it is in great need to develop ecologically sustainable antimicrobial agents. In the study, the minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC), and action mechanism of terpinen-4-ol against S. agalactiae was investigated to evaluate antibacterial activity of terpinen-4-ol. Results showed the MIC and MBC of terpinen-4-ol were 98 and 196 µg/mL, respectively. Time-kill curves displayed that the antibacterial activity of terpinen-4-ol was in a concentration-dependent manner. Transmission electron micrographs showed that the cell membrane and wall of S. agalactiae were damaged, and plasmolysis and chromatins were inconspicuous. Release of Ca2+ and Mg2+ proved that terpinen-4-ol could increase cell membrane permeability. And the release of lactate dehydrogenase (LDH) suggested that cell wall was destroyed. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and 4',6-diamidino-2-phenylindole (DAPI) staining results showed that terpinen-4-ol could affect the synthesis of protein and DNA. These results suggested that terpinen-4-ol might be used as candidate for treating S. agalactiae infection.

Molecular mechanism of activation of human musk receptors OR5AN1 and OR1A1 by (R)-muscone and diverse other musk-smelling compounds.

Understanding olfaction at the molecular level is challenging due to the lack of crystallographic models of odorant receptors (ORs). To better understand the molecular mechanism of OR activation, we focused on chiral (R)-muscone and other musk-smelling odorants due to their great importance and widespread use in perfumery and traditional medicine, as well as environmental concerns associated with bioaccumulation of musks with estrogenic/antiestrogenic properties. We experimentally and computationally examined the activation of human receptors OR5AN1 and OR1A1, recently identified as specifically responding to musk compounds. OR5AN1 responds at nanomolar concentrations to musk ketone and robustly to macrocyclic sulfoxides and fluorine-substituted macrocyclic ketones; OR1A1 responds only to nitromusks. Structural models of OR5AN1 and OR1A1 based on quantum mechanics/molecular mechanics (QM/MM) hybrid methods were validated through direct comparisons with activation profiles from site-directed mutagenesis experiments and analysis of binding energies for 35 musk-related odorants. The experimentally found chiral selectivity of OR5AN1 to (R)- over (S)-muscone was also computationally confirmed for muscone and fluorinated (R)-muscone analogs. Structural models show that OR5AN1, highly responsive to nitromusks over macrocyclic musks, stabilizes odorants by hydrogen bonding to Tyr260 of transmembrane α-helix 6 and hydrophobic interactions with surrounding aromatic residues Phe105, Phe194, and Phe207. The binding of OR1A1 to nitromusks is stabilized by hydrogen bonding to Tyr258 along with hydrophobic interactions with surrounding aromatic residues Tyr251 and Phe206. Hydrophobic/nonpolar and hydrogen bonding interactions contribute, respectively, 77% and 13% to the odorant binding affinities, as shown by an atom-based quantitative structure-activity relationship model.

A Multispecific Investigation of the Metal Effect in Mammalian Odorant Receptors for Sulfur-Containing Compounds.

Metal-coordinating compounds are generally known to have strong smells, a phenomenon that can be attributed to the fact that odorant receptors for intense-smelling compounds, such as those containing sulfur, may be metalloproteins. We previously identified a mouse odorant receptor (OR), Olfr1509, that requires copper ions for sensitive detection of a series of metal-coordinating odorants, including (methylthio)methanethiol (MTMT), a strong-smelling component of male mouse urine that attracts female mice. By combining mutagenesis and quantum mechanics/molecular mechanics (QM/MM) modeling, we identified candidate binding sites in Olfr1509 that may bind to the copper-MTMT complex. However, whether there are other receptors utilizing metal ions for ligand-binding and other sites important for receptor activation is still unknown. In this study, we describe a second mouse OR for MTMT with a copper effect, namely Olfr1019. In an attempt to investigate the functional changes of metal-coordinating ORs in multiple species and to decipher additional sites involved in the metal effect, we cloned various mammalian orthologs of the 2 mouse MTMT receptors, and a third mouse MTMT receptor, Olfr15, that does not have a copper effect. We found that the function of all 3 MTMT receptors varies greatly among species and that the response to MTMT always co-occurred with the copper effect. Furthermore, using ancestral reconstruction and QM/MM modeling combined with receptor functional assay, we found that the amino acid residue R260 in Olfr1509 and the respective R261 site in Olfr1019 may be important for receptor activation.

Live-cell Measurement of Odorant Receptor Activation Using a Real-time cAMP Assay.

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. To efficiently and accurately deorphanize ORs, we combine the use of a heterologous cell line to express mammalian ORs and a genetically modified biosensor plasmid to measure cAMP production downstream of OR activation in real time. This assay can be used to screen odorants against ORs and vice versa. Positive odorant-receptor interactions from the screens can be subsequently confirmed by testing against various odor concentrations, generating concentration-response curves. Here we used this method to perform a high-throughput screening of an odorous compound against a human OR library expressed in Hana3A cells and confirmed that the positively-responding receptor is the cognate receptor for the compound of interest. We found this high-throughput detection method to be efficient and reliable in assessing OR activation and our data provide an example of its potential use in OR functional studies.

Effect of Modified Pulsatilla Powder on Enterotoxigenic Escherichia coli O101-Induced Diarrhea in Mice.

MPP can be effective in the treatment of E. coli O101-induced diarrhea in mice. MPP can improve the weight loss caused by diarrhea, increase spleen and thymus indices, and reduce the diarrhea index. MPP can reduce the number of WBC, regulate the level of cytokines, and regulate the intestinal microbial flora. These data suggest that MPP is a promising candidate for treatment of E. coli-induced diarrhea in humans and animals.

Effect of Chuanminshen violaceum polysaccharides and its sulfated derivatives on immunosuppression induced by cyclophosphamide in mice.

One hundred mice were randomly divided into five groups. The mice in one group were injected with physiological saline as the normal control group. The mice in the other four groups were injected with physiological saline, sulfated Chuanminshen violaceum polysaccharides (SCVP), Chuanminshen violaceum polysaccharide (CVP) and astragalus polysaccharide (AP) once daily for 7 d and then with cyclophosphamide (CY) in the last 3 d. The serum cytokine level, apoptosis protein expressions, spleen lymphocyte proliferation, changes in peripheral blood T-cell subsets, and immune organ index were then measured. Results showed that SCVP and CVP can overcome CY-induced immunosuppression by promoting spleen lymphocyte proliferation, raising serum IFN-γ and IL-2 levels, enlarging immune organ indexes, and decreasing excessive apoptosis. Moreover, SCVP and CVP showed the potential to treat autoimmune diseases based on CD4(+)/CD8(+) ratios. Results suggested that SCVP and CVP exhibited the potential to treat autoimmune and immunosuppression diseases.

Antiviral effect of sulfated Chuanmingshen violaceum polysaccharide in chickens infected with virulent Newcastle disease virus.

Newcastle disease virus (NDV) belonging to the Paramyxovirinae subfamily is one of the most devastating pathogens in poultry. Although vaccines are widely applied to control the infection, outbreaks of Newcastle disease (ND) repeatedly happen. Currently, there are no alternative control measures available for ND. In the present study, we found that sulfated Chuanmingshen violaceum polysaccharide (sCVPS) were potent inhibitors of NDV in specific pathogen free chickens infected with a virulent strain. With sCVPS treatment, the survival rate increased by almost 20% and virus titers in test organs, including brain, lung, spleen and thymus, were significantly decreased. The sCVPS also exhibited the ability to prevent viral transmission by reducing the amount of virus shed in saliva and feces. Higher concentrations of interferon α and γ in serum were detected in chickens treated with sCVPS, indicating that one of the antiviral mechanisms may be attributed to the property of immunoenhancement. Histopathological examination showed that sCVPS could alleviate the tissue lesions caused by NDV infection. These results suggest that sCVPS are expected to be a new alternative control measure for NDV infection and further studies could be carried out to evaluate the antiviral activity of sCVPS against other paramyxoviruses.

Greater taxol yield of fungus Pestalotiopsis hainanensis from dermatitic scurf of the giant panda (Ailuropoda melanoleuca).

While taxol yields of fungi from non-animal sources are still low, whether Pestalotiopsis hainanensis isolated from the scurf of a dermatitic giant panda, Ailuropoda melanoleuca, provides a greater taxol yield remains unknown. The objective of the study was to determine the corresponding taxol yield. The structure of the taxol produced by the fungus was evaluated by thin layer chromatography (TLC), ultraviolet (UV) spectroscopy, high-performance liquid chromatography (HPLC), (1)H and (13)C nuclear magnetic resonance spectroscopy ((1)H-NMR and (13)C-NMR), and time-of-flight mass spectrometry (TOF-MS), with standard taxol as a control. The results demonstrated that the P. hainanensis fungus produced taxol, which had the same structure as the standard taxol and yield of 1,466.87 µg/L. This fungal taxol yield from the dermatitic giant panda was significantly greater than those of fungus from non-animal sources. The taxol-producing fungus may be a potential candidate for the production of taxol on an industrial scale.