Algivirga pacifica gen. nov., sp. nov., a Novel Agar-Degrading Marine Bacterium of the Family Flammeovirgaceae Isolated from Micronesia

An aerobic, Gram-negative, coccoid to short rod-shaped and non-flagellated marine bacterial strain S354^T was isolated from seawater of Micronesia. The strain was capable to degrade agar-forming slight depression into agar plate. Growth occurred at a temperature range of 12–44 °C, a pH range of 5–9, and a salinity range of 1–7 % (w/v) NaCl. Phylogenetic analyses based on 16S rRNA gene sequences suggested that S354^T belongs to the family Flammeovirgaceae. The novel strain was most closely related to Limibacter armeniacum YM 11-185^T with similarity of 92.5 %. The DNA G+C content was 43.8 mol%. The major fatty acids (>10 %) were iso-C_15:0 and C_16:1 ω5c. The predominant isoprenoid quinone was determined to be MK-7. Polar lipid profile of S354^T consisted of phosphatidylethanolamine, unknown polar lipid, and unknown glycolipids. Based on the phenotypic, phylogenetic, biochemical, and physiological tests conducted in this study, S354^T is proposed to represent a type strain of a novel genus and species. The 16S rRNA gene sequence of S354^T is registered in GenBank under the accession number JQ639084. The type of strain Algivirga pacifica gen. nov., sp. nov. is S354^T (=KCCM 90107^T=JCM 18326^T).     

Effect of GABA Receptor Agonists or Antagonists Injected Spinally on the Blood Glucose Level in Mice

The possible roles of gamma-amino butyric acid (GABA) receptors located in the spinal cord for the regulation of the blood glucose level were studied in ICR mice. We found in the present study that intrathecal (i.t.) injection with baclofen (a GABA_B receptor agonist; 1–10 μg/5 μl) or bicuculline (a GABA_A receptor antagonist; 1–10 μg/5 μl) caused an elevation of the blood glucose level in a dose-dependent manner. The hyperglycemic effect induced by baclofen was more pronounced than that induced by bicuculline. However, muscimol (a GABA_A receptor agonist; 1–5 μg/5 μl) or phaclofen (a GABA_B receptor antagonist; 5–10 μg/5 μl) administered i.t. did not affect the blood glucose level. Baclofen–induced elevation of the blood glucose was dose-dependently attenuated by phaclofen. Furthermore, i.t. pretreatment with pertussis toxin (PTX; 0.05 or 0.1 μg/5 μl) for 6 days dose-dependently reduced the hyperglycemic effect induced by baclofen. Our results suggest that GABA_B receptors located in the spinal cord play important roles for the elevation of the blood glucose level. Spinally located PTX-sensitive G-proteins appear to be involved in hyperglycemic effect induced by baclofen. Furthermore, inactivation of GABA_A receptors located in the spinal cord appears to be responsible for tonic up-regulation of the blood glucose level.     

The Homologous Tripartite Viral RNA Polymerase of A/Swine/Korea/CT1204/2009(H1N2) Influenza Virus Synergistically Drives Efficient Replication and Promotes Respiratory Droplet Transmission in Ferrets

We previously reported that influenza A/swine/Korea/1204/2009(H1N2) virus was virulent and transmissible in ferrets in which the respiratory-droplet-transmissible virus (CT-Sw/1204) had acquired simultaneous hemagglutinin (HA_D225G) and neuraminidase (NA_S315N) mutations. Incorporating these mutations into the nonpathogenic A/swine/Korea/1130/2009(H1N2, Sw/1130) virus consequently altered pathogenicity and growth in animal models but could not establish efficient transmission or noticeable disease. We therefore exploited various reassortants of these two viruses to better understand and identify other viral factors responsible for pathogenicity, transmissibility, or both. We found that possession of the CT-Sw/1204 tripartite viral polymerase enhanced replicative ability and pathogenicity in mice more significantly than did expression of individual polymerase subunit proteins. In ferrets, homologous expression of viral RNA polymerase complex genes in the context of the mutant Sw/1130 carrying the HA_225G and NA_315N modifications induced optimal replication in the upper nasal and lower respiratory tracts and also promoted efficient aerosol transmission to respiratory droplet contact ferrets. These data show that the synergistic function of the tripartite polymerase gene complex of CT-Sw/1204 is critically important for virulence and transmission independent of the surface glycoproteins. Sequence comparison results reveal putative differences that are likely to be responsible for variation in disease. Our findings may help elucidate previously undefined viral factors that could expand the host range and disease severity induced by triple-reassortant swine viruses, including the A(H1N1)pdm09 virus, and therefore further justify the ongoing development of novel antiviral drugs targeting the viral polymerase complex subunits.     

Structural and Functional Analysis of the Natural JNK1 Inhibitor Quercetagetin

c-Jun NH2-terminal kinases (JNKs) and phosphatidylinositol 3-kinase (PI3-K) play critical roles in chronic diseases such as cancer, type II diabetes, and obesity. We describe here the binding of quercetagetin (3,3′,4′,5,6,7-hydroxyflavone), related flavonoids, and SP600125 to JNK1 and PI3-K by ATP-competitive and immobilized metal ion affinity-based fluorescence polarization assays and measure the effect of quercetagetin on JNK1 and PI3-K activities. Quercetagetin attenuated the phosphorylation of c-Jun and AKT, suppressed AP-1 and NF-κB promoter activities, and also reduced cell transformation. It attenuated tumor incidence and reduced tumor volumes in a two-stage skin carcinogenesis mouse model.Our crystallographic structure determination data show that quercetagetin binds to the ATP-binding site of JNK1. Notably, the interaction between Lys55, Asp169, and Glu73 of JNK1 and the catechol moiety of quercetagetin reorients the N-terminal lobe of JNK1, thereby improving compatibility of the ligand with its binding site. The results of a theoretical docking study suggest a binding mode of PI3-K with the hydroxyl groups of the catechol moiety forming hydrogen bonds with the side chains of Asp964 and Asp841 in the p110γ catalytic subunit. These interactions could contribute to the high inhibitory activity of quercetagetin against PI3-K. Our study suggests the potential use of quercetagetin in the prevention or therapy of cancer and other chronic diseases.     

Macro- to Microscale Strain Transfer in Fibrous Tissues is Heterogeneous and Tissue-Specific

Mechanical deformation applied at the joint or tissue level is transmitted through the macroscale extracellular matrix to the microscale local matrix, where it is transduced to cells within these tissues and modulates tissue growth, maintenance, and repair. The objective of this study was to investigate how applied tissue strain is transferred through the local matrix to the cell and nucleus in meniscus, tendon, and the annulus fibrosus, as well as in stem cell-seeded scaffolds engineered to reproduce the organized microstructure of these native tissues. To carry out this study, we developed a custom confocal microscope-mounted tensile testing device and simultaneously monitored strain across multiple length scales. Results showed that mean strain was heterogeneous and significantly attenuated, but coordinated, at the local matrix level in native tissues (35–70% strain attenuation). Conversely, freshly seeded scaffolds exhibited very direct and uniform strain transfer from the tissue to the local matrix level (15–25% strain attenuation). In addition, strain transfer from local matrix to cells and nuclei was dependent on fiber orientation and tissue type. Histological analysis suggested that different domains exist within these fibrous tissues, with most of the tissue being fibrous, characterized by an aligned collagen structure and elongated cells, and other regions being proteoglycan (PG)-rich, characterized by a dense accumulation of PGs and rounder cells. In meniscus, the observed heterogeneity in strain transfer correlated strongly with cellular morphology, where rounder cells located in PG-rich microdomains were shielded from deformation, while elongated cells in fibrous microdomains deformed readily. Collectively, these findings suggest that different tissues utilize distinct strain-attenuating mechanisms according to their unique structure and cellular phenotype, and these differences likely alter the local biologic response of such tissues and constructs in response to mechanical perturbation.     

In vitro antiviral activity of phlorotannins isolated from Ecklonia cava against porcine epidemic diarrhea coronavirus infection and hemagglutination

Despite the prepdominat agent causing severe entero-pathogenic diarrhea in swine, there are no effective therapeutical treatment of porcine epidemic diarrhea virus (PEDV). In this study, we evaluated the antiviral activity of five phlorotannins isolated from Ecklonia cava (E. cava) against PEDV. In vitro antiviral activity was tested using two different assay strategies: (1) blockage of the binding of virus to cells (simultaneous-treatment assay) and (2) inhibition of viral replication (post-treatment assay). In simultaneous-treatment assay, compounds 2–5 except compound 1 exhibited antiviral activities of a 50% inhibitory concentration (IC₅₀) with the ranging from 10.8 ± 1.4 to 22.5 ± 2.2 μM against PEDV. Compounds 1–5 were completely blocked binding of viral spike protein to sialic acids at less than 36.6 μM concentrations by hemagglutination inhibition. Moreover, compounds 4 and 5 of five phlorotannins inhibited viral replication with IC₅₀ values of 12.2 ± 2.8 and 14.6 ± 1.3 μM in the post-treatment assay, respectively. During virus replication steps, compounds 4 and 5 exhibited stronger inhibition of viral RNA and viral protein synthesis in late stages (18 and 24 h) than in early stages (6 and 12 h). Interestingly, compounds 4 and 5 inhibited both viral entry by hemagglutination inhibition and viral replication by inhibition of viral RNA and viral protein synthesis, but not viral protease. These results suggest that compounds isolated from E. cava have strong antiviral activity against PEDV, inhibiting viral entry and/or viral replication, and may be developed into natural therapeutic drugs against coronavirus infection.     

In-gel total protein quantification using a ninhydrin-based method

Precise in-gel quantification of total protein amount of bands or spots in gels is the basis of subsequent biochemical, molecular biological and immunological analyses. Though several methods have been designed to evaluate relative amounts of proteins, these methods are of limited reliability because (semi-) quantifications depend on the amount of protein migrating into the gel and different proteins may lead to different absorptions/intensities of stained bands or spots. In the present study, we described a method to quantify both, hydrophilic and hydrophobic proteins using in-gel digestion with proteinase K, subsequent extraction and acid hydrolysis followed by the use of the ninhydrin reaction. The protocol is accurate and compatible with mass spectrometric characterization of proteins. Reproducible in-gel protein quantification was performed from SDS-PAGE and IEF/SDS-PAGE gels using bovine serum albumin as a standard protein. Bacteriorhodopsin separated on SDS-PAGE gel was quantified in addition in order to show that the method is also suitable for quantification of hydrophobic protein. This protocol for reliable in-gel protein quantification, which not only provides “arbitrary units of optical density”, can also be completed in a minimum of 4 days or maximum 1 week depending on the type of electrophoresis with the disadvantage of being time consuming.     

Attenuation by a Vigna nakashimae extract of nonalcoholic fatty liver disease in high-fat diet-fed mice

A Vigna nakashimae (VN) extract has been shown to have antidiabetic and anti-obesity effects. However, the mechanism underlying the effect of a VN extract on hepatic inflammation and endoplasmic reticulum (ER) stress remains unclear. In the present study, we investigated how a VN extract protects against the development of non-alcoholic fatty liver disease (NAFLD). A VN extract for 12 weeks reduced the body weight, serum metabolic parameters, cytokines, and hepatic steatosis in high-fat diet (HFD)-fed mice. A VN extract decreased HFD-induced hepatic acetyl CoA carboxylase and glucose transporter 4 expressions. In addition to the levels of high-mobility group box 1 and receptor for advanced glycation, the hepatic expression of ATF4 and caspase-3 was also reduced by a VN extract. Thus, these data indicate that a chronic VN extract prevented NAFLD through multiple mechanisms, including inflammation, ER stress, and apoptosis in the liver.     

Identifying MicroRNA and mRNA Expression Profiles in Embryonic Stem Cells Derived from Parthenogenetic, Androgenetic and Fertilized Blastocysts

MicroRNAs (miRNAs) are a class of highly conserved small non-coding RNA molecules that play a pivotal role in several cellular functions. In this study, miRNA and messenger RNA (mRNA) profiles were examined by Illumina microarray in mouse embryonic stem cells (ESCs) derived from parthenogenetic, androgenetic, and fertilized blastocysts. The global analysis of miRNA-mRNA target pairs provided insight into the role of miRNAs in gene expression. Results showed that a total of 125 miRNAs and 2394 mRNAs were differentially expressed between androgenetic ESCs (aESCs) and fertilized ESCs (fESCs), a total of 42 miRNAs and 87 mRNAs were differentially expressed between parthenogenetic ESCs (pESCs) and fESCs, and a total of 99 miRNAs and 1788 mRNAs were differentially expressed between aESCs and pESCs. In addition, a total of 575, 5 and 376 miRNA-mRNA target pairs were observed in aESCs vs. fESCs, pESCs vs. fESCs, and aESCs vs. pESCs, respectively. Furthermore, 15 known imprinted genes and 16 putative uniparentally expressed miRNAs with high expression levels were confirmed by both microarray and real-time RT-PCR. Finally, transfection of miRNA inhibitors was performed to validate the regulatory relationship between putative maternally expressed miRNAs and target mRNAs. Inhibition of miR-880 increased the expression of Peg3, Dyrk1b, and Prrg2 mRNA, inhibition of miR-363 increased the expression of Nfat5 and Soat1 mRNA, and inhibition of miR-883b-5p increased Nfat5, Tacstd2, and Ppapdc1 mRNA. These results warrant a functional study to fully understand the underlying regulation of genomic imprinting in early embryo development.