Fine Epitope Mapping of the Central Immunodominant Region of Nucleoprotein from Crimean-Congo Hemorrhagic Fever Virus (CCHFV)

Crimean-Congo hemorrhagic fever (CCHF), a severe viral disease known to have occurred in over 30 countries and distinct regions, is caused by the tick-borne CCHF virus (CCHFV). Nucleocapsid protein (NP), which is encoded by the S gene, is the primary antigen detectable in infected cells. The goal of the present study was to map the minimal motifs of B-cell epitopes (BCEs) on NP. Five precise BCEs (E1, ²⁴⁷FDEAKK²⁵²; E2a, ²⁵⁴VEAL²⁵⁷; E2b, ²⁵⁸NGYLNKH²⁶⁴; E3, ²⁶⁷EVDKA²⁷¹; and E4, ²⁷⁴DSMITN²⁷⁹) identified through the use of rabbit antiserum, and one BCE (E5, ²⁵⁸NGYL²⁶¹) recognized using a mouse monoclonal antibody, were confirmed to be within the central region of NP and were partially represented among the predicted epitopes. Notably, the five BCEs identified using the rabbit sera were able to react with positive serum mixtures from five sheep which had been infected naturally with CCHFV. The multiple sequence alignment (MSA) revealed high conservation of the identified BCEs among ten CCHFV strains from different areas. Interestingly, the identified BCEs with only one residue variation can apparently be recognized by the positive sera of sheep naturally infected with CCHFV. Computer-generated three-dimensional structural models indicated that all the antigenic motifs are located on the surface of the NP stalk domain. This report represents the first identification and mapping of the minimal BCEs of CCHFV-NP along with an analysis of their primary and structural properties. Our identification of the minimal linear BCEs of CCHFV-NP may provide fundamental data for developing rapid diagnostic reagents and illuminating the pathogenic mechanism of CCHFV.     

Isolation and characterization of phenanthrene-degrading <Emphasis Type="Italic">Sphingomonas paucimobilis</Emphasis> strain ZX4

Phenanthrene-degrading bacterium strain ZX4 was isolated from an oil-contaminated soil, and identified as Sphingomonas paucimobilis based on 16S rDNA sequence, cellular fatty acid composition, mol% G + C and Biolog-GN tests. Besides phenanthrene, strain ZX4 could also utilize naphthalene, fluorene and other aromatic compounds. The growth on salicylic acid and catechol showed that the strain degraded phenanthrene via salicylate pathway, while the assay of catechol 2, 3-dioxygenase revealed catechol could be metabolized through meta-cleavage pathway. Three genes, including two of meta-cleavage operon genes and one of GST encoding gene were obtained. The order of genes arrangement was similar to S-type meta-pathway operons. The phylogenetic trees based on 16S rDNA sequence and meta-pathway gene both revealed that strain ZX4 is clustered with strains from genus Sphingomonas.     

Treatment of H2S using a horizontal biotrickling filter based on biological activated carbon: reactor setup and performance evaluation

Biological treatment is an emerging and prevalent technology for treating off-gases from wastewater treatment plants. The most commonly reported odorous compound in off-gases is hydrogen sulfide (H₂S), which has a very low odor threshold. A self-designed, bench-scale, cross-flow horizontal biotrickling filter (HBF) operated with bacteria immobilized activated carbon (termed biological activated carbon—BAC), was applied for the treatment of H₂S. A mixed culture of sulfide-oxidizing bacteria dominated by Acidithiobacillus thiooxidans acclimated from activated sludge was used as bacterial seed and the biofilm was developed by culturing the bacteria in the presence of carbon pellets in mineral medium. HBF performance was evaluated systematically over 120 days, depending on a series of changing factors including inlet H₂S concentration, gas retention time (GRT), pH of recirculation solution, upset and recovery, sulfate accumulation, pressure drop, gas-liquid ratio, and shock loading. The biotrickling filter system can operate at high efficiency from the first day of operation. At a volumetric loading of 900 m³ m^–3 h^–1 (at 92 ppmv H₂S inlet concentration), the BAC exhibited maximum elimination capacity (113 g H₂S/m^–3 h^–1) and a removal efficiency of 96% was observed. If the inlet concentration was kept at around 20 ppmv, high H₂S removal (over 98%) was achieved at a GRT of 4 s, a value comparable with those currently reported for biotrickling filters. The bacterial population in the acidic biofilter demonstrated capacity for removal of H₂S over a broad pH range (pH 1–7). A preliminary investigation into the different effects of bacterial biodegradation and carbon adsorption on system performance was also conducted. This study shows the HBF to be a feasible and economic alternative to physical and chemical treatments for the removal of H₂S.     

miRConnect: identifying effector genes of miRNAs and miRNA families in cancer cells

micro(mi)RNAs are small non-coding RNAs that negatively regulate expression of most mRNAs. They are powerful regulators of various differentiation stages, and the expression of genes that either negatively or positively correlate with expressed miRNAs is expected to hold information on the biological state of the cell and, hence, of the function of the expressed miRNAs. We have compared the large amount of available gene array data on the steady state system of the NCI60 cell lines to two different data sets containing information on the expression of 583 individual miRNAs. In addition, we have generated custom data sets containing expression information of 54 miRNA families sharing the same seed match. We have developed a novel strategy for correlating miRNAs with individual genes based on a summed Pearson Correlation Coefficient (sPCC) that mimics an in silico titration experiment. By focusing on the genes that correlate with the expression of miRNAs without necessarily being direct targets of miRNAs, we have clustered miRNAs into different functional groups. This has resulted in the identification of three novel miRNAs that are linked to the epithelial-to-mesenchymal transition (EMT) in addition to the known EMT regulators of the miR-200 miRNA family. In addition, an analysis of gene signatures associated with EMT, c-MYC activity, and ribosomal protein gene expression allowed us to assign different activities to each of the functional clusters of miRNAs. All correlation data are available via a web interface that allows investigators to identify genes whose expression correlates with the expression of single miRNAs or entire miRNA families. miRConnect.org will aid in identifying pathways regulated by miRNAs without requiring specific knowledge of miRNA targets.     

Biochemical basis for the requirement of kinase activity for Cbl-dependent ubiquitinylation and degradation of a target tyrosine kinase

Members of the Cbl family of ubiquitin ligases have emerged as crucial negative regulators of tyrosine kinase signaling. These proteins preferentially interact with and target activated tyrosine kinases for ubiquitinylation, thereby facilitating the lysosomal sorting of receptor tyrosine kinases or proteasomal degradation of nonreceptor tyrosine kinases. Recent work has indicated a crucial role of the target kinase activity in Cbl-dependent ubiquitinylation and degradation, but the biochemical basis for this requirement is not understood. Here, we have used the Src-family kinase Fyn, a well characterized Cbl target, to address this issue. Using defined Fyn mutants, we demonstrate that the kinase activity of Fyn is crucial for its Cbl-dependent ubiquitinylation and degradation, but a low level of ubiquitinylation and degradation of kinase-inactive Fyn mutants was consistently observed. Mutational induction of an open conformation enhanced the susceptibility of kinase-active Fyn to Cbl but was insufficient to promote the ubiquitinylation and degradation of kinase-inactive Fyn. Notably, the Cbl-dependent degradation of Fyn did not require the Fyn-mediated phosphorylation of Cbl. Finally, we show that the major determinant of the susceptibility of Fyn protein to Cbl-dependent ubiquitinylation and degradation is the extent to which it physically associates with Cbl; kinase activity of Fyn serves as a critical determinant to promote its association with Cbl, which we demonstrate is mediated by multiple protein-protein interactions. Our results strongly suggest that promotion of association with Cbl is the primary mechanism by which the kinase activity of the targets of Cbl contributes to their susceptibility to Cbl.     

Small molecules blocking the entry of severe acute respiratory syndrome coronavirus into host cells

Severe acute respiratory syndrome coronavirus (SARS-CoV) is the pathogen of SARS, which caused a global panic in 2003. We describe here the screening of Chinese herbal medicine-based, novel small molecules that bind avidly with the surface spike protein of SARS-CoV and thus can interfere with the entry of the virus to its host cells. We achieved this by using a two-step screening method consisting of frontal affinity chromatography-mass spectrometry coupled with a viral infection assay based on a human immunodeficiency virus (HIV)-luc/SARS pseudotyped virus. Two small molecules, tetra-O-galloyl-beta-D-glucose (TGG) and luteolin, were identified, whose anti-SARS-CoV activities were confirmed by using a wild-type SARS-CoV infection system. TGG exhibits prominent anti-SARS-CoV activity with a 50% effective concentration of 4.5 microM and a selective index of 240.0. The two-step screening method described here yielded several small molecules that can be used for developing new classes of anti-SARS-CoV drugs and is potentially useful for the high-throughput screening of drugs inhibiting the entry of HIV, hepatitis C virus, and other insidious viruses into their host cells.     

Intein-Mediated Protein trans-Splicing of the Recombinant Streptavidin on Magnetosomes

Molecular Biology - When expressing streptavidin recombinant polypeptide on magnetosomes (called bacterial magnetic nanoparticles, or BMPs), the presence of endogenous bacterial biotin might be...     

A novel domain-duplicated SlitFAR3 gene involved in sex pheromone biosynthesis in Spodoptera litura

Fatty acyl reductases (FARs) are key enzymes that participate in sex pheromone biosynthesis by reducing fatty acids to fatty alcohols. Lepidoptera typically harbor numerous FAR gene family members. Although FAR genes are involved in the biosynthesis of sex pheromones in moths, the key FAR gene of Spodoptera litura remains unclear. In this work, we predicted 30 FAR genes from the S. litura genome and identified a domain duplication within gene SlitFAR3, which exhibited high and preferential expression in the sexually mature female pheromone glands (PGs) and a rhythmic expression pattern during the scotophase of sex pheromone production. The molecular docking of SlitFAR3, as predicted using a 3D model, revealed a co-factor NADPH binding cavity and 2 substrate binding cavities. Functional expression in yeast cells combined with comprehensive gas chromatography indicated that the SlitFAR3 gene could produce fatty alcohol products. This study is the first to focus on the special phenomenon of FAR domain duplication, which will advance our understanding of biosynthesis-related genes from the perspective of evolutionary biology.     

Anti-cancer activities of tea epigallocatechin-3-gallate in breast cancer patients under radiotherapy

The purpose of this study was to test the hypothesis that administration of epigallocatechin-3-gallate (EGCG), a polyphenol present in abundance in widely consumed tea, inhibits cell proliferation, invasion, and angiogenesis in breast cancer patients. EGCG in 400 mg capsules was orally administered three times daily to breast cancer patients undergoing treatment with radiotherapy. Parameters related to cell proliferation, invasion, and angiogenesis were analyzed while blood samples were collected at different time points to determine efficacy of the EGCG treatment. Compared to patients who received radiotherapy alone, those given radiotherapy plus EGCG for an extended time period (two to eight weeks) showed significantly lower serum levels of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and reduced activation of metalloproteinase-9 and metalloproteinase-2 (MMP9/MMP2). Addition of sera obtained from patients treated with combination of radiotherapy and EGCG feeding for 2-8 weeks to in vitro cultures of highly-metastatic human MDA-MB-231 breast cancer cells resulted in the following significant changes: (1) suppression of cell proliferation and invasion; (2) arrest of cell cycles at the G0/G1 phase; (3) reduction of activation of MMP9/MMP2, expressions of Bcl-2/Bax, c-Met receptor, NF-κB, and the phosphorylation of Akt. MDA-MB-231 cells exposed to 5-10 μM EGCG also showed significant augmentation of the apoptosis inducing effects of γ-radiation, concomitant with reduced NF-κB protein level and AKT phosphorylation. These results provide hitherto unreported evidence that EGCG potentiated efficacy of radiotherapy in breast cancer patients, and raise the possibility that this tea polyphenol has potential to be a therapeutic adjuvant against human metastatic breast cancer.