Characterization of a Novel Picornavirus Isolate from a Diseased European Eel (Anguilla anguilla)

A novel picornavirus was isolated from specimens of a diseased European eel (Anguilla anguilla). This virus induced a cytopathic effect in eel embryonic kidney cells and high mortality in a controlled transmission study using elvers. Eel picornavirus has a genome of 7,496 nucleotides that encodes a polyprotein of 2,259 amino acids. It has a typical picornavirus genome layout, but its low similarity to known viral proteins suggests a novel species in the family Picornaviridae.     

A Novel Strategy to Isolate Ubiquitin Conjugates Reveals Wide Role for Ubiquitination during Neural Development

Ubiquitination has essential roles in neuronal development and function. Ubiquitin proteomics studies on yeast and HeLa cells have proven very informative, but there still is a gap regarding neuronal tissue-specific ubiquitination. In an organism context, direct evidence for the ubiquitination of neuronal proteins is even scarcer. Here, we report a novel proteomics strategy based on the in vivo biotinylation of ubiquitin to isolate ubiquitin conjugates from the neurons of Drosophila melanogaster embryos. We confidently identified 48 neuronal ubiquitin substrates, none of which was yet known to be ubiquitinated. Earlier proteomics and biochemical studies in non-neuronal cell types had identified orthologs to some of those but not to others. The identification here of novel ubiquitin substrates, those with no known ubiquitinated ortholog, suggests that proteomics studies must be performed on neuronal cells to identify ubiquitination pathways not shared by other cell types. Importantly, several of those newly found neuronal ubiquitin substrates are key players in synaptogenesis. Mass spectrometry results were validated by Western blotting to confirm that those proteins are indeed ubiquitinated in the Drosophila embryonic nervous system and to elucidate whether they are mono- or polyubiquitinated. In addition to the ubiquitin substrates, we also identified the ubiquitin carriers that are active during synaptogenesis. Identifying endogenously ubiquitinated proteins in specific cell types, at specific developmental stages, and within the context of a living organism will allow understanding how the tissue-specific function of those proteins is regulated by the ubiquitin system.Posttranslational modification of proteins by ubiquitin is involved in a wide range of cellular processes (1). Ubiquitination is linked to the turnover of an ever growing number of proteins; it regulates protein trafficking and is also widely used to transiently facilitate protein-protein interactions (2, 3). As the number of known ubiquitinated proteins keeps growing, the focus is turning toward identifying when, where, and how those proteins are ubiquitinated in vivo with the aim of understanding how protein function is being regulated within the context of a whole organism. The ubiquitin pathway is essential for brain development and function, and its failure is associated with a number of neurodegenerative diseases, including Parkinson and Alzheimer diseases (4–6). Ubiquitin conjugation is carried out by the sequential action of ubiquitin-activating (E1), -conjugating (E2), and -ligating (E3) enzymes and can be reversed by deubiquitinating enzyme (DUB)1 proteases. The involvement of a number of those enzymes in synaptogenesis has been documented in several model systems (7–12). In Drosophila, for example, synaptogenesis is dependent on the E3 ligase Highwire and on the DUB fat facets (13). A few proteins involved in synaptogenesis have been shown to be ubiquitin substrates, including the postsynaptic proteins Shank, GKAP, and AKAP79/150 in cultured neurons (14) and the Caenorhabditis elegans synaptic protein DLK-1 kinase, which was shown to be ubiquitinated when overexpressed in HEK293T kidney cells (9). Most neuronal targets of the ubiquitin pathway, however, remain undiscovered. Yeast and HeLa cell-based proteomics approaches have failed to provide significant insights into the neuronal mechanisms regulated by ubiquitination. With the exception of a polyubiquitin affinity-based purification that successfully identified by Western blotting three ubiquitin substrates in cultured neurons (14), no proteomics approach has been described that can identify ubiquitinated neuronal proteins. Because neuronal function and activity are highly context-dependent, rather than working on neuronal culture, we have aimed to identify which proteins are ubiquitinated in vivo within the neurons of a living organism.Herein, we describe a novel strategy for the efficient isolation of neuronal ubiquitin conjugates from flies. The approach is based on the in vivo biotinylation of ubiquitin by ectopically expressing the Escherichia coli BirA enzyme to attach a biotin molecule to a specific BirA recognition sequence (15, 16) added at the N terminus of each ubiquitin chain. With the purpose of isolating ubiquitin conjugates uniquely from the nervous system of Drosophila melanogaster, we used the GAL4/UAS system for tissue-targeted expression (17). To increase the biotinylation efficiency, we took advantage of the processing activity of endogenous DUBs to digest a linear polypeptide precursor containing six copies of the tagged ubiquitin and the BirA enzyme, which are then present in the same cellular microenvironment. Because of the strength and the specificity of the avidin-biotin interaction, we were able to isolate and enrich the neuronal ubiquitinated proteins from a multicellular organism up to levels not achieved previously by any other approach. This allowed us to identify by mass spectrometry those neuronal proteins that are ubiquitinated and to resolve by Western blotting whether they are mono- or polyubiquitinated. This was achieved in the absence of proteasome inhibitors; therefore, physiological ubiquitination levels are reported. We focused on identifying the proteins that are ubiquitinated within the neurons in the period from neurite outgrowth and axonal pathfinding to target recognition and synapse formation (18). For that purpose, we applied our strategy on postmitotic neurons during embryonic stages 13–17 (19), a 12-h period during which embryos undergo synaptogenesis. Our strategy could be used to isolate ubiquitin conjugates from other tissues from the fruit flies, from different developmental stages, and in different mutant backgrounds, and it is likely to be applicable to other model organisms.     

A Novel Strategy To Develop a Robust Infectious Hepatitis C Virus Cell Culture System Directly from a Clinical Isolate

Hepatitis C virus (HCV) infection is a leading cause of chronic liver diseases. Progress in the HCV field was greatly enhanced by constructing infectious cDNA clone of JFH-1. Since then, JFH-1-based intra- and intergenotypic recombinants have been developed, and this permitted the study of vaccines and antiviral inhibitors for all genotypes. Recently, highly efficient HCV culture systems have been established by using consensus sequence-based clones. We developed a novel strategy to construct infectious HCV cDNA clone by combining functional screening of sequences directly from a genotype 2a clinical isolate (PR63) and cell culture adaptation. Using JFH-1 cDNA as the starting backbone, we sequentially replaced the JFH-1 fragments with a sequence from the pools of PR63 sequences. Through engineering adaptive mutations that improve HCV infectivity, we finally established a full-length cell culture-derived infectious clone of PR63, named PR63cc, that could efficiently produce virus particles in Huh7-derived cells, with peak titers of 1.6 × 10⁵ focus-forming units/ml. The PR63cc could be neutralized by an anti-E2 antibody and inhibited by antiviral agents but appeared more resistant to an NS5A inhibitor than JFH-1. In summary, we developed a new approach to construct an infectious HCV cDNA clone that can produce viruses efficiently in cell culture. This approach could be applied to other viral isolates, with potential implications for individualized treatments of HCV patients.     

Thermostable Alkaline Protease from a Novel Marine Haloalkalophilic Bacillus Clausii Isolate

An oxidative and SDS-stable alkaline protease secreted by a marine haloalkalophilic Bacillus clausii isolated from the tidal mud flats of the Korean Yellow Sea near Inchon City was investigated in batch fermentation in shake flasks and in a bioreactor under a range of conditions. The isolate produced maximum protease yields (15,000 U ml⁻¹) under submerged fermentation conditions at 42 °C for 40 h with an aeration of 1.5 v/v/min and agitation of 400 rev/min in a formulated soybean—casein medium (pH 9.6) containing (w/v): soybean meal (2%), casein (1%), corn starch (0.5%), NH₄Cl (0.05%), NaCl (0.05%), KH₂PO₄(0.04%), K₂HPO₄(0.03%), MgSO₄(0.02%), yeast extract (0.01%) and Na₂CO₃(0.6%). The optimal pH and temperature of activity of the partially purified enzyme were 11.5 and 80 °C, respectively. The alkaline protease showed extreme stability towards SDS and oxidizing agents, retaining its activity above 96 and 75% on treatment for 72 h with 5% SDS and 5% H₂O₂, respectively. The inhibition profile exhibited by phenylmethanesulphonyl fluoride suggested that the protease from B. clausii belongs to the family of serine proteases.     

Characterization of a Sarcocystis neurona. Isolate (SN6) from a Naturally Infected Horse from Oregon

An isolate of Sarcocystis neurona (SN6) was obtained from the spinal cord of a horse from Oregon with neurologic signs. The parasite was isolated in cultures of bovine monocytes and equine spleen cells. The parasite divided by endopolygeny and completed at least one asexual cycle in cell cultures in three days. Two gamma interferon knockout mice inoculated with cell culture-derived merozoites became ill 35 d later and S. neurona schizonts and merozoites were found in encephalitic lesions. The parasite in tissue sections of mice reacted with S. neurona-specific antibodies and S. neurona was reisolated from the brain of knockout mice.     

Molecular and Biological Characterization of a Cryptosporidium molnari-Like Isolate from a Guppy (Poecilia reticulata)

Histological, morphological, genetic, and phylogenetic analyses of a Cryptosporidium molnari-like isolate from a guppy (Poecilia reticulata) identified stages consistent with those of C. molnari and revealed that C. molnari is genetically very distinct from all other species of Cryptosporidium. This study represents the first genetic characterization of C. molnari.     

巴西甘薯叶亲脂性成分研究

巴西甘薯叶[Ipomoea batatas Lam.(cv.Simon)]的氯仿成分经反复硅胶柱层析分离得到了8个化合物,通过理化性质和波谱方法分别鉴定为：乙酰-β香树醇(1)、木栓酮(2)、表木栓醇(3)、三十烷醇(4)、β-谷甾醇(5)、咖啡酸乙酯(6)、东莨菪素(7)和胡萝卜苷(8)。其中3、4、6、7和8为首次从该植物中分得。     

Identification and Characterization of Novel Lipophilic Antimicrobial Peptides Derived from Naturally Occurring Proteins

Microbes are increasingly developing defensive mechanisms against known drugs via mutations. There are signs of emergence of superbugs immune to most known antibiotics available. The need for a new class of drugs to counteract this problem is of paramount importance for continued general well being of mankind. A new class of drugs, antimicrobial peptides, has not been fully exploited primarily due to high cytotoxicity, poor lipophilicity preventing systemic distribution and stability. We have synthesised 9-amino acid residue cationic peptides RH01 and RH02 lipidated with myristoyl and octyl groups respectively. These peptides exhibited potent antimicrobial activity and low cytotoxicity. The lipopeptide RH01 has antimicrobial activity against a broad range of microorganisms including bacteria, yeast and filamentous fungi with greatest activity toward Gram-positive bacteria, including S. aureus MRSA stain, MIC’s ranging between 2–8 μM. The MIC for Gram-negative bacteria was higher ranging from between 30–250 μM. RH01 also had antimicrobial activity towards fungi showing good activity against the pathogenic yeast Candida albicans but was less active towards the filamentous fungi Aspergillus niger. The antimicrobial activity of RH01 as a measure of Ki₍₅₀₎ for E. coli and S. aureus was 35–60 μM and 3–7 μM, respectively. In-house data showed the compound is bactericidal even at higher bacteria concentration. The octylated lipopeptide RH02 has similar activities towards S. aureus (3.3 μM) and E coli (53.3 μM) as the myristolated RH01. There was no haemolytic activity of the lipopeptide RH01 towards human blood. Acute intravenous toxicity study in mice showed that both RH01 and RH02 induced no macroscopic abnormalities at their highest non-lethal dose of 75 mg/kg and 150 mg/kg bodyweight, respectively.Australian Peptide Conference Issue.     

Aflatoxin-producing Potential of Isolates of the Aspergillus flavus-oryzae Group from Peanuts (Arachis hypogaea)

Seventy-eight samples of farmer stock peanuts, representing peanuts grown in nine different geographical areas during 1964, were assayed for aflatoxin and examined for associated microflora. Only two samples contained more than 50 ppb of aflatoxin. Infestation by members of the Aspergillus flavus-oryzae group varied from 35 to 100% of the kernels per area and from 1 to 100% of the kernels per sample. Aflatoxin production by individual isolates ranged from 0 to 349,143 ppb under the test conditions employed. In general, the isolates produced 8 to 10 times more B₁ than B₂, and no isolate producing aflatoxins G₁ or G₂ was found. The importance of proper postharvest handling of peanuts is emphasized by the prevalence of isolates of A. flavus-oryzae capable of producing aflatoxins on farmers stock peanuts.     

外显子捕捉法快速分离新基因的原理和方法

从大片段基因组DNA中快速分离转录序列是疾病基因定位克隆(positional cloning)中的关键步骤.外显子捕捉法是一种较为成功的方法.文章对该法的基本原理、方法步骤及应用成果等作了较为详细的介绍.     

生物信息学预测与实验验证相结合策略筛选鉴定新的人分泌蛋白基因

使用生物信息学预测结合实验验证的策略筛选鉴定人新的分泌蛋白基因。用SignalP、SOSUI、PSORT和BLAST等程序对UniProt蛋白数据库进行生物信息学分析 ,筛选出用于实验验证的 1 4个功能未知基因。采用RT PCR方法 ,克隆得到 1 4个基因的全长编码序列 ,并构建到真核表达载体pcDNA3.1 ( - ) Myc His质粒。采用蛋白质印迹与免疫荧光分析 ,检测到其中 7个基因的表达。除其中一个在细胞核表达外 ,其余 6个只在细胞质中表达 ;其中的 4个基因的表达产物在细胞培养液中可被检测到 ,鉴定为 4个新的分泌蛋白基因。     

龙须菜体表附生细菌的几种分离方法比较

采用超声波粉碎法、涡旋振荡法、超声波清洗法、研磨匀浆法等4种不同的方法处理龙须菜,分离其体表的附生细菌,并对分离细菌的数量、种类、形态结构、细胞壁特性等进行了观察和分析。通过对不同方法和相间方法的不同处理所得结果比较显示,超声波清洗法和研磨匀浆法对分离细菌的数量和种类效果都较差;超声波粉碎法和涡旋振荡法效果较好,尤以超声波粉碎法的30W30s处理效果最好,该方法分离到本项目4个方法13个处理获得的16个菌株中的12个菌株,龙须菜的细菌数1．75×10~6cells/g。     

龙须菜体表附生细菌的几种分离方法比较

采用超声波粉碎法、涡旋振荡法、超声波清洗法、研磨匀浆法等4种不同的方法处理龙须菜,分离其体表的附生细菌,并对分离细菌的数量、种类、形态结构、细胞壁特性等进行了观察和分析。通过对不同方法和相间方法的不同处理所得结果比较显示,超声波清洗法和研磨匀浆法对分离细菌的数量和种类效果都较差;超声波粉碎法和涡旋振荡法效果较好,尤以超声波粉碎法的30W30s处理效果最好,该方法分离到本项目4个方法13个处理获得的16个菌株中的12个菌株,龙须菜的细菌数1．75×10⁶cells/g。     

Identification of Two Metallothioneins as Novel Inhalative Coffee Allergens Cof a 2 and Cof a 3

BackgroundDust of green coffee beans is known to be a relevant cause for occupational allergic disorders in coffee industry workers. Recently, we described the first coffee allergen (Cof a 1) establishing an allergenic potential of green coffee dust.ObjectiveOur aim was to identify allergenic components of green coffee in order to enhance inhalative coffee allergy diagnosis.MethodsA Coffea arabica pJuFo cDNA phage display library was created and screened for IgE binding with sera from allergic coffee workers. Two further coffee allergens were identified by sequence analysis, expressed in E. coli, and evaluated by Western blots. The prevalence of sensitization to recombinant Cof a 1, Cof a 2, and Cof a 3 and to commercially available extract was investigated by ELISA (enzyme-linked immunosorbent assay) respectively CAP (capacity test) screening in 18 sera of symptomatic coffee workers.ResultsIn addition to the previously described chitinase Cof a 1, two Coffea arabica cysteine-rich metallothioneins of 9 and 7 kDa were identified and included in the IUIS Allergen Nomenclature as Cof a 2 and Cof a 3. Serum IgE antibodies to at least one of the recombinant allergens were found in 8 out of 18 symptomatic coffee workers (44%). Only 2 of the analysed sera (11%) had reacted previously to the commercial allergy test.ConclusionsIn addition to the previously described Cof a 1 we have identified two further coffee proteins to be type I coffee allergens (Cof a 2 and Cof a 3) which may have a relevant potential for the specific diagnosis and/or therapy of coffee allergy.     

Microfabricated Post-Array-Detectors (mPADs): an Approach to Isolate Mechanical Forces

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts. Traction forces are generated through myosin-actin interactions and play an important role in our physiology. During development, they enable cells to move from one location to the next in order to form the early structures of tissue. Traction forces help in the healing processes. They are necessary for the proper closure of wounds or the migration and crawling of leukocytes through our body. These same forces can be detrimental to our health in the case of cancer metastasis or vascular growth towards a tumor. The most common method by which to study cells in vitro has been to use a glass or polystyrene dish. However, the rigidity of the substrates makes it impossible to physically measure cell traction forces, and there are relatively few methods to study traction forces. Our lab has developed a technique to overcome these limitations. The method is based on a vertical array of flexible cantilevers, the stiffness and size scale of which are such that individual cells spread across many cantilevers and deflect them in the process. The pillars we use are 3 μm in diameter, 10 μm tall, and are configured in a regular array with 9 μm center-to-center spacing. But these physical dimensions can be readily varied to accommodate a variety of studies. We start with a silicon master, but the final posts are made out of silicone rubber called poly (dimethyl siloxane), or PDMS. We can measure the deflections under a microscope and calculate the magnitude and direction of traction forces required to produce the observed deflections. We call these substrates microfabricated post-array-detectors, or mPADs. Here, we will show you how we fabricate and use the mPADs to assess modulations of cellular contractility.     

CCAST: A Model-Based Gating Strategy to Isolate Homogeneous Subpopulations in a Heterogeneous Population of Single Cells

A model-based gating strategy is developed for sorting cells and analyzing populations of single cells. The strategy, named CCAST, for Clustering, Classification and Sorting Tree, identifies a gating strategy for isolating homogeneous subpopulations from a heterogeneous population of single cells using a data-derived decision tree representation that can be applied to cell sorting. Because CCAST does not rely on expert knowledge, it removes human bias and variability when determining the gating strategy. It combines any clustering algorithm with silhouette measures to identify underlying homogeneous subpopulations, then applies recursive partitioning techniques to generate a decision tree that defines the gating strategy. CCAST produces an optimal strategy for cell sorting by automating the selection of gating markers, the corresponding gating thresholds and gating sequence; all of these parameters are typically manually defined. Even though CCAST is optimized for cell sorting, it can be applied for the identification and analysis of homogeneous subpopulations among heterogeneous single cell data. We apply CCAST on single cell data from both breast cancer cell lines and normal human bone marrow. On the SUM159 breast cancer cell line data, CCAST indicates at least five distinct cell states based on two surface markers (CD24 and EPCAM) and provides a gating sorting strategy that produces more homogeneous subpopulations than previously reported. When applied to normal bone marrow data, CCAST reveals an efficient strategy for gating T-cells without prior knowledge of the major T-cell subtypes and the markers that best define them. On the normal bone marrow data, CCAST also reveals two major mature B-cell subtypes, namely CD123+ and CD123- cells, which were not revealed by manual gating but show distinct intracellular signaling responses. More generally, the CCAST framework could be used on other biological and non-biological high dimensional data types that are mixtures of unknown homogeneous subpopulations.     

Quantitative PCR Confirms Purity of Strain GT, a Novel Trichloroethene-to-Ethene-Respiring Dehalococcoides Isolate

A novel Dehalococcoides isolate capable of metabolic trichloroethene (TCE)-to-ethene reductive dechlorination was obtained from contaminated aquifer material. Growth studies and 16S rRNA gene-targeted analyses suggested culture purity; however, the careful quantitative analysis of Dehalococcoides 16S rRNA gene and chloroethene reductive dehalogenase gene (i.e., vcrA, tceA, and bvcA) copy numbers revealed that the culture consisted of multiple, distinct Dehalococcoides organisms. Subsequent transfers, along with quantitative PCR monitoring, yielded isolate GT, possessing only vcrA. These findings suggest that commonly used qualitative 16S rRNA gene-based procedures are insufficient to verify purity of Dehalococcoides cultures. Phylogenetic analysis revealed that strain GT is affiliated with the Pinellas group of the Dehalococcoides cluster and shares 100% 16S rRNA gene sequence identity with two other Dehalococcoides isolates, strain FL2 and strain CBDB1. The new isolate is distinct, as it respires the priority pollutants TCE, cis-1,2-dichloroethene (cis-DCE), 1,1-dichloroethene (1,1-DCE), and vinyl chloride (VC), thereby producing innocuous ethene and inorganic chloride. Strain GT dechlorinated TCE, cis-DCE, 1,1-DCE, and VC to ethene at rates up to 40, 41, 62, and 127 μmol liter⁻¹ day⁻¹, respectively, but failed to dechlorinate PCE. Hydrogen was the required electron donor, which was depleted to a consumption threshold concentration of 0.76 ± 0.13 nM with VC as the electron acceptor. In contrast to the known TCE dechlorinating isolates, strain GT dechlorinated TCE to ethene with very little formation of chlorinated intermediates, suggesting that this type of organism avoids the commonly observed accumulation of cis-DCE and VC during TCE-to-ethene dechlorination.