Preparation of nuclear matrices from cultured cells: subfractionation of nuclei in situ

Analyses of the different structural systems of the nucleus and the proteins associated with them pose many problems. Because these systems are largely overlapping, in situ localization studies that preserve the in vivo location of proteins and cellular structures often are not satisfactory. In contrast, biochemical cell fractionation may provide artifactual results due to cross-contamination of extracts and structures. To overcome these problems, we have developed a method that combines biochemical cell fractionation and in situ localization and leads to the preparation of a residual cellular skeleton (nuclear matrix and cytoskeletal elements) from cultured cells. This method's main feature is that cell fractionation is performed in situ. Therefore, structures not solubilized in a particular extraction step remain attached to the substrate and retain their morphology. Before and after each extraction step they can be analyzed for the presence and location of the protein under study by using immunological or cytochemical techniques. Thereby the in vivo origin of a protein solubilized in a particular extraction step is determined. The solubilized protein then may be further characterized biochemically. In addition, to allow analyses of proteins associated with the residual cellular skeleton, we have developed conditions for its solubilization that do not interfere with enzymatic and immunological studies

Semi-automated curation of protein subcellular localization: a text mining-based approach to Gene Ontology (GO) Cellular Component curation

Background: Manual curation of experimental data from the biomedical literature is an expensive and time-consuming endeavor. Nevertheless, most biological knowledge bases still rely heavily on manual curation for data extraction and entry. Text mining software that can semi- or fully automate information retrieval from the literature would thus provide a significant boost to manual curation efforts. Results: We employ the Textpresso category-based information retrieval and extraction system http://www.textpresso.org webcite, developed by WormBase to explore how Textpresso might improve the efficiency with which we manually curate C. elegans proteins to the Gene Ontology's Cellular Component Ontology. Using a training set of sentences that describe results of localization experiments in the published literature, we generated three new curation task-specific categories (Cellular Components, Assay Terms, and Verbs) containing words and phrases associated with reports of experimentally determined subcellular localization. We compared the results of manual curation to that of Textpresso queries that searched the full text of articles for sentences containing terms from each of the three new categories plus the name of a previously uncurated C. elegans protein, and found that Textpresso searches identified curatable papers with recall and precision rates of 79.1% and 61.8%, respectively (F-score of 69.5%), when compared to manual curation. Within those documents, Textpresso identified relevant sentences with recall and precision rates of 30.3% and 80.1% (F-score of 44.0%). From returned sentences, curators were able to make 66.2% of all possible experimentally supported GO Cellular Component annotations with 97.3% precision (F-score of 78.8%). Measuring the relative efficiencies of Textpresso-based versus manual curation we find that Textpresso has the potential to increase curation efficiency by at least 8-fold, and perhaps as much as 15-fold, given differences in individual curatorial speed. Conclusion: Textpresso is an effective tool for improving the efficiency of manual, experimentally based curation. Incorporating a Textpresso-based Cellular Component curation pipeline at WormBase has allowed us to transition from strictly manual curation of this data type to a more efficient pipeline of computer-assisted validation. Continued development of curation task-specific Textpresso categories will provide an invaluable resource for genomics databases that rely heavily on manual curation

Manual pressing of nannochloropsis oculata dried biomass for enhanced lipid extraction

Microalgae offer significant potential to produce high value products and biofuels, whilst simultaneously being used to bio-remediate water or capture carbon dioxide (CO2). Microalgal cell disruption processes are often necessary to increase lipid extraction from microalgae before conventional solvent extraction processes are used to isolate lipids. The extracted lipids can be processed to produce biofuels. The combinations of hydraulic pressing with liquid nitrogen (LN2) treatment were applied to samples of dried Nannochloropsis oculata in the presented study to enhance the cellular destruction and lipid yields. The results indicated higher lipid extraction with LN2 treatment (0.159 g/g dry algae) compared to the LN2 untreated samples (0.070 g/g dry algae). The corresponding cell disruptions were found to be seventy-eight and fifty percent, respectively, at the same 10 bar (145 psi) pressure level. The control sample (without any treatment) lipid yield was 0.006 g/g dry algae, while the lipid yield varied between 0.192-0.213 g/g dry algae with LN2 treated biomass with pressure loadings of 70-100 bar (1015-1450 psi) and with a corresponding cell disruption of 93-98 percent. The presence of palmitate, oleate and linoleate found in the fatty acid methyl ester composition of the extracted lipids, shows a favourable profile to produce biodiesel

Optimization of DNA extraction from human urinary samples for mycobiome community profiling.

IntroductionRecent data suggest the urinary tract hosts a microbial community of varying composition, even in the absence of infection. Culture-independent methodologies, such as next-generation sequencing of conserved ribosomal DNA sequences, provide an expansive look at these communities, identifying both common commensals and fastidious organisms. A fundamental challenge has been the isolation of DNA representative of the entire resident microbial community, including fungi.Materials and methodsWe evaluated multiple modifications of commonly-used DNA extraction procedures using standardized male and female urine samples, comparing resulting overall, fungal and bacterial DNA yields by quantitative PCR. After identifying protocol modifications that increased DNA yields (lyticase/lysozyme digestion, bead beating, boil/freeze cycles, proteinase K treatment, and carrier DNA use), all modifications were combined for systematic confirmation of optimal protocol conditions. This optimized protocol was tested against commercially available methodologies to compare overall and microbial DNA yields, community representation and diversity by next-generation sequencing (NGS).ResultsOverall and fungal-specific DNA yields from standardized urine samples demonstrated that microbial abundances differed significantly among the eight methods used. Methodologies that included multiple disruption steps, including enzymatic, mechanical, and thermal disruption and proteinase digestion, particularly in combination with small volume processing and pooling steps, provided more comprehensive representation of the range of bacterial and fungal species. Concentration of larger volume urine specimens at low speed centrifugation proved highly effective, increasing resulting DNA levels and providing greater microbial representation and diversity.ConclusionsAlterations in the methodology of urine storage, preparation, and DNA processing improve microbial community profiling using culture-independent sequencing methods. Our optimized protocol for DNA extraction from urine samples provided improved fungal community representation. Use of this technique resulted in equivalent representation of the bacterial populations as well, making this a useful technique for the concurrent evaluation of bacterial and fungal populations by NGS

Modularization for the Cell Ontology

One of the premises of the OBO Foundry is that development of an orthogonal set of ontologies will increase domain expert contributions and logical interoperability, and decrease maintenance workload. For these reasons, the Cell Ontology (CL) is being re-engineered. This process requires the extraction of sub-modules from existing OBO ontologies, which presents a number of practical engineering challenges. These extracted modules may be intended to cover a narrow or a broad set of species. In addition, applications and resources that make use of the Cell Ontology have particular modularization requirements, such as the ability to extract custom subsets or unions of the Cell Ontology with other OBO ontologies. These extracted modules may be intended to cover a narrow or a broad set of species, which presents unique complications.

We discuss some of these requirements, and present our progress towards a customizable simple-to-use modularization tool that leverages existing OWL-based tools and opens up their use for the CL and other ontologies

Evaluation of four different strategies to characterize plasma membrane proteins from banana roots

Plasma membrane proteins constitute a very important class of proteins. They are involved in the transmission of external signals to the interior of the cell and selective transport of water, nutrients and ions across the plasma membrane. However, the study of plasma membrane proteins is challenging because of their poor solubility in aqueous media and low relative abundance. In this work, we evaluated four different strategies for the characterization of plasma membrane proteins from banana roots: (i) the aqueous-polymer two-phase system technique (ATPS) coupled to gelelectrophoresis (gel-based), and (ii) ATPS coupled to LC-MS/MS (gel free), (iii) a microsomal fraction and (iv) a full proteome, both coupled to LC-MS/ MS. Our results show that the gel-based strategy is useful for protein visualization but has major limitations in terms of time reproducibility and efficiency. From the gel-free strategies, the microsomal-based strategy allowed the highest number of plasma membrane proteins to be identified, followed by the full proteome strategy and by the ATPS based strategy. The high yield of plasma membrane proteins provided by the microsomal fraction can be explained by the enrichment of membrane proteins in this fraction and the high throughput of the gel-free approach combined with the usage of a fast high-resolution mass spectrometer for the identification of proteins