Springer protocols feed by Microbiology 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Methods for Isolation and Cultivation of Filamentous Fungi
Filamentous fungi are important organisms for basic discovery, industry, and human health. Their natural growth environments are extremely variable, a fact reflected by the numerous methods developed for their isolation and cultivation. Fungal culture in the laboratory is usually carried out on agar plates, shake flasks, and bench top fermenters starting with an inoculum that typically features fungal spores. Here we discuss the most popular methods for the isolation and cultivation of filamentous fungi for various purposes with the emphasis on enzyme production and molecular microbiology. (Source: Springer protocols feed by Microbiology)
Source: Springer protocols feed by Microbiology - January 1, 2014 Category: Microbiology Source Type: news
Capsid Modification Strategies for Detargeting Adenoviral Vectors
Adenoviral vectors hold immense potential for a wide variety of gene therapy based applications; however, their efficacy and toxicity is dictated by “off target” interactions that preclude cell specific targeting to sites of disease. A number of “off target” interactions have been described in the literature that occur between the three major capsid proteins (hexon, penton, and fiber) and components of the circulatory system, including cells such as erythrocytes, white blood cells, and platelets, as well as circulatory proteins including complement proteins, coagulation factors, von Willebrand Facto...
Source: Springer protocols feed by Microbiology - October 24, 2013 Category: Microbiology Source Type: news
Use of Dodecahedron “VLPs” as an Alternative to the Whole Adenovirus
We report here how these particles are expressed and purified, the labeling method for trafficking studies as well as their use in molecular interaction studies. The potential of these particles for biotechnological applications is under evaluation, making their study a “niche” along side traditional adenoviral vectors. (Source: Springer protocols feed by Microbiology)
Source: Springer protocols feed by Microbiology - October 24, 2013 Category: Microbiology Source Type: news
Study of Adenovirus and CAR Axonal Transport in Primary Neurons
Vectors derived from the canine adenovirus serotype 2 (CAV-2) possess a high neurotropism and efficient retrograde transport that lead to widespread neuronal transduction in the central nervous system (CNS) of various animals. These abilities are due to the engagement of virions to the coxsackievirus and adenovirus receptor at the surface of neurons, which is linked to the endocytic and axonal transport machineries. The trafficking of CAV-2 and the coxsackievirus and adenovirus receptor (CAR) can be visualized ex vivo by incubating primary neurons (e.g., motoneurons and hippocampal neurons) with fluorescently labeled virio...
Source: Springer protocols feed by Microbiology - October 24, 2013 Category: Microbiology Source Type: news
The Use of Chromatin Immunoprecipitation (ChIP) to Study the Binding of Viral Proteins to the Adenovirus Genome In Vivo
The encapsidation of adenovirus (Ad) DNA into virus particles depends on cis-acting sequences located at the left end of the viral genome. Repeated DNA sequences in the packaging domain contribute to viral DNA encapsidation and several viral proteins bind to these repeats when analyzed using in vitro DNA–protein binding assays. In this chapter, we describe a chromatin immunoprecipitation (ChIP) approach to study the binding of viral proteins to packaging sequences in vivo. This assay permits accurate quantification over a wide range of DNA concentrations. The use of formaldehyde cross-linking to stabilize DNA–p...
Source: Springer protocols feed by Microbiology - October 24, 2013 Category: Microbiology Source Type: news
DNA Microarray to Analyze Adenovirus–Host Interactions
Defining the molecular toxicity of viral vectors that are or will be in use for clinical trials is a prerequisite for their safe application in humans. DNA chips allow high-throughput evaluation of the profile of transduced cells and have contributed to underlining specific aspects of vector toxicity both in in vitro and in vivo assets. With gene chips we have been able to identify vector-specific properties, such as the cell cycle alteration induced by vector genomic DNA, along with the activation of specific innate immune pathways that can be ascribed to viral particles. We herein describe a detailed protocol for the use...
Source: Springer protocols feed by Microbiology - October 24, 2013 Category: Microbiology Source Type: news
Determination of the Transforming Activities of Adenovirus Oncogenes
The last 50 years of molecular biological investigations into human adenoviruses (Ads) have contributed enormously to our understanding of the basic principles of normal and malignant cell growth. Much of this knowledge stems from analyses of the Ad productive infection cycle in permissive host cells. Also, initial observations concerning the transforming potential of human Ads subsequently revealed decisive insights into the molecular mechanisms of the origins of cancer and established Ads as a model system for explaining virus-mediated transformation processes. Today it is well established that cell transformation by hum...
Source: Springer protocols feed by Microbiology - October 24, 2013 Category: Microbiology Source Type: news
Oncolytic Adenovirus Characterization: Activity and Immune Responses
Virotherapy in one of the main current applications of recombinant adenoviruses. Oncolytic adenovirus are designed to target tumors, replicate selectively in tumor cells, and elicit immune responses against tumor antigens. Transgene expression in replication-competent oncolytic vectors allows to explore multiple strategies to enhance the potential of virotherapy. In this chapter we describe common in vivo and in vitro techniques used to evaluate the potency and biodistribution of oncolytic viruses. Monitoring immune responses against viral and tumor antigens is crucial as the immune system determines the outcome of virothe...
Source: Springer protocols feed by Microbiology - October 24, 2013 Category: Microbiology Source Type: news
Genetic Interruption of Target Genes for Investigation of Virulence Factors
Recently, more emphasis has been given to understand molecular genetics and the contribution of a gene in the disease process. In fact, increased understanding of bacterial pathogenesis and intracellular communication has revealed many potential strategies for development of novel agents to treat bacterial infection. Therefore, to study the function and the involvement of a particular gene in pathogenesis, the inactivation or interruption is very important. In this section, various methods leading to inactivation of the gene in Staphylococcus aureus will be discussed. (Source: Springer protocols feed by Microbiology)
Source: Springer protocols feed by Microbiology - October 6, 2013 Category: Microbiology Source Type: news
Staphylococcal Cassette Chromosome mec (SCCmec) Analysis of MRSA
Methicillin-susceptible S. aureus (MSSA) changes to methicillin-resistant S. aureus upon the acquisition of Staphylococcal Cassette Chromosome mec (SCCmec), a genomic island that encodes methicillin resistance. All SCCmec elements reported to date share four common characteristics: (1) carrying the mec gene complex (mec); (2) carrying the ccr gene complex (ccr); (3) being flanked by characteristic nucleotide sequences, inverted repeats, and direct repeats, at both ends; and (4) being integrated at the integration site sequence (ISS) for SCC, which is located at the 3′-end of orfX or at the extremity of the SCC elemen...
Source: Springer protocols feed by Microbiology - October 6, 2013 Category: Microbiology Source Type: news
Multilocus Sequence Typing (MLST) of Staphylococcus aureus
MLST is a widely accepted method of sequence-based typing that relies on analysis of relatively conserved genes that encode essential proteins. For Staphylococcus aureus the level of discrimination provided by MLST is sufficient to provide a relatively detailed picture of the global dissemination of the pathogen. The method is not restrictive in the precise methodology used to acquire the sequences, but the method of assigning types requires that the data be of high quality. Excellent web-based tools have been developed and are curated by the groups that launched MLST. These tools have allowed the scheme to be maintained a...
Source: Springer protocols feed by Microbiology - October 6, 2013 Category: Microbiology Source Type: news
Pulsed-Field Gel Electrophoresis Typing of Staphylococcus aureus Isolates
Pulsed-field gel electrophoresis (PFGE) is the most applied and effective genetic typing method for epidemiological studies and investigation of foodborne outbreaks caused by different pathogens, including Staphylococcus aureus. The technique relies on analysis of large DNA fragments generated by the cleavage of intact bacterial chromosomes with a rare cutting restriction enzyme, subsequently resolved by pulsed-field electrophoresis with periodic changes of the orientation of the electrical field across the gel. The high discriminatory power, improved reproducibility due to standardization of experimental protocols and dat...
Source: Springer protocols feed by Microbiology - October 6, 2013 Category: Microbiology Source Type: news
Internal Transcribed Spacer (ITS)-PCR Identification of MRSA
Polymerase chain reaction (PCR) analysis of the 16S–23S rRNA gene internal transcribed spacer (ITS) followed by microchip gel electrophoresis was useful for identification of staphylococci and for strain delineation of Staphylococcus aureus. In the study presented in this chapter, 74 ITS patterns were demonstrated among 1,188 isolated colonies of S. aureus: 55 patterns for methicillin-susceptible S. aureus (MSSA), 4 patterns for methicillin-resistant S. aureus (MRSA), and 15 patterns for both MSSA and MRSA, highlighting the inability of ITS pattern analysis to differentiate the MSSA and MRSA strains. To overcome this...
Source: Springer protocols feed by Microbiology - October 6, 2013 Category: Microbiology Source Type: news
Immunofluorescence Microscopy for the Detection of Surface Antigens in Methicillin-Resistant Staphylococcus aureus (MRSA)
Immunofluorescence microscopy is a widely used laboratory method which allows detection and visualization of specific antigens. The method employs the specificity of antibodies to deliver fluorophore to a specific target and then visualize it with a microscope. The power of the technique is that it requires relatively little manipulation and relatively few bacterial cells, enabling the detection of antigen expression where other methods cannot, such as during an actual infection in an animal. Here, we apply the method to follow antigen expression on the surface of MRSA cells over time in in vivo infection models. (Source: ...
Source: Springer protocols feed by Microbiology - October 6, 2013 Category: Microbiology Source Type: news
Rapid Methods for Detection of MRSA in Clinical Specimens
Antimicrobial susceptibility test methods such as disk diffusion, broth microdilution, and oxacillin screen plate require 24 h of incubation after having the organism growing in pure culture. Rapid and accurate identification of MRSA isolates is essential not only for patient care, but also for effective infection control programs to limit the spread of MRSA. In the last few years, several commercial rapid tests for detection of MRSA directly from nasal swabs and blood cultures have been developed for use in clinical laboratories. Real-time PCR and other molecular tests are gaining popularity as MRSA screening tests to ide...
Source: Springer protocols feed by Microbiology - October 6, 2013 Category: Microbiology Source Type: news
