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Past Issue in 2019
Volume: 9, Issue: 3
Biochemistry
Measurement of Redox States of the β3 Integrin Disulfide Bonds by Mass Spectrometry
Functional disulfide bonds mediate a change in protein function in which they reside when cleaved or formed. To elucidate how a functional disulfide bond controls protein activity, it is critical that the redox state of the bond in the population of protein molecules is known. Measurement of changes in disulfide bond redox state relies on thiol probes and immunoblotting. Such technique only offers a qualitative indication of a change in redox state but not the identity of cysteines involved. A differential cysteine alkylation and mass spectrometry technique is described here that affords precise quantification of protein disulfide bond redox state. The utility of the technique is demonstrated by quantifying the redox state of 24 of the 28 disulfide bonds in human β3 integrin from purified platelets.
Cancer Biology
Calvarial Bone Implantation and in vivo Imaging of Tumor Cells in Mice
Bone is one of common metastasis sites for many types of cancer. In bone metastatic microenvironment, tumor-bone interactions play a significant role in the regulation of osteolytic or osteoblastic bone metastasis. In order to investigate the direct interaction between tumor cells and bone tissue, it is essential to generate appropriate animal models that mimic the behavior of tumor cells in bone metastatic lesions. Calvarial implantation model (bone invasion model) is a newly-established animal model that accurately recapitulates the behavior of tumor cells in the tumor-bone microenvironment. The surgical technique for tumor cell implantation is simpler than intracardiac, intra-arterial, or intraosseous injection techniques. This model can be useful for the identification of key factors driving tumor-induced osteolytic or osteoblastic changes.
In situ, Cell-free Protein Expression on Microarrays and Their Use for the Detection of Immune Responses
Until recently, whole-proteome microarrays for comprehensive studies of protein interactions were mostly produced by individual cloning and cellular expression of very many open reading frames, followed by protein isolation and purification as well as array production. To overcome this cumbersome process, we have developed a method to generate microarrays representing entire proteomes by a combination of multiple spotting and on-chip, cell-free protein expression. Here, we describe the protocol for the production of bacterial protein microarrays. With slight adaptations, however, the procedure can be applied to the proteome of any organism. Expression constructs of each gene are generated by PCR on bacterial genomic DNA followed by a common secondary amplification that is adding relevant regulative elements to either end of the constructs. The unpurified PCR-products are spotted onto the microarray surface. Full-length proteins are directly expressed in situ in a cell-free manner and stay attached to the surface without further action. As an example of a typical application, we describe here the proteome-wide analysis of the immune response to a bacterial infectious agent by characterizing the binding profiles of the antibodies in patient sera.
Cell Biology
Imaging Higher-order Chromatin Structures in Single Cells Using Stochastic Optical Reconstruction Microscopy
Higher-order chromatin organization shaped by epigenetic modifications influence the chromatin environment and subsequently regulate gene expression. Direct visualization of the higher-order chromatin structure at their epigenomic states is of great importance for understanding chromatin compaction and its subsequent effect on gene expression and various cellular processes. With the recent advances in super-resolution microscopy, the higher-order chromatin structure can now be directly visualized in situ down to the scale of ~30 nm. This protocol provides detailed description of super-resolution imaging of higher-order chromatin structure using stochastic optical reconstruction microscopy (STORM). We discussed fluorescence staining methods of DNA and histone proteins and crucial technical factors to obtain high-quality super-resolution images.
Immunology
Induction and Analysis of Anti-CD40-induced Colitis in Mice
Colon inflammation or colitis affects more than 1 million people worldwide. Several pre-clinical models, including chemical-induced (i.e., DSS, TNBS) or pathogen-induced (i.e., Citrobacter rodentium) have been used to study mechanisms involved in the development and regulation of colitis. Anti-CD40 induced colitis model has gained acceptance to study the roles of innate immune cells during acute intestinal inflammation. Here we describe a rapid, robust and reproducible protocol to induce and analyze anti-CD40 mediated colitis in mice.
Microbiology
Measuring Homologous Recombination Rates between Chromosomal Locations in Salmonella
Homologous recombination between two similar DNA molecules, plays an important role in the repair of double-stranded DNA breaks. Recombination can occur between two sister chromosomes, or between two locations of similar sequence identity within the same chromosome. The assay described here is designed to measure the rate of homologous recombination between two locations with sequence similarity within the same bacterial chromosome. For this purpose, a selectable/counter-selectable genetic cassette is inserted into one of the locations and homologous recombination repair rates are measured as a function of recombinational removal of the inserted cassette. This recombinational repair process is called gene conversion, non-reciprocal recombination. We used this method to measure the recombination rates between genes within gene families and to study the stability of mobile genetic elements inserted into members of gene families.
Molecular Biology
In vitro Protein-DNA Binding Assay (AlphaScreen® Technology)
Identification of specific DNA binding sites of transcription factors is important in understanding their functions. Recent techniques allow us to investigate genome-wide in vivo binding positions by chromatin immunoprecipitation combined with high-throughput sequencing. However, to further explore the binding motifs of transcription factors, in-depth biochemical analysis is required. Here, we describe an efficient protocol of protein-DNA interactions based on a combination of our in vitro transcription/translation system and AlphaScreen® technology. The in vitro transcription/translation system supports an efficient and quick way of protein synthesis by alleviating cumbersome cloning steps. In addition, AlphaScreen® system provides a highly sensitive, quick, and easy handling platform to investigate the protein-DNA interactions in vitro. Thus, our method largely contributes to comprehensive analysis of the biochemical properties of transcription factors.
Flip-flop Mediated Conditional Gene Inactivation in Drosophila
Mosaic analysis in Drosophila, an important tool to assess cellular phenotypes of mutants in an otherwise heterozygous background, relies on mitosis. Hence, it cannot be used to inactivate gene function in mitotically inactive, terminally differentiated cells such as neurons. To address this issue, we developed “Flip-flop”, a novel, Flippase-dependent in vivo cassette-inversion method that functions independent of mitosis, and therefore can be used for gene inactivation in both mitotic as well as post-mitotic cells. This method allows tagging protein-coding genes with EGFP and generates mutant cells that are marked with mCherry upon cassette inversion. Here, we describe protocols for generation and validation of fly lines that can be used for conditional gene inactivation in mitotic as well as post-mitotic cells. We provide typical examples of Flip-flop mediated mosaic analysis in SNF4Aγ and Trim9. Use of Flip-flop mediated functional analysis will permit a detailed investigation of the role of genes previously recalcitrant to mosaic analysis.
pNP Transgenic RNAi System Manual in Drosophila
Much of our knowledge about the mechanisms underlying biological processes relies on genetic approaches, whereby gene activity is reduced and the phenotypic consequences of perturbation are analyzed in detail. For functional genomic studies, a specific, systematic, and cost-effective manner is critical. Transgenic RNAi system is the top priority choice to study gene functions due to its simple and practical characteristics in Drosophila. We established a novel system that works well in both soma and germ cells which is efficient and specific. With this system, we can precisely and efficiently modulate highly expressed genes, and simultaneously knock down multiple genes in one step. In this study, we provide a detailed protocol of the pNP system, which replaces other transgenic systems, and expect it can provide some help to researchers who are using this system.
Neuroscience
Assessing Spatial Working Memory Using the Spontaneous Alternation Y-maze Test in Aged Male Mice
The global population is aging and the prevalence of age-related diseases, such as Alzheimer's disease and vascular dementia is increasing. Understanding functional impairments and disease processes is of vital importance in order to develop effective therapeutics. Using the natural exploratory behavior of mice, the spontaneous alternation y-maze can assess short-term spatial working memory. The protocol for y-maze testing is straightforward and requires minimal resources, as well as animal training and output. Therefore, it can be broadly applied to study short-term memory in aged rodent models.
A Novel Hygrotaxis Assay for Assessing Thirst Perception and Water Sensation in Drosophila
Water is essential for the survival of terrestrial animals. Animals obtain water from their food, from metabolism and, more importantly, by drinking. It is therefore vital for dehydrated animals to efficiently locate water sources in the wild. Traditional paradigm for studying the genetic basis of humidity sensation and preference use an apparatus consisting of two chambers providing a binary choice between flows of dry and moist air that maintain steep humidity gradients. Here, we introduce a novel assay to study water-seeking behavior (hygrotaxis) in Drosophila, in which dehydrated flies detect a humidity gradient and rapidly aggregate near an inaccessible water source. Our previous work using this method demonstrated that hygrotactic behavior relies on both specific sensory organs and central brain neurons.
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