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This protocol provides detailed instructions on how to perform fluorescent polysome profiling in C. elegans to collect actively translated mRNA, to quantify changes in global translation, and to detect ribosomal binding partners.Respiratory dysfunction is among the main cause of severe and fatal pathologies worldwide. The use of effective experimental models and methodologies for the study of the pulmonary pathophysiology is necessary to prevent, control and cure these diseases. Plethysmography, a technique for the assessment of lung function, has been widely applied in mice for the characterization of respiratory physiology. However, classical plethysmography methods present technical limitations such as the use of anesthesia and animal immobilization. Whole-body plethysmography (WBP) avoids these issues providing a non-invasive approach for the assessment of the respiratory function in conscious animals. WBP relies on the recording of pressure changes that are produced by the spontaneous breathing activity of an animal placed inside an airtight chamber. During normal respiration, pressure variation is directly proportional to the respiratory pattern of the animal allowing the measurement of the respiratory rate and tidal volume. These parameters are commonly used to evaluate pulmonary function in different physiological and disease models. In contrast to classical plethysmography methods, WBP technique allows reproducible serial measurements as it avoids animal restraint or the use of anesthesia. These key features rend WBP a suitable approach for longitudinal studies allowing the assessment of progressive respiratory alterations in physiological and pathological conditions. This protocol describes the procedures for the measurement of the breathing patterns in mice using the WBP method, the data analysis and results interpretation.Bacteria blight diseases of rice due to several genera of pathogenic bacteria are one of the major constraints worldwide for rice production. The disease can be best managed through host plant resistance sources. For most of these bacteria such as Xanthomonas oryzae pv. oryzae, X. oryzae pv. oryzicola, Pseudomonas fuscovaginae, Burkholderia glumae, Burkholderia gladioli and Acidovorax avenae subsp. avenae, specific diagnostic techniques that include molecular and pathogenicity tests have been developed. However, for Pantoea spp., information on pathogenicity assay is very limited and protocols used are not uniform. Most authors use the leaf clipping method. In this paper, we describe the protocol for mechanical inoculation of rice seedlings aged 35 days. The method consists of infiltrating bacterial suspensions at concentrations of 108 CFU/ml, with a needleless syringe into the intercellular and interveinal spaces of rice leaves underside at about 4-5 cm below the leaf tip. This method can be used for a standardized pathogenicity assessment, germplasm resistance evaluation for identifying and characterizing resistance sources.Genetic transformation is crucial for both investigating gene functions and for engineering of crops to introduce new traits. Rice (Oryza sativa L.) is an important model in plant research, since it is the staple food for more than half of the world's population. As a result, numerous transformation methods have been developed for both indica and japonica rice. Since breeders continuously develop new rice varieties, transformation protocols have to be adapted for each new variety. Here we provide an optimized transformation protocol with detailed tips and tricks for a new African variety Komboka using immature embryos. In Komboka, we obtained an apparent transformation rate of up to 48% for GUS/GFP reporter gene constructs using this optimized protocol. This protocol is also applicable for use with other elite indica rice varieties.Depending on its concentration and cellular origin the production of reactive oxygen species (ROS) in the organism serves a variety of functions. While high concentrations during an oxidative burst are used to fight pathogens, low to moderate amounts of ROS act as signaling molecules important for several physiological processes such as regulation of immune responses. The ROS-sensitive dye 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) is an inexpensive and well-established tool for measuring intracellular ROS levels. However, it needs to be carefully controlled to be able to draw firm conclusions on the nature of ROS species produced and the cellular source of ROS generation such as the enzyme complex NADPH-oxidase 2 (NOX-2). Y-27632 research buy In this protocol, a robust method to determine low intracellular ROS production using H2DCFDA was validated by several ROS-specific as well as NOX-2-specific inhibitors. Cells were treated with inhibitors or control substances prior to treatment with the ROS-inducer of interest. H2DCFDA was added only for the last 30 min of the treatment schedule. To terminate its conversion, we used a ROS-specific inhibitor until analysis by flow cytometry within the FITC-channel (Ex 488 nm/Em 519 nm). In summary, this protocol allows the detection of signaling-relevant intracellular ROS production in cell lines and primary immune cells (e.g., Mono Mac 6 cells and Bone marrow-derived dendritic cells, respectively). Using this method in combination with specific inhibitors, we were able to validate even exceptionally low amounts of ROS produced by NOX-2 and relevant for immune-regulatory signaling.Contact-dependent interbacterial competition is a common strategy used by bacteria to fight for their ecological niches. Interbacterial competition is monitored by a competition assay involving co-culturing the attacker and the recipient bacterial cells on agar, followed by recovery of the surviving recipient cells. Conventional interbacterial competition assays rely on serial dilution, plate spreading, and colony counting experiments for the readout. The high demand for time and labor in a competition assay limits its use for large-scale screening. However, a high-throughput interbacterial competition screening method is required to screen genetic factors involved in an interbacterial competition. Here, using Agrobacterium tumefaciens as an attacker and Escherichia coli as a recipient, we developed a robust, fast, efficient, and high-throughput type VI secretion system-dependent interbacterial competition screening platform. This system allows for 96 simultaneous competition assays without the need for serial dilution and plate spreading.
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