Chapmanjeppesen1269

MNase is also applied to release NET-bound fungal elements for counting. This protocol can be applied to assay NET killing of other biofilm-forming organisms.Cell-based functional assays are an important part of compound screening and drug lead optimization, and they can also play a crucial role in the determination of the residues involved in ligand binding and signaling for a particular G-protein-coupled receptor. Conventional methods used for Gαq/15-coupled receptors rely on the use of fluorescent probes for Ca++ sensing (such as Fura-2 and Fluo-4) or on the incorporation of [3H]-inositol into inositol 1,4,5- triphosphate (IP3). Pamapimod concentration However, these methods are not suitable for screening large libraries of compounds or for screening several mutants of the same receptor. In contrast, the IP-One assay by Cisbio is a TR-FRET assay suitable for large compound library screening when using stable cell lines that express a specific 7TMR. However, when using transiently transfected mutants of a 7TMR, this assay is not ideal, as it requires a two-step protocol of cell culture. Therefore, we have optimized the IP-One assay protocol using the reverse transfection method in 384-well plates. This offers a time- and resource-efficient alternative to the two-step protocol previously used for the screening of several mutants of Gαq/15-coupled 7TMRs.Microtubule dynamic instability is driven by the hydrolysis of the GTP bound to the β-subunit of the α-β tubulin heterodimer. Nucleotide analogues are commonly used to mimic the different steps of the tubulin GTPase cycle, but most of them are poor microtubule nucleators. Usually, microtubule assembly is seeded by guanylyl-(α, β)-methylene-diphosphonate (GMPCPP) or glycerol that can be limiting factors in monitoring the effect of other nucleotide analogs on their polymerization. Here, we describe a protocol that allows the assembly of microtubules in the presence of nucleotide analogues without the need of heterogeneous seeds and at a low final glycerol concentration. Microtubules are first assembled in the presence of the analogue of interest and glycerol to promote assembly. These microtubules are then sonicated to produce seeds that will be used to assemble microtubules in the absence of glycerol. This strategy produces homogeneous nucleotide-bound microtubules that can be further analyzed by biochemical or structural methods such as cryo-electron microscopy.The nucleotides involved in RNA-RNA interaction can be tagged by chemical- or UV-induced crosslinking, and further identified by classical or modern high throughput techniques. The contacts of mRNA with 18S rRNA that occur along the mRNA channel of 40S subunit have been mapped by site-specific UV crosslinking followed by reverse transcriptase termination sites (RTTS) using radioactive or fluorescent oligonucleotides. However, the sensitivity of this technique is restricted to the detection of those fragments that resulted from the most frequent crosslinkings. Here, we combined RTTS with RNAseq to map the mRNA-18S rRNA contacts with a much deeper resolution. Although aimed to detect the interaction of mRNA with the ES6S region of 18S rRNA, this technique can also be applied to map the interaction of mRNA with other non-coding RNA molecules (e.g., snRNAs, microRNAs and lncRNAs) during transcription, splicing or RNA-mediated postranscriptional regulation.The quantitative measurement of water flow-induced swimming of fish species using a swimmill is a powerful method to evaluate motor ability of individual fish. Zebrafish is a commonly used vertebrate that enables the study of morphological, physiological and behavioral characteristics associated with genes. We here established a reproducible method that allows to measure the body length and the critical swimming speed of adult zebrafish using a swimmill.Paper nanobiosensors have been established as an excellent platform for analysis of veterinary and human pathogens causing various diseases. Especially, lateral flow assays or biosensors ideal for sensitive, rapid, robust and accurate analysis in laboratory setups and on-site analysis. Viral RNA detection is of great importance for public health as well as animal health protection. In that aspect, the present protocol focuses on the development of functionalized gold nanoparticle-based lateral flow biosensor for fish nervous necrosis virus (Nodavirus) nucleic acids detection. Total viral RNA, isolated from fish samples was subjected to reverse transcription PCR amplification and the amplification products were mixed with specific oligonucleotide probe. A red test line was formed when nodavirus product was present. The proposed assay has great implications on basic research since it eliminates the need for time-consuming, cumbersome electrophoresis protocols and could be adjusted for use on the site of fish culture by fish farmers. Disease monitoring by such bioanalytical platforms without time consuming and costly procedures would have great impact on the aquaculture and environmental safety.We have previously described the development of two specialized Escherichia coli strains for high-level recombinant membrane protein (MP) production. These engineered strains, termed SuptoxD and SuptoxR, are capable of suppressing the cytotoxicity caused by MP overexpression and of producing greatly enhanced MP yields. Here, we present a Bio-protocol that describes gene overexpression and culturing conditions that maximize the accumulation of membrane-integrated and well-folded recombinant MPs in these strains.In order for the brain to function properly, a carefully orchestrated homeostasis must be maintained. To help regulate this delicate balance, the brain has developed a highly selective blood-brain barrier (BBB). Under normal conditions, the BBB excludes harmful blood-borne material from the brain parenchyma. However, numerous neuropathological conditions can disrupt this barrier, causing BBB permeability and subsequent CNS dysfunction. Understanding the mechanisms involved in BBB permeability are essential to elucidating the pathology of various neurological disorders as well as identifying methods for drug delivery to the CNS. Here, we describe several in vivo methods to measure BBB permeability in mice using an array of diverse sized tracers including exogenous 376 Da fluorescein salt, 66.5 kDa bovine serum albumin, and 70 kDa dextran as well as endogenous 160 kDa mouse IgG. When administered intravenously, these substances are excluded from a healthy brain by the BBB. However, BBB dysfunction can allow entry of these tracers into the brain and this accumulation can be measured using spectrophotometry, fluorescent microscopy, and immunohistochemistry. We also describe a method to induce BBB permeability using Clostridium perfringens epsilon toxin. Finally, we include a short discussion about the advantages and disadvantages of each method and their appropriate downstream applications.According to the World Health Organization (WHO), nearly 1.13 billion people worldwide have hypertension, a major factor responsible for premature death globally. The inherent multifactorial nature of hypertension makes its study difficult since the chronic rise in blood pressure depends on the intricate connection  between  dietary,  genetic  and  environmental  factors.  Therefore, the pathophysi-ology of hypertension is not completely understood. For these reasons, there is an ongoing search for animal models that better mimic changes resulting from this disease. Because of its complexity, the use of animal models aimed at elucidating the pathogenesis of hypertension and to evaluate new therapeutic possibilities is an important tool for understanding this disease since it enables consistent experimental strategies that are impractical in humans. Over time, many animal models have been developed for the study of chronic increases in blood pressure ranging from genetic models that include the spontaneously hypertensive rat (SHR) and genetic manipulations, such as the TGR (mRen2) rat, as well as neurogenic or endocrine models. One of the most commonly used hypertensive rat models today is that of hypertension induced by treatment with deoxycorticosterone acetate associated with high sodium intake, i.e., the DOCA-salt model. This model is known to have a neurogenic component linked to increased sympathetic nervous system activity, and as such the DOCA-salt model promotes cross-talk between endocrine and neural components that lead to increased blood pressure, and may impact the functioning of other organs.The organization and distribution of proteins, lipids, and nucleic acids in eukaryotic cells is an essential process for cell function. Retrograde trafficking from the plasma membrane to the Golgi and endoplasmic reticulum can greatly modify cell membrane composition and intracellular protein dynamics, and thus typifies a key sorting step. However, methods to efficiently quantify the extent or kinetics of these events are currently limited. link2 Here, we describe a novel quantitative and effectively real-time single-cell flow cytometry assay to directly measure retrograde membrane transport. The assay takes advantage of the well-known retrograde trafficking of cholera toxin engineered with split-fluorescent proteins to generate novel tools for immediate monitoring of intracellular trafficking. This approach will greatly extend the ability to study the underlying biology of intracellular membrane trafficking, and how trafficking systems can adapt to the physiologic needs of different cell types and cell states.Small extracellular vesicles (sEVs) encompass a variety of distinct vesicles that are secreted to the extracellular space. Many methodologies currently used for EV isolation (e.g., differential ultracentrifugation concluding in a high-speed pellet, precipitation by macromolecular crowding agents or size excusion chromatography-SEC) do not fractionate distinct sEV sub-populations. Samples obtained by the aforementioned methods are usually used for characterization and physiological studies. However the fraction that contains the molecule of interest or is the carrier of a specific activity is unknown. Therefore isolating distinct sEV sub-populations is critical to understand EV function. The goal of this procedure is to purify distinct sEV sub-populations based on slight differences in their buoyant density. Moreover, this technique also allows sEVs purification from vesicle-free RNA-protein complexes co-isolating in the high-speed pellet or by the use of crowding agents. This protocol describes cultivation of mammalian cells for sEV collection, sEV sedimentation, buoyant density fractionation of sEV sub-populations and immunoblots for sEV markers. link3 This protocol can be used to fractionate distinct sEV sub-populations produced by a variety of mammalian cells.ATPases are the enzymes that breakdown ATP to ADP and release inorganic phosphate (Pi). Here we provide a detailed protocol to determine the ATPase activity of a recombinant AAA+-ATPase protein (GENERAL CONTROL NON-REPRESSIBLE-4 [GCN4]) by spectrophotometric absorption at 360 nm to measure the accumulated inorganic phosphate. In general, the substrate 2-amino-6-mercapto-7-methylpurine riboside (methylthioguanosine, a guanosine analog MESG) is enzymatically converted in the presence of Pi by purine nucleoside phosphorylase (PNP) to ribose 1-phosphate and 2-amino-6-mercapto-7-methylpurine. The spectrophotometric shift in maximum absorbance at 330 nm for the MESG substrate and subsequent conversion product at 360 nm due to enzymatic conversion was measured. The GCN4-His-tagged recombinant protein was expressed in Escherichia coli BL21 cells and purified using Ni-NTA column. This purified protein was then used for the quantitation of Pi in solution or the continuous determination of Pi released due to the ATPase activity of GCN4, an AAA+-ATPase protein conserved in many eukaryotes, which in plants regulates stomatal aperture during biotic and abiotic stress in plants.