Biochemistry

The Min system determines the cell division plane of bacteria. As a cue of spatiotemporal regulation, the Min system uses wave propagation of MinD protein (Min wave). Therefore, the reconstitution of the Min wave in cell-sized closed space will lead to the creation of artificial cells capable of cell division. The Min waves emerge via coupling between the reactions among MinD, MinE, and ATP and the differences in diffusion rate on the cell membrane and in the cytoplasm. Because Min waves appear only under the balanced condition of the reaction-diffusion coupling, special attentions are needed towards several technical points for the reconstitution of Min waves in artificial cells. This protocol describes a technical method for stably generating Min waves in artificial cells.

Exosomes have emerged as an important mediator of intercellular communication. They are present in extracellular milieu and therefore, easily accessible by neighboring or distant cells. They carry mRNA, microRNAs and proteins within their vesicles and once internalized by recipient cells; they can modulate multiple signaling pathways with pleiotropic effects from inducing antiviral state to disease progression. We have previously shown that hepatitis C virus (HCV) infected hepatocytes or hepatoma cells harboring genome-length replicon secrete exosomes in culture supernatants. These exosomes are taken up by hepatic stellate cells (HSC) and activate them to induce fibrosis during HCV infection. Here, we describe detailed protocols for exosomes isolation and uptake of BODIPY labeled exosomes by hepatic stellate cells.

Xanthomonads can scavenge iron from the extracellular environment by secreting the siderophores, which are synthesized by the proteins encoded by xss (Xanthomonas siderophore synthesis) gene cluster. The siderophore production varies among xanthomonads in response to a limited supply of iron where Xanthomonas campestris pv. campestris (Xcc) produces less siderophores than Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc). Siderophore production can be measured by HPLC and with the CAS (Chrome azurol S)-agar plate assay, however HPLC is a more accurate method over CAS-agar plate assay for siderophore quantification in Xanthomonads. Here we describe how to quantify siderophores from xanthomonads using HPLC.

Efficient delivery of oligonucleotide therapeutics, i.e., siRNAs, to the central nervous system represents a significant barrier to their clinical advancement for the treatment of neurological disorders. Small, endogenous extracellular vesicles were shown to be able to transport lipids, proteins and RNA between cells, including neurons. This natural trafficking ability gives extracellular vesicles the potential to be used as delivery vehicles for oligonucleotides, i.e., siRNAs. However, robust and scalable methods for loading of extracellular vesicles with oligonucleotide cargo are lacking. We describe a detailed protocol for the loading of hydrophobically modified siRNAs into extracellular vesicles upon simple co-incubation. We detail methods of the workflow from purification of extracellular vesicles to data analysis. This method may advance extracellular vesicles-based therapies for the treatment of a broad range of neurological disorders.

Here we describe both non-extraction and solvent-extraction methods for root aliphatic suberin analysis. The non-extraction method is fast as roots are directly depolymerized using acidic transmethylation. However, suberin aliphatic components are isolated together with all the other acyl chains making up the lipids (e.g., membranes) present in roots. For the solvent-extraction method, roots are first delipidated before transmethylation. This method is longer but allows separation of soluble and polymerized root lipids. This protocol is optimized for tissue culture- or soil-grown Arabidopsis thaliana plants, but can be used with roots of other plants.

Rhamnolipids produced by Pseudomonas aeruginosa (P. aeruginosa) represent a group of biosurfactants with various applications (e.g., bioremediation of oil spills, cosmetics, detergents and cleaners). The commonly used colorimetric methods for rhamnolipid quantification, including anthrone, phenol−sulfuric acid and orcinol based quantification (Helbert and Brown, 1957; Chandrasekaran and BeMiller, 1980), are laborious and operationally hazardous because of the strong acid/chemical emanation which can cause deterioration of instruments measurements (e.g., spectrophotometer). Therefore, the methylene-blue-based analysis appears as a promising alternative to safely quantify whole rhamnolipid molecules based on chemical complexation reaction (Pinzon and Ju, 2009). Indeed, methylene blue and rhamnolipids form a complex in a water-chloroform phase system. The rhamnolipids-methylene blue complex is partitioned into the chloroform phase which will develop a blue color that can be quantified at 638 nm to deduce rhamnolipids concentration. Here, we describe a variant of methylene-blue-based rhamnolipids quantification procedure that allows spectrophotometric quantification on standard 96-well plastic microplate contrarily to original methylene blue procedure that requires specific and expensive microplate due to chloroform chemical properties.

Macrophage depletion has been used extensively to study autoimmune disease and more recently in tumor models. The clodronate-containing liposomes will be recognized as foreign particles and get engulfed by macrophages upon dosing into the animal by the chosen routes. Consequently, macrophages that have engulfed liposomes will all be destroyed by the liposomal. In the protocol presented here the clodronate-containing liposomes were used to systemically deplete macrophages in mice.

Outer membrane vesicles (OMVs) are spherical bilayered phospholipids of 20-200 nm in size produced from all Gram-negative bacteria and Gram-positive bacteria investigated to date. Previous biochemical and proteomic studies have revealed that the Gram-negative bacteria-derived OMVs are composed of various components like outer membrane proteins, lipopolysaccharides, outer membrane lipids, periplasmic proteins, DNA, and RNA. Here, in this protocol, we describe the method to isolate the OMVs from the culture supernatant of Escherichia coli (E. coli).

Here we describe procedures for the flower cuticular waxes extraction, modification and subsequent qualitative and quantitative analysis by gas-chromotography-mass spectrometry (GC-MS) and gas-chromotography with flame ionization detector (GC-FID), accordingly. To characterize flower cutin monomers two experimental setup are described: (i) analysis of enzymatically isolated cuticles in order to determine the relative proportions of cutin monomers; (ii) analysis of freeze-dried material for quantitative estimation of the cutin content. This report is an adaptation of the earlier published protocols developed for the chemical analysis of the cuticles in vegetative organs (Leide et al., 2007).

The primary aerial surfaces of all land plants are coated by a lipidic cuticle, which restricts non-stomatal water loss and protects the plant from pathogens, herbivores, and ultraviolet radiation. The cuticle is made up of two components: cutin, a polymer of hydroxy- and epoxy- long-chain fatty acid derivatives and glycerol, and cuticular waxes, which are derivatives of very-long-chain fatty acids. While chemical analysis of cutin can be a lengthy and technically challenging task, analysis of cuticular waxes is relatively simple, and can be routinely used to characterize different plant species, adaptations of a given species to environmental conditions, or mutant phenotypes. Here, we present a protocol tailored for the analysis of cuticular waxes on the surface of the model organism Arabidopsis thaliana. Because cuticular waxes are found on the outermost surface of the plant, the wax extraction process is very simple, and sample processing can be completed in less than one day. Chemical analysis involves quantitation of wax monomers by gas chromatography coupled with flame ionization detection (GC/FID), and identification of wax monomers by either mass spectrometry or comparison of retention times of individual wax components to those of known standards.