Methods in molecular biology最新文献

Neuronal mitochondria are essential organelles to maintain synaptic activity due to the high calcium buffering capacity and ATP production. In neurons, mitochondria transport occurs along the microtubules mediated by motor proteins, kinesins and dynein, to drive mitochondria toward the synapses. Disruption of axonal transport is an early pathogenic event in neurodegenerative disorders and growing evidence supports that it may precede neurodegeneration. Here, we describe a method to label mitochondria with fluorescent proteins to monitor their movement along the axons in hiPSC-derived medium spiny neuron-like cells. We also included a detailed protocol for differentiation of hiPSC that produces electrophysiologically mature GABAergic striatal neurons with low amount of glial population.

Mitochondrial functional assays using MitoPlates™ S-1 allow us to characterize mitochondria in terms of substrate metabolism. MitoPlates™ are 96-well microplates pre-coated with a diverse set of substrates. The electron flow from NADH and FADH₂ producing mitochondrial substrates is measured based on the reduction of redox dye, that acts as a terminal electron acceptor. Here, we describe the application of MitoPlates™ to characterize the metabolism of synaptic mitochondria enclosed in isolated pre- and postsynaptic terminals (synaptoneurosomes).

Molecular dynamics (MD) simulations enable in silico investigation of the dynamic behavior of proteins and protein complexes. Here, we describe MD simulations of the SNARE bundle forming the complex with the neuronal proteins Synaptotagmin-1 (Syt1) and Complexin (Cpx). Syt1 is the synaptic vesicle (SV) protein that serves as the neuronal calcium sensor and triggers synaptic fusion upon calcium binding, and this process is promoted and accelerated by Cpx. The fusion depends on the Syt1 interactions with the SNARE-Cpx complex and with the lipid bilayer of the presynaptic membrane (PM). The MD simulations of the PM-Syt1-SNARE-Cpx-SV molecular system described here enabled us to investigate how this protein-lipid complex promotes the merging of SV and PM, triggering synaptic fusion.

Hypothyroidism, which is characterized by insufficient production of thyroid hormones, and malaria, a mosquito-borne infectious disease caused by Plasmodium parasites, are significant global health challenges. Studying how these two conditions interact could provide valuable insights into their complex relationship and potential treatment options.To induce hypothyroidism in the research, scientists used drugs to block the production of thyroid hormones. Then, they infected mice with Plasmodium berghei ANKA to mimic cerebral malaria infection. The spleen is essential in the body's immune response to malaria. It is involved in both innate and adaptive immunity, iron recycling, and the removal of old red blood cells or damaged cells infected with Plasmodium. Monitoring disease progression in male mice is crucial for early detection, and techniques like flow cytometry can help identify specific immune system populations within the spleen that are relevant to the research.

This chapter details protocols for determining plasma thyroid hormone (TH) levels and tissue TH content by competitive radioimmunoassays (RIAs). These protocols include: an initial test of the chromatographic performance, isotopic labeling to produce high activity ¹²⁵I-T3 and ¹²⁵I-T4, free iodide estimation of the labeled products, purification of tracers from iodide by paper electrophoresis, extraction of THs from plasma and tissue samples, and the RIA procedures. The RIA involves the competition between radioactive labeled and unlabeled hormones for specific antibody binding, and due to its high sensitivity is capable of detecting a minimum of 2.5 pg of T4 and 0.4 pg of T3.Drs. María Jesús Obregon and Gabriella Morreale de Escobar improved these protocols in the 1970s, enhancing sensitivity and accuracy. Their improvements enabled the detection of TH content in tissues, providing crucial insights into maternal THs role in fetal development and the importance of iodine intake during gestation. Their research also facilitated the early detection of congenital hypothyroidism, preventing neurological impairments in newborns. Internationally and in Spain, their contributions are widely acknowledged, leading to substantial public health impacts, including the implementation of nationwide neonatal screening programs. Despite progress, thyroid diseases remain prevalent, underscoring the need for continued research into thyroid physiology and associated disorders, employing highly sensitive techniques like the RIA outlined herein that we continue using in our research.

High Resolution-Magic Angle Spinning (HR-MAS) solid-state NMR spectroscopy is finding increasing application in the analysis of solid foods, bypassing the need for complicated solvent extraction procedures. In the present protocol, we report a simple analytical approach based on HR-MAS NMR spectroscopy for the phenolic profiling of olive fruits, flesh, or skin. This approach allows the facile characterization of phenolic compounds in olive fruits cultivated for extra-virgin olive oil production as a function of maturation and variety, in addition to processing technology for table olives.

Bacterial small RNAs (sRNAs) are well known for their ability to modulate gene expression at the post-transcriptional level. Their rather simple and modular organization provides the user with defined building blocks for synthetic biology approaches. In this chapter, we introduce a plasmid series for Escherichia coli and describe protocols for fast and efficient construction of synthetic sRNA expression plasmids based on Golden Gate assembly. In addition, we present the G-GArden tool, which assists with the design of oligodeoxynucleotides and overhangs for scarless assembly strategies. We propose that the presented procedures are suitable for many applications in different bacteria, which are related to E. coli and beyond.

Cholesterol is a critical lipid that is present at high concentrations in the plasma membranes of animal cells. Most of the membrane cholesterol is sequestered by other membrane lipids and the transmembrane domains of proteins. Cholesterol in excess of such sequestration forms a pool that is referred to as "accessible cholesterol." This pool of cholesterol plays a crucial role in maintaining lipid homeostasis and in controlling cell growth. The accessible cholesterol pool can also be exploited by bacteria and viruses to promote infection and host immune responses rapidly lower levels of this pool to confer protection. We had previously developed a bacterial toxin sensor called ALOD4 to monitor and quantify accessible cholesterol in cultured cells. Here, we report the characterization of a modified version of ALOD4 that is specialized to detect and monitor accessible cholesterol levels in primary immune cells by flow cytometry analysis.

Studies on the mechanisms and regulation of functional assemblies of SNARE proteins mediating membrane fusion essentially make use of recombinant proteins and artificial phospholipid bilayers. We have developed an easy-to-use in vivo system reconstituting membrane fusion in living bacteria. It relies on the formation of caveolin-dependent intracytoplasmic cisternae followed by the controlled synthesis of members of the synaptic SNARE machinery. Only when a SNARE complex is formed with its intact components does the docking and subsequent fusion occur between the cisternae and the plasma membrane that is accompanied by the disappearance of the former. The phenotypic response of the bacterial cell to fusion events is a remarkable increase in cell body length due to an expansion of the plasma membrane. Therefore, such an easy-to-observe phenotype makes this system amenable to structure-function studies of SNAREs. We describe here the specific ways to produce caveolin and the SNARE proteins from compatible plasmids upon bacterial transformation and to obtain the elongated cell phenotype. We also provide protocols to carry out the preparation of cell culture samples suitable for biochemical and light microscopy analysis.

The actin cytoskeleton serves an important, but poorly characterized, role in controlling granule exocytosis. The dynamic nature of actin remodeling allows it to act both as a barrier to prevent indiscriminate granule release and as a facilitator of membrane fusion. In its capacity to promote exocytosis, filamentous actin binds to components of the exocytotic machinery through actin binding proteins, but also through direct interactions with SNAREs. The platelet is an excellent cellular model to evaluate SNARE-actin interactions because of the marked reorganization of its actin cytoskeleton that occurs with activation and because of its abundance of secretory granules. This chapter will describe methods to evaluate SNARE-actin interactions in platelets using isolated platelet actin cytoskeleton, granule-enriched membrane fractions in a cell-free secretory system, and purified actin and recombinant SNAREs.