At dilute concentrations, the grand-canonical partition function of the ligand provides a simple formulation of the protein's equilibrium shifts. The model's projections of spatial distribution and response probability fluctuate with varying ligand concentrations, and its thermodynamic conjugates are readily comparable to macroscopic measurements. This attribute makes it a highly valuable tool for the interpretation of experimental data at the atomic level. The theory's demonstration and explanation are highlighted through the lens of general anesthetics and voltage-gated channels, for which structural data are readily available.
A quantum/classical polarizable continuum model is implemented through the use of multiwavelets, as detailed herein. The solvent model, unlike many existing continuum solvation models, employs a flexible solute-solvent boundary and a variable permittivity dependent on position. With adaptive refinement strategies in our multiwavelet implementation, we can precisely incorporate both surface and volume polarization effects into the quantum/classical coupling. Solvent environments of intricate complexity are accommodated by the model, obviating the need for a posteriori volume polarization corrections. Our results, when compared against a sharp-boundary continuum model, display a strong correlation to the polarization energies calculated for the entries in the Minnesota solvation database.
For measuring basal and insulin-stimulated glucose uptake in murine tissues, an in-vivo procedure is presented here. Steps for the intraperitoneal administration of 2-deoxy-D-[12-3H]glucose, with or without insulin, are presented. We now detail the steps of tissue sampling, tissue preparation for quantification of 3H counts on a scintillation counter, and the procedure for data analysis. This protocol's applicability extends to other glucoregulatory hormones, genetic mouse models, and other animal species. For detailed instructions on employing and executing this protocol, see the work by Jiang et al. (2021).
In order to fully understand protein-mediated cellular processes, a thorough understanding of protein-protein interactions is necessary; however, the examination of transient and unstable interactions in live cells remains a complex challenge. This paper outlines a protocol that examines the interaction of an intermediate assembly form of a bacterial outer membrane protein with constituents of the bacterial barrel assembly machinery complex. A detailed protocol for expressing a protein target, combining chemical and in vivo photo-crosslinking techniques, and subsequently detecting the crosslinks, including immunoblotting, is presented. This protocol's capacity to analyze interprotein interactions in other processes is significant. Miyazaki et al. (2021) elaborates on the protocol's operational details and execution specifics.
Understanding aberrant myelination, a key feature in neuropsychiatric and neurodegenerative diseases, demands an in vitro platform that allows for the study of neuron-oligodendrocyte interaction, specifically myelination. Three-dimensional (3D) nanomatrix plates provide the platform for a controlled, direct co-culture protocol, specifically designed for hiPSC-derived neurons and oligodendrocytes. The protocol for differentiating hiPSCs into cortical neuron and oligodendrocyte cell types on 3D nanofiber arrays is provided here. We detail, in the subsequent sections, the process of detaching and isolating the oligodendrocyte lineage, which is subsequently followed by a neuron-oligodendrocyte co-culture experiment within the three-dimensional microenvironment.
Mitochondrial regulation of bioenergetics and cell death is fundamental to the adaptive responses of macrophages to infectious stimuli. To examine mitochondrial function in macrophages during bacterial infection, we present this protocol. This report details a methodology for assessing mitochondrial polarization, cellular death, and bacterial infection in live, human primary macrophages, employing a single-cell analysis approach for infected specimens. Employing Legionella pneumophila as a model organism is examined in detail within our study. check details This protocol's application can be modified for the investigation of mitochondrial functions in different environments. For a thorough explanation of this protocol's operation and procedure, see the publication by Escoll et al. (2021).
Injury to the atrioventricular conduction system (AVCS), the vital electrical connection between atrial and ventricular compartments, can result in a diversity of cardiac conduction problems. This paper outlines a protocol for targeting the mouse AVCS's structure, thus enabling analysis of its response to injury. check details Cellular ablation by tamoxifen, along with electrocardiographic AV block detection and the quantification of histological and immunofluorescence markers, serve to analyze the AVCS. The mechanisms behind AVCS injury repair and regeneration are open to study through the application of this protocol. To gain complete insight into the utilization and execution of this protocol, please refer to the work of Wang et al. (2021).
The innate immune response depends critically on cyclic guanosine monophosphate (cGMP)-AMP synthase (cGAS), a pivotal dsDNA recognition receptor. Sensing DNA, activated cGAS catalyzes the formation of cGAMP, a secondary messenger that activates downstream signaling, which, in turn, induces the synthesis of interferons and inflammatory cytokines. In this report, we identify ZYG11B, a member of the Zyg-11 family, as a potent contributor to cGAS-mediated immune responses. Disruption of ZYG11B's function hinders cGAMP creation, leading to impeded interferon and inflammatory cytokine transcription. The underlying mechanism by which ZYG11B acts is to amplify the attraction of cGAS to DNA, intensify the compaction of the cGAS-DNA complex, and bolster the structural integrity of this complex. Consequently, the infection of cells with herpes simplex virus 1 (HSV-1) causes a degradation of ZYG11B, independent of any cGAS mechanism. check details Our findings implicate ZYG11B's prominent involvement in the early phase of DNA-induced cGAS activation, and moreover, suggest a viral strategy to attenuate the innate immune system's function.
The remarkable capacity of hematopoietic stem cells for self-renewal and the subsequent differentiation into various blood cell lineages underscores their significance in blood production. Sex/gender differences are present in HSCs and the cells they produce through differentiation. Despite their fundamental significance, the specific mechanisms involved remain largely unstudied. Our previous research showcased an improvement in hematopoietic stem cell (HSC) survival and proliferative potential following the removal of latexin (Lxn) in female mice. Hematopoiesis and HSC function remain unchanged in Lxn knockout (Lxn-/-) male mice, irrespective of the presence or absence of myelosuppressive conditions. We have discovered that Thbs1, a downstream target of Lxn in female hematopoietic stem cells, displays repression in the male counterpart. Male-specific high expression of miR98-3p (microRNA 98-3p) facilitates the suppression of Thbs1 in male hematopoietic stem cells (HSCs), thus negating the functional effects of Lxn on male HSCs and hematopoiesis. These research findings expose a regulatory mechanism, involving a sex-chromosome-linked microRNA, which differentially regulates Lxn-Thbs1 signaling during hematopoiesis, thereby shedding light on the process responsible for sex-based differences in both normal and cancerous hematopoiesis.
Endogenous cannabinoid signaling is indispensable for key brain functions, and the identical pathways can be pharmacologically adjusted for pain, epilepsy, and post-traumatic stress disorder management. The impact of endocannabinoids on excitability is predominantly a consequence of presynaptic 2-arachidonoylglycerol (2-AG) interacting with the canonical cannabinoid receptor, CB1. We demonstrate a neocortical pathway where anandamide (AEA), a substantial endocannabinoid, effectively inhibits somatically measured voltage-gated sodium channel (VGSC) currents in the majority of neurons, a phenomenon not seen with 2-AG. An intracellular CB1 receptor, activated within this pathway by anandamide, decreases the propensity for recurrent action potential generation. By simultaneously activating CB1 receptors and inhibiting VGSC currents, WIN 55212-2 exemplifies this pathway's function in mediating the effects of exogenous cannabinoids on neuronal excitability. Functional separation of CB1 and VGSC actions is indicated by the absence of coupling at nerve terminals and 2-AG's ineffectiveness in blocking somatic VGSC currents.
Alternative splicing and chromatin regulation, as key mechanisms, play a vital role in guiding gene expression. Evidence suggests that histone modifications contribute to alternative splicing decisions, but the influence of alternative splicing on chromatin structure requires additional study. Downstream of T-cell signaling cascades, we observe alternative splicing of multiple genes encoding histone-modifying enzymes, including HDAC7, a gene previously connected to the modulation of gene expression and T-cell differentiation. CRISPR-Cas9 gene editing, coupled with cDNA expression, reveals that varying inclusion of HDAC7 exon 9 impacts the interaction between HDAC7 and protein chaperones, which, in turn, alters histone modifications and subsequently impacts gene expression. Of particular note, the more extended isoform, resulting from induction by the RNA-binding protein CELF2, bolsters the expression of pivotal T-cell surface proteins, especially CD3, CD28, and CD69. We demonstrate that variations in HDAC7 splicing have a global effect on histone modifications and gene expression, which, in turn, plays a role in the progression of T cell development.
Progressing from gene discovery in autism spectrum disorders (ASDs) to the understanding of the related biological processes is a key hurdle to overcome. In zebrafish mutants, we concurrently assess the in vivo functional effects of 10 ASD genes at the behavioral, structural, and circuit levels, demonstrating both unique and overlapping consequences of gene loss-of-function.