Gotowa bibliografia na temat „Ion-RNA Interactions”

Combined experimental-theoretical investigation of ultrafast hydration dynamics of an A-form RNA double helix in water reveals an ordered arrangement of water molecules and provides boundary conditions for the ion atmosphere around the polyanionic RNA.

RNA molecules cannot fold in the absence of counterions. Experiments are typically performed in the presence of monovalent and divalent cations. How to treat the impact of a solution containing a mixture of both ion types on RNA folding has remained a challenging problem for decades. By exploiting the large concentration difference between divalent and monovalent ions used in experiments, we develop a theory based on the reference interaction site model (RISM), which allows us to treat divalent cations explicitly while keeping the implicit screening effect due to monovalent ions. Our theory captures both the inner shell and outer shell coordination of divalent cations to phosphate groups, which we demonstrate is crucial for an accurate calculation of RNA folding thermodynamics. The RISM theory for ion–phosphate interactions when combined with simulations based on a transferable coarse-grained model allows us to predict accurately the folding of several RNA molecules in a mixture containing monovalent and divalent ions. The calculated folding free energies and ion-preferential coefficients for RNA molecules (pseudoknots, a fragment of the rRNA, and the aptamer domain of the adenine riboswitch) are in excellent agreement with experiments over a wide range of monovalent and divalent ion concentrations. Because the theory is general, it can be readily used to investigate ion and sequence effects on DNA properties.

Background: Cellular iron uptake, utilization, and storage are tightly controlled through the action of iron regulatory proteins (IRPs). IRPs achieve this control by binding to IREs-mRNA in the 5'- or 3'-end of mRNAs that encode proteins involved in iron metabolism. The interaction of iron regulatory proteins with mRNAs containing an iron responsive element plays a central role in this regulation. The IRE RNA family of mRNA regulatory structures combines absolutely conserved protein binding sites with phylogenetically conserved base pairs that are specific to each IREs and influence RNA/protein stability. Our previous result revealed the binding and kinetics of IRE RNA with IRP1. The aim of the present study is to gain further insight into the differences in protein/RNA stability as a function of pH and ionic strength. Objective: To determine the extent to which the binding affinity and stability of protein/RNA complex was affected by ionic strength and pH. Methods: Fluorescence spectroscopy was used to characterize IRE RNA-IRP protein interaction. Results: Scatchard analysis revealed that the IRP1 protein binds to a single IRE RNA molecule. The binding affinity of two IRE RNA/IRP was significantly changed with the change in pH. The data suggests that the optimum binding of RNA/IRP complex occurred at pH 7.6. Dissociation constant for two IRE RNA/IRP increased with an increase in ionic strength, with a larger effect for FRT IRE RNA. This suggests that numerous electrostatic interactions occur in the ferritin IRE RNA/IRP than ACO2 IRE RNA/IRP complex. Iodide quenching shows that the majority of the tryptophan residues in IRP1 are solvent-accessible, assuming that most of the tryptophan residues contribute to protein fluorescence. Conclusion: The results obtained from this study clearly indicate that IRE RNA/IRP complex is destabilized by the change in pH and ionic strength. These observations suggest that both pH and ion are important for the assembly and stability of the IRE RNA/IRP complex formation.

Abstract DNA and RNA sequences rich in guanine can fold into noncanonical structures called G-quadruplexes (GQs), which exhibit a common stem structure of Hoogsteen hydrogen-bonded guanine tetrads and diverse loop structures. GQ sequence motifs are overrepresented in promoters, origins of replication, telomeres, and untranslated regions in mRNA, suggesting roles in modulating gene expression and preserving genomic integrity. Given these roles and unique aspects of different structures, GQs are attractive targets for drug design, but greater insight into GQ folding pathways and the interactions stabilizing them is required. Here, we performed molecular dynamics simulations to study two bimolecular GQs, a telomeric DNA GQ and the analogous telomeric repeat-containing RNA (TERRA) GQ. We applied the Drude polarizable force field, which we show outperforms the additive CHARMM36 force field in both ion retention and maintenance of the GQ folds. The polarizable simulations reveal that the GQs bind bulk K+ ions differently, and that the TERRA GQ accumulates more K+ ions, suggesting different ion interactions stabilize these structures. Nucleobase dipole moments vary as a function of position and also contribute to ion binding. Finally, we show that the TERRA GQ is more sensitive than the telomeric DNA GQ to water-mediated modulation of ion-induced dipole-dipole interactions.

Cations are essential for ribonucleic acids (RNA), as they neutralize the negatively charged phosphate backbone. Divalent metals play important roles in the folding and function of RNA. The relationship between RNA and divalent cations magnesium (Mg(II)) and iron (Fe(II)) has been investigated. Mg(II) is involved in tertiary interactions of many large RNAs, and necessary for ribozyme activity. The influence of Mg(II) on RNA secondary and tertiary structure is investigated experimentally. Mg(II) binding to A-form RNA is accompanied by changes in CD spectra, indicating that Mg-RNA interactions influence the helical structure of RNA duplexes and helical regions of unfolded RNAs. Quantum mechanics calculations are used to probe the energetics of Mg(II)-chelation with phosphate oxygen atoms of nucleic acids. We identify the specific forces that contribute to stability of Mg(II)-chelation complexes in RNA. Fe(II) can serve as a substitute for Mg(II) in RNA folding and function. Fe(II) was abundant on early earth, it is plausible that RNA folding and function was mediated by Fe(II) instead of, or in combination with, Mg(II) in the anoxic environment of early earth. We have investigated oxidoreductase catalytic activity observed in RNA when in combination with Fe(II). This activity, only observed in the presence of Fe(II) and absence of Mg(II)appears to be a resurrection of ancient RNA capabilities that were extinguished upon the depletion of Fe(II) from the environment during the rise of oxygen after the great oxidation event. Finally, metal-ion based cleavage of RNA is used to identify the binding sites of Mg(II) and Fe(II). We observe that both metals cleave RNA in similar positions, providing further support for Fe(II) as a substitute for Mg(II) in RNA.

Les aptamères sont des acides nucléiques capables de se lier sélectivement à un ligand ou à une famille de molécules. Les aptamères sont la partie sensible des riboswitches, qui sont des segments régulateurs de l'ARN messager impliqués dans l'expression génétique. Les aptamères ont aussi des applications prometteuses comme sondes artificielles et capteurs Pour ces technologies, il est crucial de comprendre comment la liaison se produit, de la quantifier, et de comprendre comment les changements conformationnels sont induits par les ligands. Les objectifs de cette thèse sont d'explorer l'applicabilité de la spectrometrie de mobilité ionique (IMS) couplée à la spectrométrie de masse (SM) native aux aptamères d'ADN et d'ARN, d'abord dans la quantification de liaison, ensuite dans la détection du changement conformationnel lors de la liaison du ligand.Dans la première partie, nous avons évalué la détermination des valeurs de constantes d’équilibre de dissociation (KD) par MS, en tenant compte des facteurs de réponse relatifs (Rx) des aptamères libres et liés. Les titrages en SM sont comparés, pour validation, avec la calorimétrie par titrage isotherme (ITC). Deux aptamères d'ARN sont pris comme modèles : l'aptamère du vert de malachite, largement étudié par ITC, et l'aptamère de la riboflavine mononucléotide , un cas réaliste d'ARN Mg2+-dépendant pour la liaison du ligand. Nous avons observé que l'acétate d'ammonium et l'acétate de triméthyl ammonium conviennent à l'étude des aptamères et leurs complexes, et que les valeurs de KD obtenues par ITC et SM native sont comparables. Les aptamères ARN de la néomycine et de la tobramycine ont été choisis pour tester la limite de détection en SM native. Nous concluons que la SM native est adaptée pour déterminer des valeurs de KD comprises entre 50 nM et 30 µM. La correction apportée par Rx est relativement modeste dans tous les cas, en suggerant que la liaison du ligand n'est pas associée à une différence conformationnelle significative lors de l'ionisation. Pour ces aptamères, nous concluons que l'hypothèse de Rx égaux est acceptable.Dans la deuxième partie, nous avons évalué si le mécanisme de "liaison adaptative" des aptamères peut être révélé par IMS. À cette fin, en plus des systèmes énumérés ci-dessus, nous avons étudié l'aptamère ARN de la tétracycline et une série d'aptamères ADN capables de lier la cocaïne, pour lesquels le changement conformationnel par liaison du ligand est largement documenté dans la littérature. Pour tous les aptamères à l'exception de l'aptamère de la tétracycline, nous n'avons pas observé de différences significatives dans la conformation en phase gazeuse des ions liés aux ligands ou Mg2+. Cependant, nous avons observé un changement significatif dans la mobilité des ions de l'aptamère de la tétracycline. Le Mg2+ (100 µM) s’avère essentiel pour la liaison du ligand. Pour la série des aptamères de la cocaïne, même si nous ayons observé dans des conditions douces de pré IMS des ions compacts aussi bien pour les aptamères libres que pour les aptamères liés, une extension conformationnelle est visible à haute activation pre-IMS, bien révélée par l'état de charge 7-, qui suggère des réarrangements de phase gazeuse. Pour mieux étudier ces réarrangements, nous avons modifié les séquences avec des extensions dA, afin de comparer des systèmes ayant un nombre similaire de degrés de liberté sans modifier la structure cœur. Nous proposons également de nouvelles façons de présenter ces données, mieux adaptées quand la dissociation du ligand, la perte d’aduits et le dépliement d’ion arrivent dans les mêmes gammes d’énergie. L'augmentation graduelle de l'activation collisionnelle avant l'IMS, a révélé que l’energie de dépliement est corrélée au contenu en paires de bases, ce qui suggère que les paires de bases sont conservées dans les structures en phase gazeuse. Nous avons également observé que le ligand se perd à des énergies inférieures à celles du dépliage
Aptamers are single-stranded nucleic acids capable to bind selectively to a ligand or to a family of molecules. Aptamers are the sensing part of riboswitches, which are regulatory segments of messenger RNA involved in gene expression. Aptamers are also promising artificial probes, sensors and stimuli-responsive elements. In the development of aptamer-based technology, it is crucial to understand how binding is occurring, to quantify affinities, and ligand-induced conformational changes. The objective of this thesis is to explore the applicability of native IM-MS to DNA and RNA aptamers to quantify binding and to detect conformational change upon binding.In the first part, we evaluated the quantitative determination of equilibrium dissociation constants (KD) by mass spectrometry (MS), and the necessity of including a correction for relative response factors of free and bound aptamers. We compared isothermal titration calorimetry and MS titrations to validate the quantifications. Two RNA aptamers were taken as models: the malachite green aptamer, extensively studied by ITC, and the riboflavin mononucleotide aptamer, a case of Mg2+-dependent ligand binding. We observed that typical volatile electrolytes ammonium acetate and trimethyl ammonium acetate are suitable to study RNA aptamer binding, and that comparable KD values are obtained from ITC and native MS. The neomycin and tobramycin RNA aptamers were chosen to test the limit of detection of native MS. We found that native MS is appropriate to determine KD values in the range from 50 nM to 30 µM. The relative response factor correction was relatively modest in all cases, suggesting that the ligand binding is not associated to a significant conformational difference upon ionization. For these aptamers, we conclude that assuming equal response factors is acceptable.In the second part, we evaluated whether the aptamers’ “adaptive binding” mechanism can be revealed by ion mobility spectrometry (IMS). To this aim, in addition to the systems listed above we studied the tetracycline RNA aptamer and a series of cocaine-binding DNA aptamers, for which the conformational change upon binding is reported in literature. For all aptamers except the tetracycline aptamer, we did not observe a significant difference in the shape of the gas-phase structure upon ligand or Mg2+ binding. However, a significant change was observed in tetracycline RNA aptamer’s ion mobilities, at biologically relevant concentration of Mg2+ (100 µM), and we found that Mg2+ is essential for ligand binding, in agreement with previous solution studies. For the cocaine-binding DNA aptamer series, although we observed similar compactness for the free and bound aptamers in soft pre-IMS conditions, a conformational extension occurs at high pre-IMS activation, best revealed by charge state 7-, suggesting gas-phase rearrangements. To better investigate whether the energetics of these rearrangements depend on pre-folding or on ligand binding, we modified the sequences with dA overhangs, to compare systems with similar numbers of degrees of freedom without altering the core structure. We also propose new ways of presenting the data, adapted to the cases where ligand dissociation, declustering and unfolding occur at similar voltages. The gradual increase of the pre-IMS collisional activation revealed that the unfolding energetics is correlated with the base pairs content, suggesting that base pairs are conserved in the gas-phase structures. We also found that ligand is lost at lower energies than unfolding.In summary, gas-phase compaction occur for both the free aptamers and bound aptamers, and memories of the solution-phase structures can only be revealed in some particular cases. However, the compaction towards similar shapes might constitute an advantage for the quantification, because molecular systems of similar shapes have similar electrospray responses. Consequently, native MS provides reliable estimations of KD values

RNase P is an essential ribonuclease responsible for removal of the 5’ leader of tRNA precursors. Bacterial RNase P consists of an RNA subunit and a small basic protein. The catalytic activity is associated with the RNA subunit, i.e. bacterial RNase P RNA is a ribozyme. The protein subunit is, however, essential for activity in vivo. RNase P RNA, as well as the holoenzyme, requires the presence of divalent metal ions for activity. The aim of this thesis was to increase our understanding of the catalytic mechanism of RNase P RNA mediated cleavage. The importance of the nucleotides close to the cleavage site and the roles of divalent metal ions in RNase P RNA-catalyzed reaction were investigated. Escherichia coli RNase P RNA (M1 RNA) was used as a model system. It was shown that different metal ions have differential effects on cleavage site recognition. Cleavage activity was rescued by mixing metal ions that do not promote cleavage activity by themselves. This suggests that efficient and correct cleavage is the result of metal ion cooperativity in the RNase P RNA-mediated cleavage reaction. The results suggested that one of the metal ions involved in this cooperativity is positioned in the vicinity of a well-known interaction between RNase P RNA and its substrate. Based on my studies on how different metal ions bind to RNA and influence its activity we raise the interesting possibility that the activity of biocatalysts that depend on RNA for activity are up- or downregulated depending on the intracellular concentrations of the bulk biological metal ions Mg2+ and Ca2+. The nucleotides upstream of the cleavage site in the substrate were found to influence the cleavage efficiency. This was not exclusively due to intermolecular base pairing within the substrate but also dependent on the identities of the nucleotides at position –2 and –1. The strength of the base pair at position –1/+73 was demonstrated to affect cleavage efficiency. These observations are in keeping with previous suggestion that the nucleotides close to the cleavage site are important for RNase P cleavage. We conclude that the residue at -1 is a positive determinant for cleavage by RNase P. Hence, my studies extend our understanding of the RNase P cleavage site recognition process.

Thesis (Ph. D.)--Florida State University, 2008.
Advisors: Nancy L. Greenbaum [and] Geoffrey F. Strouse, Florida State University, College of Arts and Sciences, Dept. of Chemistry & Biochemistry. Title and description from dissertation home page (viewed June 20, 2008). Document formatted into pages; contains xv, 120 pages. Includes bibliographical references.

Thesis (Ph.D.)--State University of New York at Buffalo, 2008.
Title from PDF title page (viewed on Jan. 22, 2009) Available through UMI ProQuest Digital Dissertations. Thesis adviser: Morrow, Janet R. Includes bibliographical references.

Riboswitches are noncoding RNA molecules that can control gene expression upon cognate ligand binding. Riboswitches are primarily present in bacteria and are crucial for bacteria's survival, which makes riboswitches attractive targets for discovering new antimicrobials. Designing drugs that target a riboswitch function can be accelerated by understanding the effect of physicochemical factors (like ions, temperature, pressure, cosolvents, and pH) on the riboswitch folding and cognate ligand binding. The magnesium (Mg2+) ions possess the unique capability to exhibit site-specific binding along the RNA chain and modulate the population of functionally relevant RNA tertiary structures. In this thesis, I have discussed the effect of Mg2+ on the folding thermodynamics and kinetics of the riboswitches and how cations assist riboswitches in attaining specific tertiary structures. I have provided insight into how the anionic cognate ligands bind to the polyanionic RNA backbone. Further, I have discussed the properties that contribute to cations' binding at specific riboswitch sites. I then highlighted, how riboswitch responds to the cognate ligand binding and transmits the ligand binding information to the gene expression machinery.

碩士
國立臺灣大學
化學研究所
101
Ion pairing interactions play important roles in protein stability and RNA recognition. Ion pairs are formed between a pair of oppositely charged amino acids. Interestingly, natural charged amino acids have different number of hydrophobic methylenes on their side chains. For negatively charged residues, Asp has one methylene and Glu has two methylenes. The analogous non-encoded negatively charged amino acid, Aad, contains three methylenes. To study the effect of negatively charged amino acid side chain length on cross strand ion pairs in β-sheets stability, a basic β-hairpin model HPTZbbArg was designed. Zbb denotes the negatively charged residues. The hairpin structure for peptides HPTAspArg, HPTGluArg, and HPTAadArg were confirmed by NMR methods. The fraction folded of the peptides was determined using chemical shift data involving the fully unfolded and the fully folded reference peptides. The interaction free energy followed the trend: Aad-Arg > Glu-Arg ≈ AspArg. Apparently, the longer the negatively charged residue side chain length, the stronger the ion pairing interaction. HIV-1 Tat protein contains an arginine-rich sequence (Tat49-57), which binds specifically to the trans-activating responsive (TAR) element and plays an important role in nuclear localization. The binding of HIV-1 Tat protein and TAR RNA is essential for HIV-1 virus genome replication. To study the effect of arginine dimethylation on RNA recognition and cellular uptake, each arginine residue in Tat49-57 was replaced with a dimethylated arginine including ADMA and SDMA, the asymmetric and symmetric dimethylated forms, respectively. The dissociation constant for the Tat derived peptide-TAR RNA complexes was determined by gel shift assay. The cellular uptake efficiency of these Tat derived peptides into Jurkat cells was assessed by flow cytometry.

This thesis provides biophysical bases of the interactions between two proteins involved in microRNA (miRNA)-mediated silencing: CNOT1 and the silencing domain of GW182. The regulation of gene expression at the post-transcriptional level involves the crucial CCR4-NOT deadenylase complex, which deadenylates mRNA, and can also inhibit translation in an independent fashion. In miRNA-mediated silencing, the CCR4-NOT complex is brought into the vicinity of the target mRNA by the successive actions of the miRNA, the Argonaute protein and finally, the GW182 protein, which interacts directly with CCR4-NOT. In the case of silencing of mRNAs containing AU-rich elements, the same action is performed by the protein called tristetraprolin involved in the regulation of inflammatory processes. The interactions and interplay between all of these high molecular weight proteins are relatively poorly understood. In particular, the interaction sites between GW182 and the CCR4-NOT complex were previously unknown. Molecular biology experiments allowed the identification of CCR4-NOT interaction motifs on GW182. One of them is crucial for deadenylation, while the other is vital in mediating the interaction with CCR4-NOT via CNOT1, the scaffolding subunit of the CCR4-NOT complex. Biophysical experiments based on hydrogen-deuterium exchange mass spectrometry allowed the identification of the corresponding binding site on CNOT1(800-999). Surprisingly, the binding site of the GW182 silencing domain was found to be at the same CNOT1(800-999) surface region as the binding site of tristetraprolin. Biochemical experiments excluded their simultaneous binding to CNOT1. The GW182 and tristetraprolin proteins share a common motif, RLPXφ, that interacts with CNOT1 in a very similar, but not identical, manner. This sequence has been proposed to act as a short linear motif. Thus, the two different gene silencing pathways: miRNA-mediated silencing and ARE-mediated silencing intersect at CNOT1, which serves as a molecular hub. The structural dynamics of the GW182 silencing domain and the CNOT1(800-999) fragments were also studied. The GW182 silencing domain was experimentally proved to be natively unstructured except for an RNA-recognition motif (RRM) domain. The GW182 RRM domain was found to be a loose structure, contrary to the CNOT1(800-999) structure that was found to be very rigid. Experiments performed in this thesis have led to the discovery of the interaction sites between the natively disordered GW182 silencing domain and the helical CNOT1(800-999) protein fragment, contributing to the understanding of the molecular mechanisms of recognition within protein complexes involved in gene silencing in different physiological processes.
Niniejsza praca doktorska dotyczy biofizycznych podstaw oddziaływania między białkami zaangażowanymi w wyciszanie ekspresji genów przez mikro-RNA (miRNA), a mianowicie pomiędzy białkiem CNOT1 a domeną wyciszającą białka GW182. W procesie wyciszania ekspresji genów przez miRNA, cząsteczki te wiążą się z białkiem Argonaute i naprowadzają je na cząsteczkę mRNA, która ma ulec wyciszeniu. Z białkiem Argonaute oddziałuje białko GW182, które z kolei wiąże się z kompleksem deadenylaz CCR4-NOT. Kompleks ten deadenyluje mRNA oraz może także blokować jego translację, co łącznie prowadzi do wyciszenia ekspresji danego genu. Z kolei w wyciszaniu mRNA zawierających sekwencje bogate w adeninę i urydynę, rolę miRNA wraz z Argonaute i GW182 pełni białko o nazwie tristetraprolina, które odgrywa kluczową rolę w procesach odpowiedzi na stany zapalne. Oddziaływania pomiędzy składnikami tego skomplikowanego układu białek o wielkich masach cząsteczkowych są jeszcze stosunkowo słabo poznane. W szczególności, nieznane były miejsca odpowiedzialne za tworzenie kompleksu pomiędzy GW182 a CCR4-NOT. Doświadczenia z zakresu biologii molekularnej pozwoliły na identyfikację miejsc wiążących CCR4-NOT w sekwencji domeny wyciszającej białka GW182. Jedno z nich ma kluczowy wpływ na deadenylację, a drugie - kluczowy wpływ na oddziaływanie z kompleksem CCR4-NOT za pośrednictwem jego centralnej podjednostki CNOT1. Badania biofizyczne metodą wymiany wodór-deuter sprzężoną ze spektrometrią mas pozwoliły z kolei na identyfikację miejsca oddziaływania GW182 na białku CNOT1 (we fragmencie 800-999), które, niespodziewanie, okazało się bardzo dobrze pokrywać z miejscem oddziaływania CNOT1(800-999) z tristetraproliną. Eksperymenty biochemiczne wykazały, że białka te konkurują o miejsce oddziaływania na CNOT1(800-999). Białka GW182 i tristetraprolina oddziałują z CNOT1 wykorzystując ten sam motyw sekwencji, RLPXφ, w bardzo podobny, jednak nie identyczny sposób. Sekwencja ta prawdopodobnie działa jako tzw. krótki motyw liniowy (z ang. short linear motif, SLiM). Zatem te dwa szlaki kontroli nad ekspresją genów krzyżują się. W pracy zbadano także dynamikę strukturalną białka CNOT1(800-999) oraz domeny wyciszającej białka GW182. Wykazano eksperymentalnie, że białko GW182 ma nieustrukturyzowany charakter, oprócz domeny wiążącej RNA (RRM), która ma strukturę bardzo dynamiczną. Natomiast białko CNOT1(800-999) charakteryzuje się stabilną, ściśle upakowaną strukturą. Przeprowadzone badania doprowadziły do odkrycia miejsc oddziaływania pomiędzy natywnie nieustrukturyzowaną domeną wyciszającą GW182, a helikalnym fragmentem białka CNOT1(800 999), przyczyniając się do zrozumienia molekularnych mechanizmów rozpoznawania w kompleksach białkowych odpowiedzialnych za regulację ekspresji genów w różnych procesach komórkowych.

The aim of this book is to provide the clinician with a comprehensive and clinical relevant survey of emerging concepts on the organization and function of the nervous system and neurologic disease mechanisms, at the molecular, cellular, and system levels. The content of is based on the review of information obtained from recent advances in genetic, molecular, and cell biology techniques; electrophysiological recordings; brain mapping; and mouse models, emphasizing the clinical and possible therapeutic implications. Many chapters of this book contain information that will be relevant not only to clinical neurologists but also to psychiatrists and physical therapists. The scope includes the mechanisms and abnormalities of DNA/RNA metabolism, proteostasis, vesicular biogenesis, and axonal transport and mechanisms of neurodegeneration; the role of the mitochondria in cell function and death mechanisms; ion channels, neurotransmission and mechanisms of channelopathies and synaptopathies; the functions of astrocytes, oligodendrocytes, and microglia and their involvement in disease; the local circuits and synaptic interactions at the level of the cerebral cortex, thalamus, basal ganglia, cerebellum, brainstem, and spinal cord transmission regulating sensory processing, behavioral state, and motor functions; the peripheral and central mechanisms of pain and homeostasis; and networks involved in emotion, memory, language, and executive function.

Abstract The chapter’s Narrative outlines the core molecular dynamics of present-day organisms, with a consideration of DNA and RNA, protein structure and protein-protein interactions in three dimensions, enzymes as catalysts, the biophysics of membrane channels and ion gradients, biochemical cascades and signal transduction cascades, and cellular homeostasis. The chapter’s Reflections include a consideration of reductionism vs. holism. The “grunge theory of matter” is replaced by a paean to matter for all the incredible things it has achieved, followed by a consideration of the distress we may feel in considering our materiality and an invitation to invoke the spiritual practice of assent.