RESUMEN
Thiolases are CoA-dependent enzymes that catalyze the thiolytic cleavage of 3-ketoacyl-CoA, as well as its reverse reaction, which is the thioester-dependent Claisen condensation reaction. Thiolases are dimers or tetramers (dimers of dimers). All thiolases have two reactive cysteines: (a) a nucleophilic cysteine, which forms a covalent intermediate, and (b) an acid/base cysteine. The best characterized thiolase is the Zoogloea ramigera thiolase, which is a bacterial biosynthetic thiolase belonging to the CT-thiolase subfamily. The thiolase active site is also characterized by two oxyanion holes, two active site waters, and four catalytic loops with characteristic amino acid sequence fingerprints. Three thiolase subfamilies can be identified, each characterized by a unique sequence fingerprint for one of their catalytic loops, which causes unique active site properties. Recent insights concerning the thiolase reaction mechanism, as obtained from recent structural studies, as well as from classical and recent enzymological studies, are addressed, and open questions are discussed.
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Coenzima A , Cisteína , Coenzima A/química , Coenzima A/metabolismo , Cisteína/metabolismo , Modelos Moleculares , Acetil-CoA C-Acetiltransferasa/química , Acetil-CoA C-Acetiltransferasa/metabolismo , Dominio CatalíticoRESUMEN
Metals are essential components in life processes and participate in many important biological processes. Dysregulation of metal homeostasis is correlated with many diseases. Metals are also frequently incorporated into diagnosis and therapeutics. Understanding of metal homeostasis under (patho)physiological conditions and the molecular mechanisms of action of metallodrugs in biological systems has positive impacts on human health. As an emerging interdisciplinary area of research, metalloproteomics involves investigating metal-protein interactions in biological systems at a proteome-wide scale, has received growing attention, and has been implemented into metal-related research. In this review, we summarize the recent advances in metalloproteomics methodologies and applications. We also highlight emerging single-cell metalloproteomics, including time-resolved inductively coupled plasma mass spectrometry, mass cytometry, and secondary ion mass spectrometry. Finally, we discuss future perspectives in metalloproteomics, aiming to attract more original research to develop more advanced methodologies, which could be utilized rapidly by biochemists or biologists to expand our knowledge of how metal functions in biology and medicine.
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Investigación Biomédica , Metaloproteínas , Humanos , Metaloproteínas/análisis , Metaloproteínas/química , Metaloproteínas/genética , Metales/análisis , Metales/química , Proteoma/genética , Proteómica/métodosRESUMEN
The lack of tools to observe drug-target interactions at cellular resolution in intact tissue has been a major barrier to understanding in vivo drug actions. Here, we develop clearing-assisted tissue click chemistry (CATCH) to optically image covalent drug targets in intact mammalian tissues. CATCH permits specific and robust in situ fluorescence imaging of target-bound drug molecules at subcellular resolution and enables the identification of target cell types. Using well-established inhibitors of endocannabinoid hydrolases and monoamine oxidases, direct or competitive CATCH not only reveals distinct anatomical distributions and predominant cell targets of different drug compounds in the mouse brain but also uncovers unexpected differences in drug engagement across and within brain regions, reflecting rare cell types, as well as dose-dependent target shifts across tissue, cellular, and subcellular compartments that are not accessible by conventional methods. CATCH represents a valuable platform for visualizing in vivo interactions of small molecules in tissue.
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Química Clic , Imagen Óptica , Animales , Encéfalo , Sistemas de Liberación de Medicamentos , Mamíferos , Ratones , Imagen Óptica/métodosRESUMEN
Small molecule covalent drugs provide desirable therapeutic properties over noncovalent ones for treating challenging diseases. The potential of covalent protein drugs, however, remains unexplored due to protein's inability to bind targets covalently. We report a proximity-enabled reactive therapeutics (PERx) approach to generate covalent protein drugs. Through genetic code expansion, a latent bioreactive amino acid fluorosulfate-L-tyrosine (FSY) was incorporated into human programmed cell death protein-1 (PD-1). Only when PD-1 interacts with PD-L1 did the FSY react with a proximal histidine of PD-L1 selectively, enabling irreversible binding of PD-1 to only PD-L1 in vitro and in vivo. When administrated in immune-humanized mice, the covalent PD-1(FSY) exhibited strikingly more potent antitumor effect over the noncovalent wild-type PD-1, attaining therapeutic efficacy equivalent or superior to anti-PD-L1 antibody. PERx should provide a general platform technology for converting various interacting proteins into covalent binders, achieving specific covalent protein targeting for biological studies and therapeutic capability unattainable with conventional noncovalent protein drugs.
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Preparaciones Farmacéuticas/metabolismo , Proteínas/uso terapéutico , Secuencia de Aminoácidos , Animales , Antineoplásicos/metabolismo , Antígeno B7-H1/química , Antígeno B7-H1/metabolismo , Membrana Celular/metabolismo , Proliferación Celular , Células Dendríticas/metabolismo , Humanos , Cinética , Ligandos , Activación de Linfocitos/inmunología , Ratones , Monocitos/metabolismo , Fenotipo , Proteínas/química , Receptores Quiméricos de Antígenos/metabolismo , Linfocitos T/citología , Linfocitos T/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The discovery of organic ligands that bind specifically to proteins is a central problem in chemistry, biology, and the biomedical sciences. The encoding of individual organic molecules with distinctive DNA tags, serving as amplifiable identification bar codes, allows the construction and screening of combinatorial libraries of unprecedented size, thus facilitating the discovery of ligands to many different protein targets. Fundamentally, one links powers of genetics and chemical synthesis. After the initial description of DNA-encoded chemical libraries in 1992, several experimental embodiments of the technology have been reduced to practice. This review provides a historical account of important milestones in the development of DNA-encoded chemical libraries, a survey of relevant ongoing research activities, and a glimpse into the future.
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Descubrimiento de Drogas/métodos , Biblioteca de Genes , Bibliotecas de Moléculas Pequeñas , Animales , Técnicas Químicas Combinatorias , Humanos , Ligandos , Biblioteca de PéptidosRESUMEN
Copper-binding metallophores, or chalkophores, play a role in microbial copper homeostasis that is analogous to that of siderophores in iron homeostasis. The best-studied chalkophores are members of the methanobactin (Mbn) family-ribosomally produced, posttranslationally modified natural products first identified as copper chelators responsible for copper uptake in methane-oxidizing bacteria. To date, Mbns have been characterized exclusively in those species, but there is genomic evidence for their production in a much wider range of bacteria. This review addresses the current state of knowledge regarding the function, biosynthesis, transport, and regulation of Mbns. While the roles of several proteins in these processes are supported by substantial genetic and biochemical evidence, key aspects of Mbn manufacture, handling, and regulation remain unclear. In addition, other natural products that have been proposed to mediate copper uptake as well as metallophores that have biologically relevant roles involving copper binding, but not copper uptake, are discussed.
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Proteínas Bacterianas/metabolismo , Quelantes/metabolismo , Cobre/metabolismo , Imidazoles/metabolismo , Oligopéptidos/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Fenómenos Biofísicos , Quelantes/química , Genoma Bacteriano , Homeostasis , Imidazoles/química , Methylosinus trichosporium/genética , Methylosinus trichosporium/metabolismo , Modelos Biológicos , Estructura Molecular , Oligopéptidos/química , Oligopéptidos/genética , Operón , Transporte de ProteínasRESUMEN
S-adenosylmethionine (AdoMet) has been referred to as both "a poor man's adenosylcobalamin (AdoCbl)" and "a rich man's AdoCbl," but today, with the ever-increasing number of functions attributed to each cofactor, both appear equally rich and surprising. The recent characterization of an organometallic species in an AdoMet radical enzyme suggests that the line that differentiates them in nature will be constantly challenged. Here, we compare and contrast AdoMet and cobalamin (Cbl) and consider why Cbl-dependent AdoMet radical enzymes require two cofactors that are so similar in their reactivity. We further carry out structural comparisons employing the recently determined crystal structure of oxetanocin-A biosynthetic enzyme OxsB, the first three-dimensional structural data on a Cbl-dependent AdoMet radical enzyme. We find that the structural motifs responsible for housing the AdoMet radical machinery are largely conserved, whereas the motifs responsible for binding additional cofactors are much more varied.
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S-Adenosilmetionina/metabolismo , Vitamina B 12/metabolismo , Animales , Sitios de Unión , Coenzimas/química , Coenzimas/metabolismo , Electroquímica , Enzimas/química , Enzimas/metabolismo , Radicales Libres/química , Radicales Libres/metabolismo , Humanos , Modelos Moleculares , Estructura Molecular , S-Adenosilmetionina/química , Vitamina B 12/análogos & derivados , Vitamina B 12/químicaRESUMEN
The formation of ordered nanostructures by molecular self-assembly of proteins and peptides represents one of the principal directions in nanotechnology. Indeed, polyamides provide superior features as materials with diverse physical properties. A reductionist approach allowed the identification of extremely short peptide sequences, as short as dipeptides, which could form well-ordered amyloid-like ß-sheet-rich assemblies comparable to supramolecular structures made of much larger proteins. Some of the peptide assemblies show remarkable mechanical, optical, and electrical characteristics. Another direction of reductionism utilized a natural noncoded amino acid, α-aminoisobutryic acid, to form short superhelical assemblies. The use of this exceptional helix inducer motif allowed the fabrication of single heptad repeats used in various biointerfaces, including their use as surfactants and DNA-binding agents. Two additional directions of the reductionist approach include the use of peptide nucleic acids (PNAs) and coassembly techniques. The diversified accomplishments of the reductionist approach, as well as the exciting future advances it bears, are discussed.
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Nanoestructuras/química , Péptidos/química , Proteínas Amiloidogénicas/química , Animales , Biotecnología , Humanos , Modelos Moleculares , Nanotecnología/métodos , Oligopéptidos/química , Ácidos Nucleicos de Péptidos/química , Ingeniería de ProteínasRESUMEN
Small-molecule fluorophores, such as fluorescein and rhodamine derivatives, are critical tools in modern biochemical and biological research. The field of chemical dyes is old; colored molecules were first discovered in the 1800s, and the fluorescein and rhodamine scaffolds have been known for over a century. Nevertheless, there has been a renaissance in using these dyes to create tools for biochemistry and biology. The application of modern chemistry, biochemistry, molecular genetics, and optical physics to these old structures enables and drives the development of novel, sophisticated fluorescent dyes. This critical review focuses on an important example of chemical biology-the melding of old and new chemical knowledge-leading to useful molecules for advanced biochemical and biological experiments.
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Fluoresceínas/síntesis química , Colorantes Fluorescentes/síntesis química , Sondas Moleculares/síntesis química , Etiquetas de Fotoafinidad/síntesis química , Rodaminas/síntesis química , Animales , Bacterias/metabolismo , Técnicas de Química Sintética , Fluoresceínas/historia , Fluoresceínas/metabolismo , Colorantes Fluorescentes/historia , Colorantes Fluorescentes/metabolismo , Historia del Siglo XIX , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Microscopía Fluorescente , Sondas Moleculares/historia , Sondas Moleculares/metabolismo , Etiquetas de Fotoafinidad/historia , Etiquetas de Fotoafinidad/metabolismo , Rodaminas/historia , Rodaminas/metabolismoRESUMEN
Hydrogen peroxide (H2O2) is a prime member of the reactive oxygen species (ROS) family of molecules produced during normal cell function and in response to various stimuli, but if left unchecked, it can inflict oxidative damage on all types of biological macromolecules and lead to cell death. In this context, a major source of H2O2 for redox signaling purposes is the NADPH oxidase (Nox) family of enzymes, which were classically studied for their roles in phagocytic immune response but have now been found to exist in virtually all mammalian cell types in various isoforms with distinct tissue and subcellular localizations. Downstream of this tightly regulated ROS generation, site-specific, reversible covalent modification of proteins, particularly oxidation of cysteine thiols to sulfenic acids, represents a prominent posttranslational modification akin to phosphorylation as an emerging molecular mechanism for transforming an oxidant signal into a dynamic biological response. We review two complementary types of chemical tools that enable (a) specific detection of H2O2 generated at its sources and (b) mapping of sulfenic acid posttranslational modification targets that mediate its signaling functions, which can be used to study this important chemical signal in biological systems.
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Peróxido de Hidrógeno/metabolismo , NADPH Oxidasas/metabolismo , Transducción de Señal , Animales , Humanos , Oxidación-Reducción , Ácidos Sulfénicos/metabolismoRESUMEN
Unraveling the complexity of the brain requires sophisticated methods to probe and perturb neurobiological processes with high spatiotemporal control. The field of chemical biology has produced general strategies to combine the molecular specificity of small-molecule tools with the cellular specificity of genetically encoded reagents. Here, we survey the application, refinement, and extension of these hybrid small-molecule:protein methods to problems in neuroscience, which yields powerful reagents to precisely measure and manipulate neural systems.
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Neurociencias , EncéfaloRESUMEN
Genetic code expansion and reprogramming enable the site-specific incorporation of diverse designer amino acids into proteins produced in cells and animals. Recent advances are enhancing the efficiency of unnatural amino acid incorporation by creating and evolving orthogonal ribosomes and manipulating the genome. Increasing the number of distinct amino acids that can be site-specifically encoded has been facilitated by the evolution of orthogonal quadruplet decoding ribosomes and the discovery of mutually orthogonal synthetase/tRNA pairs. Rapid progress in moving genetic code expansion from bacteria to eukaryotic cells and animals (C. elegans and D. melanogaster) and the incorporation of useful unnatural amino acids has been aided by the development and application of the pyrrolysyl-transfer RNA (tRNA) synthetase/tRNA pair for unnatural amino acid incorporation. Combining chemoselective reactions with encoded amino acids has facilitated the installation of posttranslational modifications, as well as rapid derivatization with diverse fluorophores for imaging.
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Escherichia coli/genética , Código Genético , Aminoácidos/química , Aminoacil-ARNt Sintetasas/química , Animales , Caenorhabditis elegans , Drosophila melanogaster , Evolución Molecular , Eliminación de Gen , Genoma , Ingeniería de Proteínas/métodos , ARN de Transferencia/química , Ribosomas/química , Saccharomyces cerevisiae/genéticaRESUMEN
The CRISPR-Cas12a system shows unique features compared with widely used Cas9, making it an attractive and potentially more precise alternative. However, the adoption of this system has been hindered by its relatively low editing efficiency. Guided by physical chemical principles, we covalently conjugated 5' terminal modified CRISPR RNA (crRNA) to a site-specifically modified Cas12a through biorthogonal chemical reaction. The genome editing efficiency of the resulting conjugated Cas12a complex (cCas12a) was substantially higher than that of the wild-type complex. We also demonstrated that cCas12a could be used for precise gene knockin and multiplex gene editing in a chimeric antigen receptor T cell preparation with efficiency much higher than that of the wild-type system. Overall, our findings indicate that covalently linking Cas nuclease and crRNA is an effective approach to improve the Cas12a-based genome editing system and could potentially provide an insight into engineering other Cas family members with low efficiency as well.
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Proteínas Bacterianas/genética , Proteínas Asociadas a CRISPR/genética , Sistemas CRISPR-Cas , Endodesoxirribonucleasas/genética , Edición Génica , Receptores Quiméricos de Antígenos/metabolismo , Acidaminococcus , Animales , ADN/química , ADN/metabolismo , Endonucleasas/metabolismo , Escherichia coli/metabolismo , Técnicas de Sustitución del Gen , Técnicas Genéticas , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Técnicas In Vitro , Células K562 , Ratones , Mutagénesis , ARN/metabolismo , Espectrometría de Masas en TándemRESUMEN
Enzymes can usually be unambiguously assigned to one of seven classes specifying the basic chemistry of their catalyzed reactions. Less frequently, two or more reaction classes are catalyzed by a single enzyme within one active site. Two examples are an isomerohydrolase and an isomero-oxygenase that catalyze isomerization-coupled reactions crucial for production of vision-supporting 11-cis-retinoids. In these enzymes, isomerization is obligately paired and mechanistically intertwined with a second reaction class. A handful of other enzymes carrying out similarly coupled isomerization reactions have been described, some of which have been subjected to detailed structure-function analyses. Herein we review these rarefied enzymes, focusing on the mechanistic and structural basis of their reaction coupling with the goal of revealing catalytic commonalities.
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Isomerasas , Isomerasas/metabolismo , Isomerasas/química , Humanos , Isomerismo , Biocatálisis , Dominio Catalítico , Catálisis , Animales , Modelos MolecularesRESUMEN
Neurohypophysial peptides are ancient and evolutionarily highly conserved neuropeptides that regulate many crucial physiological functions in vertebrates and invertebrates. The human neurohypophysial oxytocin/vasopressin (OT/VP) signaling system with its four receptors has become an attractive drug target for a variety of diseases, including cancer, pain, cardiovascular indications, and neurological disorders. Despite its promise, drug development faces hurdles, including signaling complexity, selectivity and off-target concerns, translational interspecies differences, and inefficient drug delivery. In this review we dive into the complexity of the OT/VP signaling system in health and disease, provide an overview of relevant pharmacological probes, and discuss the latest trends in therapeutic lead discovery and drug development.
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Oxitocina , Vasopresinas , Animales , Humanos , Receptores de VasopresinasRESUMEN
Drug discovery is adapting to novel technologies such as data science, informatics, and artificial intelligence (AI) to accelerate effective treatment development while reducing costs and animal experiments. AI is transforming drug discovery, as indicated by increasing interest from investors, industrial and academic scientists, and legislators. Successful drug discovery requires optimizing properties related to pharmacodynamics, pharmacokinetics, and clinical outcomes. This review discusses the use of AI in the three pillars of drug discovery: diseases, targets, and therapeutic modalities, with a focus on small-molecule drugs. AI technologies, such as generative chemistry, machine learning, and multiproperty optimization, have enabled several compounds to enter clinical trials. The scientific community must carefully vet known information to address the reproducibility crisis. The full potential of AI in drug discovery can only be realized with sufficient ground truth and appropriate human intervention at later pipeline stages.
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Inteligencia Artificial , Médicos , Animales , Humanos , Reproducibilidad de los Resultados , Descubrimiento de Drogas , TecnologíaRESUMEN
Lipid distribution in an organism is mediated by the interplay between lipoprotein particles, lipoprotein receptors and class B scavenger receptors of the CD36 family. CD36 is a multifunctional protein mediating lipid uptake, mobilization and signaling at the plasma membrane and inside of the cell. The CD36 protein family has 14 members in Drosophila melanogaster, which allows for the differentiated analysis of their functions. Here, we unravel a role for the so far uncharacterized scavenger receptor Bez in lipid export from Drosophila adipocytes. Bez shares the lipid binding residue with CD36 and is expressed at the plasma membrane of the embryonic, larval and adult fat body. Bez loss of function lowers the organismal availability of storage lipids and blocks the maturation of egg chambers in ovaries. We demonstrate that Bez interacts with the APOB homolog Lipophorin at the plasma membrane of adipocytes and trace the Bez-dependent transfer of an alkyne-labeled fatty acid from adipocytes to Lipophorin. Our study demonstrates how lipids are distributed by scavenger receptor-lipoprotein interplay and contribute to the metabolic control of development.
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Antígenos CD36 , Proteínas de Drosophila , Drosophila melanogaster , Cuerpo Adiposo , Metabolismo de los Lípidos , Animales , Femenino , Adipocitos/metabolismo , Antígenos CD36/metabolismo , Antígenos CD36/genética , Membrana Celular/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Cuerpo Adiposo/metabolismo , Lipoproteínas/metabolismo , Ovario/metabolismo , Receptores Depuradores/metabolismo , Receptores Depuradores/genéticaRESUMEN
Peptide formation from amino acids is thermodynamically unfavorable but a recent study provided evidence that the reaction occurs at the air/solution interfaces of aqueous microdroplets. Here, we show that i) the suggested amino acid complex in microdroplets undergoes dehydration to form oxazolone; ii) addition of water to oxazolone forms the dipeptide; and iii) reaction of oxazolone with other amino acids forms tripeptides. Furthermore, the chirality of the reacting amino acids is preserved in the oxazolone product, and strong chiral selectivity is observed when converting the oxazolone to tripeptide. This last fact ensures that optically impure amino acids will undergo chain extension to generate pure homochiral peptides. Peptide formation in bulk by wet-dry cycling shares a common pathway with the microdroplet reaction, both involving the oxazolone intermediate.
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Oxazolona , Péptidos , Péptidos/química , Aminoácidos/química , Dipéptidos , Agua/químicaRESUMEN
A fundamental question associated with chirality is how mixtures containing equal amounts of interconverting enantiomers can spontaneously convert to systems enriched in only one of them. Enantiomers typically have similar chemical properties, but can exhibit distinct reactivity under specific conditions, and these differences can be used to bias the system's composition in favor of one enantiomer. Transport properties are also expected to differ for enantiomers in chiral solvents, but the role of such differences in chiral symmetry breaking has not been clarified yet. In this work, we develop a theoretical framework to show that asymmetry in diffusion properties can trigger a spontaneous and selective symmetry breaking in mixtures of enantiomers. We derive a generic evolution equation for the enantiomeric excess in a chiral solvent. This equation shows that the relative stability of homochiral domains is dictated by the difference of diffusion coefficients of the two enantiomers. Consequently, deracemization toward a specific enantiomeric excess can be achieved when this difference is large enough. These results hold significant implications for our understanding of chiral symmetry breaking.
RESUMEN
Pyrite is the most common sulfide mineral in hydrothermal ore-forming systems. The ubiquity and abundance of pyrite, combined with its ability to record and preserve a history of fluid evolution in crustal environments, make it an ideal mineral for studying the genesis of hydrothermal ore deposits, including those that host critical metals. However, with the exception of boiling, few studies have been able to directly link changes in pyrite chemistry to the processes responsible for bonanza-style gold mineralization. Here, we report the results of high-resolution secondary-ion mass spectrometry and electron microprobe analyses conducted on pyrite from the Brucejack epithermal gold deposit, British Columbia. Our δ34S and trace element results reveal that the Brucejack hydrothermal system experienced abrupt fluctuations in fluid chemistry, which preceded and ultimately coincided with the onset of ultra-high-grade mineralization. We argue that these fluctuations, which include the occurrence of extraordinarily negative δ34S values (e.g., -36.1) in zones of auriferous, arsenian pyrite, followed by sharp increases of δ34S values in syn-electrum zones of nonarsenian pyrite, were caused by vigorous, fault valve-induced episodic boiling (flashing) and subsequent inundation of the hydrothermal system by seawater. We conclude that the influx of seawater was the essential step to forming bonanza-grade electrum mineralization by triggering, through the addition of cationic flocculants and cooling, the aggregation of colloidal gold suspensions. Moreover, our study demonstrates the efficacy of employing high-resolution, in situ analytical techniques to map out individual ore-forming events in a hydrothermal system.