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We asked researchers from a range of disciplines across biology, engineering, and medicine to describe a current technological need. The goal is to provide a sample of the various technological gaps that exist and inspire future research projects.
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HumanosRESUMEN
Mammalian bodies have more than a billion cells per cubic centimeter, which makes whole-body cell (WBC) profiling of an organism one of the ultimate challenges in biology and medicine. Recent advances in tissue-clearing technology have enabled rapid and comprehensive cellular analyses in whole organs and in the whole body by a combination of state-of-the-art technologies of optical imaging and image informatics. In this review, we focus mainly on the chemical principles in currently available techniques for tissue clearing and staining to facilitate our understanding of their underlying mechanisms. Tissue clearing is usually conducted by the following steps: (a) fixation, (b) permeabilization, (c) decolorizing, and (d) refractive index (RI) matching. To phenotype individual cells after tissue clearing, it is important to visualize genetically encoded fluorescent reporters and/or to stain tissues with fluorescent dyes, fluorescent labeled antibodies, or nucleic acid probes. Although some technical challenges remain, the chemical principles in tissue clearing and staining for WBC profiling will enable various applications, such as identifying cellular circuits across multiple organs and measuring their dynamics in stochastic and proliferative cellular processes, for example, autoimmune and malignant neoplastic diseases.
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Células/metabolismo , Coloración y Etiquetado , Fijación del Tejido/métodos , Animales , Fluorescencia , Humanos , Permeabilidad , RefractometríaRESUMEN
The covalent conjugation of ubiquitin family proteins is a widespread post-translational protein modification. In the ubiquitin family, the ATG8 subfamily is exceptional because it is conjugated mainly to phospholipids. However, it remains unknown whether other ubiquitin family proteins are also conjugated to phospholipids. Here, we report that ubiquitin is conjugated to phospholipids, mainly phosphatidylethanolamine (PE), in yeast and mammalian cells. Ubiquitinated PE (Ub-PE) accumulates at endosomes and the vacuole (or lysosomes), and its level increases during starvation. Ub-PE is also found in baculoviruses. In yeast, PE ubiquitination is catalyzed by the canonical ubiquitin system enzymes Uba1 (E1), Ubc4/5 (E2), and Tul1 (E3) and is reversed by Doa4. Liposomes containing Ub-PE recruit the ESCRT components Vps27-Hse1 and Vps23 in vitro. Ubiquitin-like NEDD8 and ISG15 are also conjugated to phospholipids. These findings suggest that the conjugation to membrane phospholipids is not specific to ATG8 but is a general feature of the ubiquitin family.
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Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Animales , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Liposomas/metabolismo , Mamíferos/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfolípidos/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , UbiquitinaciónRESUMEN
The development of whole-body imaging at single-cell resolution enables system-level approaches to studying cellular circuits in organisms. Previous clearing methods focused on homogenizing mismatched refractive indices of individual tissues, enabling reductions in opacity but falling short of achieving transparency. Here, we show that an aminoalcohol decolorizes blood by efficiently eluting the heme chromophore from hemoglobin. Direct transcardial perfusion of an aminoalcohol-containing cocktail that we previously termed CUBIC coupled with a 10 day to 2 week clearing protocol decolorized and rendered nearly transparent almost all organs of adult mice as well as the entire body of infant and adult mice. This CUBIC-perfusion protocol enables rapid whole-body and whole-organ imaging at single-cell resolution by using light-sheet fluorescent microscopy. The CUBIC protocol is also applicable to 3D pathology, anatomy, and immunohistochemistry of various organs. These results suggest that whole-body imaging of colorless tissues at high resolution will contribute to organism-level systems biology.
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Amino Alcoholes/análisis , Análisis de la Célula Individual/métodos , Imagen de Cuerpo Entero/métodos , Animales , Diabetes Mellitus/patología , Imagenología Tridimensional/métodos , Islotes Pancreáticos/patología , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Systems-level identification and analysis of cellular circuits in the brain will require the development of whole-brain imaging with single-cell resolution. To this end, we performed comprehensive chemical screening to develop a whole-brain clearing and imaging method, termed CUBIC (clear, unobstructed brain imaging cocktails and computational analysis). CUBIC is a simple and efficient method involving the immersion of brain samples in chemical mixtures containing aminoalcohols, which enables rapid whole-brain imaging with single-photon excitation microscopy. CUBIC is applicable to multicolor imaging of fluorescent proteins or immunostained samples in adult brains and is scalable from a primate brain to subcellular structures. We also developed a whole-brain cell-nuclear counterstaining protocol and a computational image analysis pipeline that, together with CUBIC reagents, enable the visualization and quantification of neural activities induced by environmental stimulation. CUBIC enables time-course expression profiling of whole adult brains with single-cell resolution.
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Neuroimagen/métodos , Animales , Encéfalo/citología , Callithrix , Indicadores y Reactivos/química , Ratones , Microscopía/métodosRESUMEN
The concept of chirality is of great relevance in nature, from chiral molecules such as sugar to parity transformations in particle physics. In condensed matter physics, recent studies have demonstrated chiral fermions and their relevance in emergent phenomena closely related to topology1-3. The experimental verification of chiral phonons (bosons) remains challenging, however, despite their expected strong impact on fundamental physical properties4-6. Here we show experimental proof of chiral phonons using resonant inelastic X-ray scattering with circularly polarized X-rays. Using the prototypical chiral material quartz, we demonstrate that circularly polarized X-rays, which are intrinsically chiral, couple to chiral phonons at specific positions in reciprocal space, allowing us to determine the chiral dispersion of the lattice modes. Our experimental proof of chiral phonons demonstrates a new degree of freedom in condensed matter that is both of fundamental importance and opens the door to exploration of new emergent phenomena based on chiral bosons.
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Neurotransmitter receptors are essential components of synapses for communication between neurons in the brain. Because the spatiotemporal expression profiles and dynamics of neurotransmitter receptors involved in many functions are delicately governed in the brain, in vivo research tools with high spatiotemporal resolution for receptors in intact brains are highly desirable. Covalent labeling by chemical reaction (chemical labeling) of proteins without genetic manipulation is now a powerful method for analyzing receptors in vitro. However, selective target receptor labeling in the brain has not yet been achieved. This study shows that ligand-directed alkoxyacylimidazole (LDAI) chemistry can be used to selectively tether synthetic probes to target endogenous receptors in living mouse brains. The reactive LDAI reagents with negative charges were found to diffuse well over the whole brain and could selectively label target endogenous receptors, including AMPAR, NMDAR, mGlu1, and GABAAR. This simple and robust labeling protocol was then used for various applications: three-dimensional spatial mapping of endogenous receptors in the brains of healthy and disease-model mice; multi-color receptor imaging; and pulse-chase analysis of the receptor dynamics in postnatal mouse brains. Here, results demonstrated that bioorthogonal receptor modification in living animal brains may provide innovative molecular tools that contribute to the in-depth understanding of complicated brain functions.
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Neuronas , Proteínas , Ratones , Animales , Indicadores y Reactivos , Ligandos , EncéfaloRESUMEN
Organoids are self-organizing in vitro 3D cultures that are histologically similar to a variety of human organs. A recent study by Rosselot et al (2021) shows that mature intestinal organoids possess species-specific circadian clocks similar to their respective in vivo context, suggesting organoids as promising platforms to study circadian medicine.
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Relojes Circadianos , Garrapatas , Animales , Relojes Circadianos/genética , Humanos , Intestinos , OrganoidesRESUMEN
Direct evidence for the requirement of delay in feedback repression in the mammalian circadian clock has been elusive. Cryptochrome 1 (Cry1), an essential clock component, displays evening-time expression and serves as a strong repressor at morning-time elements (E box/E' box). In this study, we reveal that a combination of day-time elements (D box) within the Cry1-proximal promoter and night-time elements (RREs) within its intronic enhancer gives rise to evening-time expression. A synthetic composite promoter produced evening-time expression, which was further recapitulated by a simple phase-vector model. Of note, coordination of day-time with night-time elements can modulate the extent of phase delay. A genetic complementation assay in Cry1(-/-):Cry2(-/-) cells revealed that substantial delay of Cry1 expression is required to restore circadian rhythmicity, and its prolonged delay slows circadian oscillation. Taken together, our data suggest that phase delay in Cry1 transcription is required for mammalian clock function.
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Relojes Circadianos , Criptocromos/metabolismo , Retroalimentación , Animales , Ritmo Circadiano , Elementos de Facilitación Genéticos , Intrones , Ratones , Regiones Promotoras Genéticas , Elementos Reguladores de la Transcripción , Análisis de la Célula IndividualRESUMEN
Many mammalian proteins have circadian cycles of production and degradation, and many of these rhythms are altered posttranscriptionally. We used ribosome profiling to examine posttranscriptional control of circadian rhythms by quantifying RNA translation in the liver over a 24-h period from circadian-entrained mice transferred to constant darkness conditions and by comparing ribosome binding levels to protein levels for 16 circadian proteins. We observed large differences in ribosome binding levels compared to protein levels, and we observed delays between peak ribosome binding and peak protein abundance. We found extensive binding of ribosomes to upstream open reading frames (uORFs) in circadian mRNAs, including the core clock gene Period2 (Per2). An increase in the number of uORFs in the 5'UTR was associated with a decrease in ribosome binding in the main coding sequence and a reduction in expression of synthetic reporter constructs. Mutation of the Per2 uORF increased luciferase and fluorescence reporter expression in 3T3 cells and increased luciferase expression in PER2:LUC MEF cells. Mutation of the Per2 uORF in mice increased Per2 mRNA expression, enhanced ribosome binding on Per2, and reduced total sleep time compared to that in wild-type mice. These results suggest that uORFs affect mRNA posttranscriptionally, which can impact physiological rhythms and sleep.
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Ritmo Circadiano , Perfilado de Ribosomas , Sueño , Animales , Ratones , Ritmo Circadiano/genética , Luciferasas/genética , Sistemas de Lectura Abierta/genética , ARN Mensajero/genética , Sueño/genética , Proteínas Circadianas Period/genéticaRESUMEN
Light-sheet microscopy has emerged as the preferred means for high-throughput volumetric imaging of cleared tissues. However, there is a need for a flexible system that can address imaging applications with varied requirements in terms of resolution, sample size, tissue-clearing protocol, and transparent sample-holder material. Here, we present a 'hybrid' system that combines a unique non-orthogonal dual-objective and conventional (orthogonal) open-top light-sheet (OTLS) architecture for versatile multi-scale volumetric imaging. We demonstrate efficient screening and targeted sub-micrometer imaging of sparse axons within an intact, cleared mouse brain. The same system enables high-throughput automated imaging of multiple specimens, as spotlighted by a quantitative multi-scale analysis of brain metastases. Compared with existing academic and commercial light-sheet microscopy systems, our hybrid OTLS system provides a unique combination of versatility and performance necessary to satisfy the diverse requirements of a growing number of cleared-tissue imaging applications.
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Microscopía , Animales , Ratones , Microscopía/métodosRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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State-of-the-art tissue-clearing methods provide subcellular-level optical access to intact tissues from individual organs and even to some entire mammals. When combined with light-sheet microscopy and automated approaches to image analysis, existing tissue-clearing methods can speed up and may reduce the cost of conventional histology by several orders of magnitude. In addition, tissue-clearing chemistry allows whole-organ antibody labelling, which can be applied even to thick human tissues. By combining the most powerful labelling, clearing, imaging and data-analysis tools, scientists are extracting structural and functional cellular and subcellular information on complex mammalian bodies and large human specimens at an accelerated pace. The rapid generation of terabyte-scale imaging data furthermore creates a high demand for efficient computational approaches that tackle challenges in large-scale data analysis and management. In this Review, we discuss how tissue-clearing methods could provide an unbiased, system-level view of mammalian bodies and human specimens and discuss future opportunities for the use of these methods in human neuroscience.
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Técnicas Histológicas/métodos , Microscopía/métodos , Sistema Nervioso/citología , Animales , Técnicas Histológicas/instrumentación , Humanos , Imagenología Tridimensional/métodos , Mamíferos , Microscopía/instrumentación , NeurocienciasRESUMEN
The first issue of Nature Reviews Neuroscience was published 20 years ago, in 2000. To mark this anniversary, in this Viewpoint article we asked a selection of researchers from across the field who have authored pieces published in the journal in recent years for their thoughts on notable and interesting developments in neuroscience, and particularly in their areas of the field, over the past two decades. They also provide some thoughts on current lines of research and questions that excite them.
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Neurociencias/historia , Historia del Siglo XXI , HumanosRESUMEN
The reduced sleep duration previously observed in Camk2b knockout mice revealed a role for Ca2+/calmodulin-dependent protein kinase II (CaMKII)ß as a sleep-promoting kinase. However, the underlying mechanism by which CaMKIIß supports sleep regulation is largely unknown. Here, we demonstrate that activation or inhibition of CaMKIIß can increase or decrease sleep duration in mice by almost 2-fold, supporting the role of CaMKIIß as a core sleep regulator in mammals. Importantly, we show that this sleep regulation depends on the kinase activity of CaMKIIß. A CaMKIIß mutant mimicking the constitutive-active (auto)phosphorylation state promotes the transition from awake state to sleep state, while mutants mimicking subsequent multisite (auto)phosphorylation states suppress the transition from sleep state to awake state. These results suggest that the phosphorylation states of CaMKIIß differently control sleep induction and maintenance processes, leading us to propose a "phosphorylation hypothesis of sleep" for the molecular control of sleep in mammals.
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Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Calcio , Animales , Calcio/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Mamíferos/metabolismo , Ratones , Ratones Noqueados , Fosforilación , SueñoRESUMEN
Temperature compensation is a striking feature of the circadian clock. Here we investigate biochemical mechanisms underlying temperature-compensated, CKIδ-dependent multi-site phosphorylation in mammals. We identify two mechanisms for temperature-insensitive phosphorylation at higher temperature: lower substrate affinity to CKIδ-ATP complex and higher product affinity to CKIδ-ADP complex. Inhibitor screening of ADP-dependent phosphatase activity of CKIδ identified aurintricarboxylic acid (ATA) as a temperature-sensitive kinase activator. Docking simulation of ATA and mutagenesis experiment revealed K224D/K224E mutations in CKIδ that impaired product binding and temperature-compensated primed phosphorylation. Importantly, K224D mutation shortens behavioral circadian rhythms and changes the temperature dependency of SCN's circadian period. Interestingly, temperature-compensated phosphorylation was evolutionary conserved in yeast. Molecular dynamics simulation and X-ray crystallography demonstrate that an evolutionally conserved CKI-specific domain around K224 can provide a structural basis for temperature-sensitive substrate and product binding. Surprisingly, this domain can confer temperature compensation on a temperature-sensitive TTBK1. These findings suggest the temperature-sensitive substrate- and product-binding mechanisms underlie temperature compensation.
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Adenosina Trifosfato/metabolismo , Quinasa Idelta de la Caseína/metabolismo , Relojes Circadianos , Ritmo Circadiano , Núcleo Supraquiasmático/enzimología , Temperatura , Animales , Sitios de Unión , Quinasa Idelta de la Caseína/química , Quinasa Idelta de la Caseína/genética , Dominio Catalítico , Cristalografía por Rayos X , Genotipo , Células HEK293 , Humanos , Hidrólisis , Cinética , Locomoción , Ratones Transgénicos , Modelos Biológicos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Mutación , Fenotipo , Fosforilación , Unión Proteica , Dominios Proteicos , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Serina , Relación Estructura-Actividad , Especificidad por Sustrato , Técnicas de Cultivo de Tejidos , TransfecciónRESUMEN
To conduct comprehensive characterization of molecular properties in organisms, we established an efficient method to produce knockout (KO)-rescue mice within a single generation. We applied this method to produce 20 strains of almost completely embryonic stem cell (ESC)-derived mice ("ES mice") rescued with wild-type and mutant Cry1 gene under a Cry1-/-:Cry2-/- background. A series of both phosphorylation-mimetic and non-phosphorylation-mimetic CRY1 mutants revealed that multisite phosphorylation of CRY1 can serve as a cumulative timer in the mammalian circadian clock. KO-rescue ES mice also revealed that CRY1-PER2 interaction confers a robust circadian rhythmicity in mice. Surprisingly, in contrast to theoretical predictions from canonical transcription/translation feedback loops, the residues surrounding the flexible P loop and C-lid domains of CRY1 determine circadian period without changing the degradation rate of CRY1. These results suggest that CRY1 determines circadian period through both its degradation-dependent and -independent pathways.
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Relojes Circadianos , Ritmo Circadiano , Criptocromos/metabolismo , Células Madre Embrionarias/metabolismo , Animales , Conducta Animal , Criptocromos/química , Criptocromos/deficiencia , Criptocromos/genética , Genotipo , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Moleculares , Actividad Motora , Mutación , Células 3T3 NIH , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Fenotipo , Fosforilación , Conformación Proteica , Transducción de Señal , Relación Estructura-Actividad , Factores de Tiempo , TransfecciónRESUMEN
SignificanceHuman sleep phenotypes are diversified by genetic and environmental factors, and a quantitative classification of sleep phenotypes would lead to the advancement of biomedical mechanisms underlying human sleep diversity. To achieve that, a pipeline of data analysis, including a state-of-the-art sleep/wake classification algorithm, the uniform manifold approximation and projection (UMAP) dimension reduction method, and the density-based spatial clustering of applications with noise (DBSCAN) clustering method, was applied to the 100,000-arm acceleration dataset. This revealed 16 clusters, including seven different insomnia-like phenotypes. This kind of quantitative pipeline of sleep analysis is expected to promote data-based diagnosis of sleep disorders and psychiatric disorders that tend to be complicated by sleep disorders.
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Bancos de Muestras Biológicas , Trastornos del Sueño-Vigilia , Aceleración , Humanos , Fenotipo , Sueño , Reino UnidoRESUMEN
The majority of low-grade isocitrate dehydrogenase-mutant (IDHmt) gliomas undergo malignant progression (MP), but their underlying mechanism remains unclear. IDHmt gliomas exhibit global DNA methylation, and our previous report suggested that MP could be partly attributed to passive demethylation caused by accelerated cell cycles. However, during MP, there is also active demethylation mediated by ten-eleven translocation, such as DNA hydroxymethylation. Hydroxymethylation is reported to potentially contribute to gene expression regulation, but its role in MP remains under investigation. Therefore, we conducted a comprehensive analysis of hydroxymethylation during MP of IDHmt astrocytoma. Five primary/malignantly progressed IDHmt astrocytoma pairs were analyzed with oxidative bisulfite and the Infinium EPIC methylation array, detecting 5-hydroxymethyl cytosine at over 850,000 locations for region-specific hydroxymethylation assessment. Notably, we observed significant sharing of hydroxymethylated genomic regions during MP across the samples. Hydroxymethylated CpGs were enriched in open sea and intergenic regions (p < 0.001), and genes undergoing hydroxymethylation were significantly associated with cancer-related signaling pathways. RNA sequencing data integration identified 91 genes with significant positive/negative hydroxymethylation-expression correlations. Functional analysis suggested that positively correlated genes are involved in cell-cycle promotion, while negatively correlated ones are associated with antineoplastic functions. Analyses of The Cancer Genome Atlas clinical data on glioma were in line with these findings. Motif-enrichment analysis suggested the potential involvement of the transcription factor KLF4 in hydroxymethylation-based gene regulation. Our findings shed light on the significance of region-specific DNA hydroxymethylation in glioma MP and suggest its potential role in cancer-related gene expression and IDHmt glioma malignancy.
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Neoplasias Encefálicas , Metilación de ADN , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Glioma , Isocitrato Deshidrogenasa , Factor 4 Similar a Kruppel , Mutación , Humanos , Isocitrato Deshidrogenasa/genética , Glioma/genética , Glioma/patología , Glioma/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Islas de CpG/genética , Femenino , Masculino , Astrocitoma/genética , Astrocitoma/patología , Astrocitoma/metabolismo , Persona de Mediana Edad , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , AdultoRESUMEN
Electroencephalogram (EEG) and electromyogram (EMG) are fundamental tools in sleep research. However, investigations into the statistical properties of rodent EEG/EMG signals in the sleep-wake cycle have been limited. The lack of standard criteria in defining sleep stages forces researchers to rely on human expertise to inspect EEG/EMG. The recent increasing demand for analysing large-scale and long-term data has been overwhelming the capabilities of human experts. In this study, we explored the statistical features of EEG signals in the sleep-wake cycle. We found that the normalized EEG power density profile changes its lower and higher frequency powers to a comparable degree in the opposite direction, pivoting around 20-30 Hz between the NREM sleep and the active brain state. We also found that REM sleep has a normalized EEG power density profile that overlaps with wakefulness and a characteristic reduction in the EMG signal. Based on these observations, we proposed three simple statistical features that could span a 3D space. Each sleep-wake stage formed a separate cluster close to a normal distribution in the 3D space. Notably, the suggested features are a natural extension of the conventional definition, making it useful for experts to intuitively interpret the EEG/EMG signal alterations caused by genetic mutations or experimental treatments. In addition, we developed an unsupervised automatic staging algorithm based on these features. The developed algorithm is a valuable tool for expediting the quantitative evaluation of EEG/EMG signals so that researchers can utilize the recent high-throughput genetic or pharmacological methods for sleep research.