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The Bin/Amphiphysin/Rvs (BAR) domain protein FAM92A1 is a multifunctional protein engaged in regulating mitochondrial ultrastructure and ciliogenesis, but its physiological role in the brain remains unclear. Here, we show that FAM92A1 is expressed in neurons starting from embryonic development. FAM92A1 knockout in mice results in altered brain morphology and age-associated cognitive deficits, potentially due to neuronal degeneration and disrupted synaptic plasticity. Specifically, FAM92A1 deficiency impairs diverse neuronal membrane morphology, including the mitochondrial inner membrane, myelin sheath, and synapses, indicating its roles in membrane remodeling and maintenance. By determining the crystal structure of the FAM92A1 BAR domain, combined with atomistic molecular dynamics simulations, we uncover that FAM92A1 interacts with phosphoinositide- and cardiolipin-containing membranes to induce lipid-clustering and membrane curvature. Altogether, these findings reveal the physiological role of FAM92A1 in the brain, highlighting its impact on synaptic plasticity and neural function through the regulation of membrane remodeling and endocytic processes.
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Encéfalo , Cognición , Ratones Noqueados , Plasticidad Neuronal , Neuronas , Sinapsis , Animales , Encéfalo/metabolismo , Neuronas/metabolismo , Sinapsis/metabolismo , Plasticidad Neuronal/fisiología , Ratones , Cognición/fisiología , Membrana Celular/metabolismo , Simulación de Dinámica Molecular , Humanos , Fosfatidilinositoles/metabolismo , Cardiolipinas/metabolismo , MasculinoRESUMEN
Organisms generate shapes across size scales. Whereas patterning and morphogenesis of macroscopic tissues has been extensively studied, the principles underlying the formation of micrometric and submicrometric structures remain largely enigmatic. Individual cells of polychaete annelids, so-called chaetoblasts, are associated with the generation of chitinous bristles of highly stereotypic geometry. Here we show that bristle formation requires a chitin-producing enzyme specifically expressed in the chaetoblasts. Chaetoblasts exhibit dynamic cell surfaces with stereotypical patterns of actin-rich microvilli. These microvilli can be matched with internal and external structures of bristles reconstructed from serial block-face electron micrographs. Individual chitin teeth are deposited by microvilli in an extension-disassembly cycle resembling a biological 3D printer. Consistently, pharmacological interference with actin dynamics leads to defects in tooth formation. Our study reveals that both material and shape of bristles are encoded by the same cell, and that microvilli play a role in micro- to submicrometric sculpting of biomaterials.
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Quitina , Microvellosidades , Microvellosidades/ultraestructura , Animales , Quitina/metabolismo , Quitina/química , Poliquetos/ultraestructura , Actinas/metabolismo , MorfogénesisRESUMEN
Viruses target mitochondria to promote their replication, and infection-induced stress during the progression of infection leads to the regulation of antiviral defenses and mitochondrial metabolism which are opposed by counteracting viral factors. The precise structural and functional changes that underlie how mitochondria react to the infection remain largely unclear. Here we show extensive transcriptional remodeling of protein-encoding host genes involved in the respiratory chain, apoptosis, and structural organization of mitochondria as herpes simplex virus type 1 lytic infection proceeds from early to late stages of infection. High-resolution microscopy and interaction analyses unveiled infection-induced emergence of rough, thin, and elongated mitochondria relocalized to the perinuclear area, a significant increase in the number and clustering of endoplasmic reticulum-mitochondria contact sites, and thickening and shortening of mitochondrial cristae. Finally, metabolic analyses demonstrated that reactivation of ATP production is accompanied by increased mitochondrial Ca2+ content and proton leakage as the infection proceeds. Overall, the significant structural and functional changes in the mitochondria triggered by the viral invasion are tightly connected to the progression of the virus infection.
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Herpes Simple , Herpesvirus Humano 1 , Mitocondrias , Mitocondrias/metabolismo , Herpesvirus Humano 1/fisiología , Herpesvirus Humano 1/metabolismo , Humanos , Herpes Simple/metabolismo , Herpes Simple/virología , Herpes Simple/patología , Animales , Infecciones por Herpesviridae/metabolismo , Infecciones por Herpesviridae/virología , Infecciones por Herpesviridae/patología , Progresión de la Enfermedad , Chlorocebus aethiopsRESUMEN
Peroxisomes are membrane-enclosed organelles with important roles in fatty acid breakdown, bile acid synthesis and biosynthesis of sterols and ether lipids. Defects in peroxisomes result in severe genetic diseases, such as Zellweger syndrome and neonatal adrenoleukodystrophy. However, many aspects of peroxisomal biogenesis are not well understood. Here we investigated delivery of tail-anchored (TA) proteins to peroxisomes in mammalian cells. Using glycosylation assays we showed that peroxisomal TA proteins do not enter the endoplasmic reticulum (ER) in both wild type (WT) and peroxisome-lacking cells. We observed that in cells lacking the essential peroxisome biogenesis factor, PEX19, peroxisomal TA proteins localize mainly to mitochondria. Finally, to investigate peroxisomal TA protein targeting in cells with fully functional peroxisomes we used a proximity biotinylation approach. We showed that while ER-targeted TA construct was exclusively inserted into the ER, peroxisome-targeted TA construct was inserted to both peroxisomes and mitochondria. Thus, in contrast to previous studies, our data suggest that some peroxisomal TA proteins do not insert to the ER prior to their delivery to peroxisomes, instead, mitochondria can be involved.
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Proteínas de la Membrana , Peroxisomas , Animales , Peroxisomas/metabolismo , Proteínas de la Membrana/metabolismo , Retículo Endoplásmico/metabolismo , Membranas Intracelulares/metabolismo , Mitocondrias/metabolismo , Mamíferos/metabolismoRESUMEN
Aging is associated with progressive phenotypic changes. Virtually all cellular phenotypes are produced by proteins, and their structural alterations can lead to age-related diseases. However, we still lack comprehensive knowledge of proteins undergoing structural-functional changes during cellular aging and their contributions to age-related phenotypes. Here, we conducted proteome-wide analysis of early age-related protein structural changes in budding yeast using limited proteolysis-mass spectrometry (LiP-MS). The results, compiled in online ProtAge catalog, unraveled age-related functional changes in regulators of translation, protein folding, and amino acid metabolism. Mechanistically, we found that folded glutamate synthase Glt1 polymerizes into supramolecular self-assemblies during aging, causing breakdown of cellular amino acid homeostasis. Inhibiting Glt1 polymerization by mutating the polymerization interface restored amino acid levels in aged cells, attenuated mitochondrial dysfunction, and led to lifespan extension. Altogether, this comprehensive map of protein structural changes enables identifying mechanisms of age-related phenotypes and offers opportunities for their reversal.
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Senescencia Celular , Longevidad , Longevidad/genética , Polimerizacion , AminoácidosRESUMEN
Collagen is one the most abundant proteins and the main cargo of the secretory pathway, contributing to hepatic fibrosis and cirrhosis due to excessive deposition of extracellular matrix. Here we investigated the possible contribution of the unfolded protein response, the main adaptive pathway that monitors and adjusts the protein production capacity at the endoplasmic reticulum, to collagen biogenesis and liver disease. Genetic ablation of the ER stress sensor IRE1 reduced liver damage and diminished collagen deposition in models of liver fibrosis triggered by carbon tetrachloride (CCl 4 ) administration or by high fat diet. Proteomic and transcriptomic profiling identified the prolyl 4-hydroxylase (P4HB, also known as PDIA1), which is known to be critical for collagen maturation, as a major IRE1-induced gene. Cell culture studies demonstrated that IRE1 deficiency results in collagen retention at the ER and altered secretion, a phenotype rescued by P4HB overexpression. Taken together, our results collectively establish a role of the IRE1/P4HB axis in the regulation of collagen production and its significance in the pathogenesis of various disease states.
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The endoplasmic reticulum (ER) is the largest organelle of the cell, composed of a continuous network of sheets and tubules, and is involved in protein, calcium (Ca2+), and lipid homeostasis. In neurons, the ER extends throughout the cell, both somal and axodendritic compartments, and is highly important for neuronal functions. A third of the proteome of a cell, secreted and membrane-bound proteins, are processed within the ER lumen and most of these proteins are vital for neuronal activity. The brain itself is high in lipid content, and many structural lipids are produced, in part, by the ER. Cholesterol and steroid synthesis are strictly regulated in the ER of the blood-brain barrier protected brain cells. The high Ca2+ level in the ER lumen and low cytosolic concentration is needed for Ca2+-based intracellular signaling, for synaptic signaling and Ca2+ waves, and for preparing proteins for correct folding in the presence of high Ca2+ concentrations to cope with the high concentrations of extracellular milieu. Particularly, ER Ca2+ is controlled in axodendritic areas for proper neurito- and synaptogenesis and synaptic plasticity and remodeling. In this review, we cover the physiologic functions of the neuronal ER and discuss it in context of common neurodegenerative diseases, focusing on pharmacological regulation of ER Ca2+ Furthermore, we postulate that heterogeneity of the ER, its protein folding capacity, and ensuring Ca2+ regulation are crucial factors for the aging and selective vulnerability of neurons in various neurodegenerative diseases. SIGNIFICANCE STATEMENT: Endoplasmic reticulum (ER) Ca2+ regulators are promising therapeutic targets for degenerative diseases for which efficacious drug therapies do not exist. The use of pharmacological probes targeting maintenance and restoration of ER Ca2+ can provide restoration of protein homeostasis (e.g., folding of complex plasma membrane signaling receptors) and slow down the degeneration process of neurons.
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Enfermedades Neurodegenerativas , Humanos , Enfermedades Neurodegenerativas/tratamiento farmacológico , Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Calcio de la Dieta/metabolismo , Lípidos , Señalización del CalcioRESUMEN
Metastasis-suppressor 1 (MTSS1) is a membrane-interacting scaffolding protein that regulates the integrity of epithelial cell-cell junctions and functions as a tumor suppressor in a wide range of carcinomas. MTSS1 binds phosphoinositide-rich membranes through its I-BAR domain and is capable of sensing and generating negative membrane curvature in vitro. However, the mechanisms by which MTSS1 localizes to intercellular junctions in epithelial cells and contributes to their integrity and maintenance have remained elusive. By carrying out EM and live-cell imaging on cultured Madin-Darby canine kidney cell monolayers, we provide evidence that adherens junctions of epithelial cells harbor lamellipodia-like, dynamic actin-driven membrane folds, which exhibit high negative membrane curvature at their distal edges. BioID proteomics and imaging experiments demonstrated that MTSS1 associates with an Arp2/3 complex activator, the WAVE-2 complex, in dynamic actin-rich protrusions at cell-cell junctions. Inhibition of Arp2/3 or WAVE-2 suppressed actin filament assembly at adherens junctions, decreased the dynamics of junctional membrane protrusions, and led to defects in epithelial integrity. Together, these results support a model in which membrane-associated MTSS1, together with the WAVE-2 and Arp2/3 complexes, promotes the formation of dynamic lamellipodia-like actin protrusions that contribute to the integrity of cell-cell junctions in epithelial monolayers.
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Actinas , Proteínas de Microfilamentos , Seudópodos , Animales , Perros , Citoesqueleto de Actina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Actinas/metabolismo , Uniones Adherentes/metabolismo , Células Epiteliales/metabolismo , Uniones Intercelulares/metabolismo , Células de Riñón Canino Madin Darby , Proteínas de la Membrana/metabolismo , Seudópodos/metabolismo , Proteínas de Microfilamentos/metabolismoRESUMEN
Cell-to-cell signalling between niche and stem cells regulates tissue regeneration. While the identity of many mediating factors is known, it is largely unknown whether stem cells optimize their receptiveness to niche signals according to the niche organization. Here, we show that Lgr5+ small intestinal stem cells (ISCs) regulate the morphology and orientation of their secretory apparatus to match the niche architecture, and to increase transport efficiency of niche signal receptors. Unlike the progenitor cells lacking lateral niche contacts, ISCs orient Golgi apparatus laterally towards Paneth cells of the epithelial niche, and divide Golgi into multiple stacks reflecting the number of Paneth cell contacts. Stem cells with a higher number of lateral Golgi transported Epidermal growth factor receptor (Egfr) with a higher efficiency than cells with one Golgi. The lateral Golgi orientation and enhanced Egfr transport required A-kinase anchor protein 9 (Akap9), and was necessary for normal regenerative capacity in vitro . Moreover, reduced Akap9 in aged ISCs renders ISCs insensitive to niche-dependent modulation of Golgi stack number and transport efficiency. Our results reveal stem cell-specific Golgi complex configuration that facilitates efficient niche signal reception and tissue regeneration, which is compromised in the aged epithelium.
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In plants, the phloem distributes photosynthetic products for metabolism and storage over long distances. It relies on specialized cells, the sieve elements, which are enucleated and interconnected through large so-called sieve pores in their adjoining cell walls. Reverse genetics identified PECTATE LYASE-LIKE 12 (PLL12) as critical for plant growth and development. Using genetic complementations, we established that PLL12 is required exclusively late during sieve element differentiation. Structural homology modeling, enzyme inactivation, and overexpression suggest a vital role for PLL12 in sieve-element-specific pectin remodeling. While short distance symplastic diffusion is unaffected, the pll12 mutant is unable to accommodate sustained plant development due to an incapacity to accommodate increasing hydraulic demands on phloem long-distance transport as the plant grows-a defect that is aggravated when combined with another sieve-element-specific mutant callose synthase 7 (cals7). Establishing CALS7 as a specific sieve pore marker, we investigated the subcellular dynamics of callose deposition in the developing sieve plate. Using fluorescent CALS7 then allowed identifying structural defects in pll12 sieve pores that are moderate at the cellular level but become physiologically relevant due to the serial arrangement of sieve elements in the sieve tube. Overall, pectin degradation through PLL12 appears subtle in quantitative terms. We therefore speculate that PLL12 may act as a regulator to locally remove homogalacturonan, thus potentially enabling further extracellular enzymes to access and modify the cell wall during sieve pore maturation.
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Arabidopsis , Arabidopsis/metabolismo , Floema/metabolismo , Glucanos/metabolismo , Plantas/metabolismoRESUMEN
Astrocytes, often considered as secondary responders to neurodegeneration, are emerging as primary drivers of brain disease. Here we show that mitochondrial DNA depletion in astrocytes affects their primary cilium, the signaling organelle of a cell. The progressive oxidative phosphorylation deficiency in astrocytes induces FOXJ1 and RFX transcription factors, known as master regulators of motile ciliogenesis. Consequently, a robust gene expression program involving motile cilia components and multiciliated cell differentiation factors are induced. While the affected astrocytes still retain a single cilium, these organelles elongate and become remarkably distorted. The data suggest that chronic activation of the mitochondrial integrated stress response (ISRmt) in astrocytes drives anabolic metabolism and promotes ciliary elongation. Collectively, our evidence indicates that an active signaling axis involving mitochondria and primary cilia exists and that ciliary signaling is part of ISRmt in astrocytes. We propose that metabolic ciliopathy is a novel pathomechanism for mitochondria-related neurodegenerative diseases.
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Astrocitos , Cilios , Mitocondrias , Astrocitos/metabolismo , Cilios/metabolismo , Cilios/patología , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Homeostasis , Mitocondrias/metabolismo , Mitocondrias/patología , Ratones , Animales , Factores de Transcripción del Factor Regulador X/genética , Factores de Transcripción del Factor Regulador X/metabolismo , ADN MitocondrialRESUMEN
Here, we describe protocols for chemical fixation and flat embedding to study the Golgi structure by thin section transmission electron microscopy (TEM) and for 3,3'-diaminobenzidine (DAB) cytochemical staining and pre-embedding immunolabelling to localize specific Golgi proteins. Furthermore, we demonstrate how the Golgi morphology can be elucidated by classifying the Golgi membranes using Microscopy Image Browser-a software that provides anonymization, modelling, and annotation.
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Aparato de Golgi , Microscopía , Microscopía/métodosRESUMEN
Autophagosome biogenesis occurs in the transient subdomains of the endoplasmic reticulum that are called omegasomes, which, in fluorescence microscopy, appear as small puncta, which then grow in diameter and finally shrink and disappear once the autophagosome is complete. Autophagosomes are formed by phagophores, which are membrane cisterns that elongate and close to form the double membrane that limits autophagosomes. Earlier electron-microscopy studies showed that, during elongation, phagophores are lined by the endoplasmic reticulum on both sides. However, the morphology of the very early phagophore precursors has not been studied at the electron-microscopy level. We used live-cell imaging of cells expressing markers of phagophore biogenesis combined with correlative light-electron microscopy, as well as electron tomography of ATG2A/B-double-deficient cells, to reveal the high-resolution morphology of phagophore precursors in three dimensions. We showed that phagophores are closed or nearly closed into autophagosomes already at the stage when the omegasome diameter is still large. We further observed that phagophore precursors emerge next to the endoplasmic reticulum as bud-like highly curved membrane cisterns with a small opening to the cytosol. The phagophore precursors then open to form more flat cisterns that elongate and curve to form the classically described crescent-shaped phagophores.
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Autofagosomas , Electrones , Autofagia , Retículo Endoplásmico , Microscopía ElectrónicaRESUMEN
Golgi membrane protein 1 (GOLM1) is a Golgi-resident type 2 transmembrane protein known to be overexpressed in several cancers, including hepatocellular carcinoma (HCC), as well as in viral infections. However, the role of GOLM1 in lipid metabolism remains enigmatic. In this study, we employed siRNA-mediated GOLM1 depletion in Huh-7 HCC cells to study the role of GOLM1 in lipid metabolism. Mass spectrometric lipidomic analysis in GOLM1 knockdown cells showed an aberrant accumulation of sphingolipids, such as ceramides, hexosylceramides, dihexosylceramides, sphinganine, sphingosine, and ceramide phosphate, along with cholesteryl esters. Furthermore, we observed a reduction in phosphatidylethanolamines and lysophosphatidylethanolamines. In addition, Seahorse extracellular flux analysis indicated a reduction in mitochondrial oxygen consumption rate upon GOLM1 depletion. Finally, alterations in Golgi structure and distribution were observed both by electron microscopy imaging and immunofluorescence microscopy analysis. Importantly, we found that GOLM1 depletion also affected cell proliferation and cell cycle progression in Huh-7 HCC cells. The Golgi structural defects induced by GOLM1 reduction might potentially affect the trafficking of proteins and lipids leading to distorted intracellular lipid homeostasis, which may result in organelle dysfunction and altered cell growth. In conclusion, we demonstrate that GOLM1 depletion affects sphingolipid metabolism, mitochondrial function, Golgi structure, and proliferation of HCC cells.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Ciclo Celular , Proliferación Celular , Ceramidas , Ésteres del Colesterol , Humanos , Metabolismo de los Lípidos , Neoplasias Hepáticas/patología , Proteínas de la Membrana/metabolismo , Fosfatos , Fosfatidiletanolaminas , ARN Interferente Pequeño/metabolismo , Esfingolípidos , EsfingosinaRESUMEN
The endoplasmic reticulum (ER) is an organelle that performs several key functions such as protein synthesis and folding, lipid metabolism and calcium homeostasis. When these functions are disrupted, such as upon protein misfolding, ER stress occurs. ER stress can trigger adaptive responses to restore proper functioning such as activation of the unfolded protein response (UPR). In certain cells, the free fatty acid palmitate has been shown to induce the UPR. Here, we examined the effects of palmitate on UPR gene expression in a human neuronal cell line and compared it with thapsigargin, a known depletor of ER calcium and trigger of the UPR. We used a Gaussia luciferase-based reporter to assess how palmitate treatment affects ER proteostasis and calcium homeostasis in the cells. We also investigated how ER calcium depletion by thapsigargin affects lipid membrane composition by performing mass spectrometry on subcellular fractions and compared this to palmitate. Surprisingly, palmitate treatment did not activate UPR despite prominent changes to membrane phospholipids. Conversely, thapsigargin induced a strong UPR, but did not significantly change the membrane lipid composition in subcellular fractions. In summary, our data demonstrate that changes in membrane lipid composition and disturbances in ER calcium homeostasis have a minimal influence on each other in neuronal cells. These data provide new insight into the adaptive interplay of lipid homeostasis and proteostasis in the cell.
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Palmitatos , Proteostasis , Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Humanos , Lípidos de la Membrana/metabolismo , Palmitatos/metabolismo , Palmitatos/farmacología , Tapsigargina/metabolismo , Tapsigargina/farmacologíaRESUMEN
Modern research in the life sciences is unthinkable without computational methods for extracting, quantifying and visualising information derived from microscopy imaging data of biological samples. In the past decade, we observed a dramatic increase in available software packages for these purposes. As it is increasingly difficult to keep track of the number of available image analysis platforms, tool collections, components and emerging technologies, we provide a conservative overview of software that we use in daily routine and give insights into emerging new tools. We give guidance on which aspects to consider when choosing the platform that best suits the user's needs, including aspects such as image data type, skills of the team, infrastructure and community at the institute and availability of time and budget.
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Procesamiento de Imagen Asistido por Computador , Programas Informáticos , Procesamiento de Imagen Asistido por Computador/métodos , Microscopía/métodosRESUMEN
We have shown the connection of hyaluronan synthesis activity with the enhanced shedding of extracellular vesicles, but detailed morphological analysis of those hyaluronan-induced EVs is still missing. In this study we utilized a comprehensive set of high-resolution imaging techniques to characterize in high detail the size and morphology of EVs originating from stable MCF7 breast cancer cell line and transiently transfected cells expressing GFP-HAS3. To avoid possible artefacts or loss of EVs resulting from the isolation process, special attention was paid to analysis of EVs in situ in monolayer and in 3D cultures. The results of this study show that GFP-HAS3 expressing MCF7 cells produce morphologically diverse EVs but also demonstrates the variation in results obtained with different experimental setup, which emphasizes the importance of comparison between different methods when interpreting the observations.
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Vesículas Extracelulares , Ácido Hialurónico , Vesículas Extracelulares/metabolismo , Humanos , Hialuronano Sintasas/metabolismo , Ácido Hialurónico/metabolismo , Células MCF-7RESUMEN
BACKGROUND AND OBJECTIVE: Advances in electron microscopy (EM) now allow three-dimensional (3D) imaging of hundreds of micrometers of tissue with nanometer-scale resolution, providing new opportunities to study the ultrastructure of the brain. In this work, we introduce a freely available Matlab-based gACSON software for visualization, segmentation, assessment, and morphology analysis of myelinated axons in 3D-EM volumes of brain tissue samples. METHODS: The software is equipped with a graphical user interface (GUI). It automatically segments the intra-axonal space of myelinated axons and their corresponding myelin sheaths and allows manual segmentation, proofreading, and interactive correction of the segmented components. gACSON analyzes the morphology of myelinated axons, such as axonal diameter, axonal eccentricity, myelin thickness, or g-ratio. RESULTS: We illustrate the use of the software by segmenting and analyzing myelinated axons in six 3D-EM volumes of rat somatosensory cortex after sham surgery or traumatic brain injury (TBI). Our results suggest that the equivalent diameter of myelinated axons in somatosensory cortex was decreased in TBI animals five months after the injury. CONCLUSION: Our results indicate that gACSON is a valuable tool for visualization, segmentation, assessment, and morphology analysis of myelinated axons in 3D-EM volumes. It is freely available at https://github.com/AndreaBehan/g-ACSON under the MIT license.
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Axones , Lesiones Traumáticas del Encéfalo , Animales , Axones/ultraestructura , Microscopía Electrónica , Vaina de Mielina/ultraestructura , Ratas , Programas InformáticosRESUMEN
Transplantation of pancreatic islet cells derived from human pluripotent stem cells is a promising treatment for diabetes. Despite progress in the generation of stem-cell-derived islets (SC-islets), no detailed characterization of their functional properties has been conducted. Here, we generated functionally mature SC-islets using an optimized protocol and benchmarked them comprehensively against primary adult islets. Biphasic glucose-stimulated insulin secretion developed during in vitro maturation, associated with cytoarchitectural reorganization and the increasing presence of alpha cells. Electrophysiology, signaling and exocytosis of SC-islets were similar to those of adult islets. Glucose-responsive insulin secretion was achieved despite differences in glycolytic and mitochondrial glucose metabolism. Single-cell transcriptomics of SC-islets in vitro and throughout 6 months of engraftment in mice revealed a continuous maturation trajectory culminating in a transcriptional landscape closely resembling that of primary islets. Our thorough evaluation of SC-islet maturation highlights their advanced degree of functionality and supports their use in further efforts to understand and combat diabetes.