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1.
Methods Mol Biol ; 2761: 67-79, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38427230

RESUMO

Cytokines have the potential to be the ideal biomarkers to track the onset and progression of immune-mediated diseases, study the development of novel therapeutic strategies, and they can serve as outcome parameters due to their crucial role in the regulation of immune and inflammatory responses. It is vital to keep track of the entire cytokine spectrum due to the complex interactions, pleiotropic effects, and redundancy in the cytokine network. The multiplex immunoassay (MIA) is, therefore, the best method for achieving that goal. This chapter addresses the key methodological processes of this technique, such as sample preparation, antibody coupling to beads, and assay procedure.


Assuntos
Anticorpos , Citocinas , Humanos , Imunoensaio/métodos , Encéfalo , Espaço Extracelular , Biomarcadores
2.
Epilepsy Res ; 201: 107337, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38461594

RESUMO

Post traumatic epilepsy (PTE) is a treatment-resistant consequence of traumatic brain injury (TBI). Recently, it has been revealed that epileptiform activity in acute chemoconvulsant seizure models is accompanied by transient shrinkages of extracellular space (ECS) called rapid volume pulsations (RVPs). Shrinkage of the ECS surrounding neurons and glia may contribute to ictogenic hyperexcitability and hypersynchrony during the chronic phase of TBI. Here, we identify the phenomenon of RVPs occurring spontaneously in rat neocortex at ≥ 3 weeks after injury in the controlled cortical impact (CCI) model for PTE. We further report that blocking the electrogenic action of the astrocytic cotransporter NBCe1 with 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) eliminates both RVPs and epileptiform activity in ex-vivo CCI neocortical brain slices. We conclude that NBCe1-mediated extracellular volume shrinkage may represent a new target for therapeutic intervention in PTE.


Assuntos
Lesões Encefálicas Traumáticas , Epilepsia Pós-Traumática , Neocórtex , Ratos , Animais , Simportadores de Sódio-Bicarbonato/metabolismo , Espaço Extracelular/metabolismo , Neocórtex/metabolismo
3.
Nature ; 626(8001): 1108-1115, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38326622

RESUMO

Psychosocial stress has profound effects on the body, including the immune system and the brain1,2. Although a large number of pre-clinical and clinical studies have linked peripheral immune system alterations to stress-related disorders such as major depressive disorder (MDD)3, the underlying mechanisms are not well understood. Here we show that expression of a circulating myeloid cell-specific proteinase, matrix metalloproteinase 8 (MMP8), is increased in the serum of humans with MDD as well as in stress-susceptible mice following chronic social defeat stress (CSDS). In mice, we show that this increase leads to alterations in extracellular space and neurophysiological changes in the nucleus accumbens (NAc), as well as altered social behaviour. Using a combination of mass cytometry and single-cell RNA sequencing, we performed high-dimensional phenotyping of immune cells in circulation and in the brain and demonstrate that peripheral monocytes are strongly affected by stress. In stress-susceptible mice, both circulating monocytes and monocytes that traffic to the brain showed increased Mmp8 expression following chronic social defeat stress. We further demonstrate that circulating MMP8 directly infiltrates the NAc parenchyma and controls the ultrastructure of the extracellular space. Depleting MMP8 prevented stress-induced social avoidance behaviour and alterations in NAc neurophysiology and extracellular space. Collectively, these data establish a mechanism by which peripheral immune factors can affect central nervous system function and behaviour in the context of stress. Targeting specific peripheral immune cell-derived matrix metalloproteinases could constitute novel therapeutic targets for stress-related neuropsychiatric disorders.


Assuntos
Transtorno Depressivo Maior , Metaloproteinase 8 da Matriz , Monócitos , Estresse Psicológico , Animais , Humanos , Camundongos , Transtorno Depressivo Maior/sangue , Transtorno Depressivo Maior/enzimologia , Transtorno Depressivo Maior/genética , Transtorno Depressivo Maior/metabolismo , Espaço Extracelular/metabolismo , Metaloproteinase 8 da Matriz/sangue , Metaloproteinase 8 da Matriz/deficiência , Metaloproteinase 8 da Matriz/genética , Metaloproteinase 8 da Matriz/metabolismo , Camundongos Endogâmicos C57BL , Monócitos/química , Monócitos/imunologia , Monócitos/metabolismo , Núcleo Accumbens/metabolismo , Núcleo Accumbens/patologia , Tecido Parenquimatoso/metabolismo , Análise da Expressão Gênica de Célula Única , Comportamento Social , Isolamento Social , Estresse Psicológico/sangue , Estresse Psicológico/genética , Estresse Psicológico/imunologia , Estresse Psicológico/metabolismo
4.
Protein Expr Purif ; 218: 106449, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38423157

RESUMO

We previously showed that the root cause of low Protein A step yield observed for certain antibodies/Fc-fusions is the presence of non-binding aggregates in cell culture harvest. A pre-assumption for the above conclusion is that the aggregates, while do not bind to the preparative Protein A column, can bind to the analytical Protein A-high performance liquid chromatography (HPLC) column used for titer measurement. In the current work, using materials from a previous case with the low yield issue, we confirmed that non-binding aggregates in preparative Protein A flow-through can indeed bind to the analytical Protein A column. In addition, we showed that this discrepancy is mainly due to the different loading densities applied under these two circumstances. We also demonstrated that aggregate bound to the analytical Protein A column slightly stronger than the monomer, as it exhibited a longer retention time. In summary, the current study not only confirmed that non-binding aggregates detected in the preparative Protein A flow-through bind to the Protein A-HPLC column and contribute to the measured titer of culture harvest but also unravelled the reason for different binding behaviors exhibited by antibody aggregates towards preparative and analytical Protein A columns.


Assuntos
Anticorpos , Fragmentos Fc das Imunoglobulinas , Cromatografia Líquida de Alta Pressão/métodos , Espaço Extracelular
5.
Comput Biol Med ; 171: 108133, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38364661

RESUMO

The brain extracellular space (ECS), an irregular, extremely tortuous nanoscale space located between cells or between cells and blood vessels, is crucial for nerve cell survival. It plays a pivotal role in high-level brain functions such as memory, emotion, and sensation. However, the specific form of molecular transport within the ECS remain elusive. To address this challenge, this paper proposes a novel approach to quantitatively analyze the molecular transport within the ECS by solving an inverse problem derived from the advection-diffusion equation (ADE) using a physics-informed neural network (PINN). PINN provides a streamlined solution to the ADE without the need for intricate mathematical formulations or grid settings. Additionally, the optimization of PINN facilitates the automatic computation of the diffusion coefficient governing long-term molecule transport and the velocity of molecules driven by advection. Consequently, the proposed method allows for the quantitative analysis and identification of the specific pattern of molecular transport within the ECS through the calculation of the Péclet number. Experimental validation on two datasets of magnetic resonance images (MRIs) captured at different time points showcases the effectiveness of the proposed method. Notably, our simulations reveal identical molecular transport patterns between datasets representing rats with tracer injected into the same brain region. These findings highlight the potential of PINN as a promising tool for comprehensively exploring molecular transport within the ECS.


Assuntos
Encéfalo , Espaço Extracelular , Ratos , Animais , Espaço Extracelular/metabolismo , Transporte Biológico , Encéfalo/diagnóstico por imagem , Encéfalo/fisiologia , Difusão , Redes Neurais de Computação
6.
Exp Mol Med ; 56(1): 66-85, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38172601

RESUMO

Mitochondria have been primarily considered intracellular organelles that are responsible for generating energy for cell survival. However, accumulating evidence suggests that mitochondria are secreted into the extracellular space under physiological and pathological conditions, and these secreted mitochondria play diverse roles by regulating metabolism, the immune response, or the differentiation/maturation in target cells. Furthermore, increasing amount of research shows the therapeutic effects of local or systemic administration of mitochondria in various disease models. These findings have led to growing interest in exploring mitochondria as potential therapeutic agents. Here, we discuss the emerging roles of mitochondria as extracellularly secreted organelles to shed light on their functions beyond energy production. Additionally, we provide information on therapeutic outcomes of mitochondrial transplantation in animal models of diseases and an update on ongoing clinical trials, underscoring the potential of using mitochondria as a novel therapeutic intervention.


Assuntos
Comunicação Celular , Mitocôndrias , Animais , Mitocôndrias/metabolismo , Diferenciação Celular , Espaço Extracelular/metabolismo
7.
Nano Lett ; 24(5): 1570-1578, 2024 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-38287297

RESUMO

Glioblastoma (GBM) is the most complex and lethal primary brain cancer. Adequate drug diffusion and penetration are essential for treating GBM, but how the spatial heterogeneity in GBM impacts drug diffusion and transport is poorly understood. Herein, we report a new method, photoactivation of plasmonic nanovesicles (PANO), to measure molecular diffusion in the extracellular space of GBM. By examining three genetically engineered GBM mouse models that recapitulate key clinical features including the angiogenic core and diffuse infiltration, we found that the tumor margin has the lowest diffusion coefficient (highest tortuosity) compared with the tumor core and surrounding brain tissue. Analysis of the cellular composition shows that tortuosity in the GBM is strongly correlated with neuronal loss and astrocyte activation. Our all-optical measurement reveals the heterogeneous GBM microenvironment and highlights the tumor margin as a diffusion barrier for drug transport in the brain, with implications for therapeutic delivery.


Assuntos
Neoplasias Encefálicas , Glioblastoma , Camundongos , Animais , Glioblastoma/patologia , Neoplasias Encefálicas/tratamento farmacológico , Encéfalo/patologia , Linhagem Celular Tumoral , Espaço Extracelular , Microambiente Tumoral
8.
Elife ; 122024 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-38252473

RESUMO

Fibroblast growth factor 2 (FGF2) exits cells by direct translocation across the plasma membrane, a type I pathway of unconventional protein secretion. This process is initiated by phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2)-dependent formation of highly dynamic FGF2 oligomers at the inner plasma membrane leaflet, inducing the formation of lipidic membrane pores. Cell surface heparan sulfate chains linked to glypican-1 (GPC1) capture FGF2 at the outer plasma membrane leaflet, completing FGF2 membrane translocation into the extracellular space. While the basic steps of this pathway are well understood, the molecular mechanism by which FGF2 oligomerizes on membrane surfaces remains unclear. In the current study, we demonstrate the initial step of this process to depend on C95-C95 disulfide-bridge-mediated FGF2 dimerization on membrane surfaces, producing the building blocks for higher FGF2 oligomers that drive the formation of membrane pores. We find FGF2 with a C95A substitution to be defective in oligomerization, pore formation, and membrane translocation. Consistently, we demonstrate a C95A variant of FGF2 to be characterized by a severe secretion phenotype. By contrast, while also important for efficient FGF2 secretion from cells, a second cysteine residue on the molecular surface of FGF2 (C77) is not involved in FGF2 oligomerization. Rather, we find C77 to be part of the interaction interface through which FGF2 binds to the α1 subunit of the Na,K-ATPase, the landing platform for FGF2 at the inner plasma membrane leaflet. Using cross-linking mass spectrometry, atomistic molecular dynamics simulations combined with a machine learning analysis and cryo-electron tomography, we propose a mechanism by which disulfide-bridged FGF2 dimers bind with high avidity to PI(4,5)P2 on membrane surfaces. We further propose a tight coupling between FGF2 secretion and the formation of ternary signaling complexes on cell surfaces, hypothesizing that C95-C95-bridged FGF2 dimers are functioning as the molecular units triggering autocrine and paracrine FGF2 signaling.


Assuntos
Espaço Extracelular , Fator 2 de Crescimento de Fibroblastos , Dimerização , ATPase Trocadora de Sódio-Potássio , Dissulfetos
9.
Fluids Barriers CNS ; 21(1): 6, 2024 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-38212833

RESUMO

BACKGROUND: The brain extracellular fluid (ECF), composed of secreted neurotransmitters, metabolites, peptides, and proteins, may reflect brain processes. Analysis of brain ECF may provide new potential markers for synaptic activity or brain damage and reveal additional information on pathological alterations. Epileptic seizure induction is an acute and harsh intervention in brain functions, and it can activate extra- and intracellular proteases, which implies an altered brain secretome. Thus, we applied a 4-aminopyridine (4-AP) epilepsy model to study the hippocampal ECF peptidome alterations upon treatment in rats. METHODS: We performed in vivo microdialysis in the hippocampus for 3-3 h of control and 4-AP treatment phase in parallel with electrophysiology measurement. Then, we analyzed the microdialysate peptidome of control and treated samples from the same subject by liquid chromatography-coupled tandem mass spectrometry. We analyzed electrophysiological and peptidomic alterations upon epileptic seizure induction by two-tailed, paired t-test. RESULTS: We detected 2540 peptides in microdialysate samples by mass spectrometry analysis; and 866 peptides-derived from 229 proteins-were found in more than half of the samples. In addition, the abundance of 322 peptides significantly altered upon epileptic seizure induction. Several proteins of significantly altered peptides are neuropeptides (Chgb) or have synapse- or brain-related functions such as the regulation of synaptic vesicle cycle (Atp6v1a, Napa), astrocyte morphology (Vim), and glutamate homeostasis (Slc3a2). CONCLUSIONS: We have detected several consequences of epileptic seizures at the peptidomic level, as altered peptide abundances of proteins that regulate epilepsy-related cellular processes. Thus, our results indicate that analyzing brain ECF by in vivo microdialysis and omics techniques is useful for monitoring brain processes, and it can be an alternative method in the discovery and analysis of CNS disease markers besides peripheral fluid analysis.


Assuntos
Epilepsia , Espaço Extracelular , Ratos , Animais , Espaço Extracelular/metabolismo , Uretana/metabolismo , Convulsões/induzido quimicamente , Epilepsia/induzido quimicamente , Epilepsia/metabolismo , Epilepsia/patologia , 4-Aminopiridina/metabolismo , 4-Aminopiridina/farmacologia , Peptídeos/química , Peptídeos/metabolismo , Amidas/metabolismo , Hipocampo/metabolismo
10.
Purinergic Signal ; 20(1): 83-89, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-37074620

RESUMO

ATP is a ubiquitous extracellular messenger released in a wide number of pathophysiological conditions. ATP is known to be present in minute amounts in the extracellular space in healthy tissues and in the blood, and to modulate a multiplicity of cell responses. Cell culture systems are widely used to explore purinergic signaling. We show here that currently used fetal bovine sera contain ATP in the 300-1300 pmol/L range. Serum ATP is associated with albumin as well as with microparticle/microvesicle fraction. Serum microparticles/microvesicles affect in vitro cell responses due to their content of miRNAs, growth factors, and other bioactive molecules. ATP is likely to be one of these bioactive factors found in a variable amount in sera of different commercial sources. ATP in serum supports ATP-dependent biochemical reactions such as the hexokinase-dependent phosphorylation of glucose to glucose 6-phosphate, and affects purinergic signaling. These findings show that cells growing in vitro in serum-supplemented media are exposed to varying levels of extracellular ATP, and thus to varying degrees of purinergic stimulation.


Assuntos
Espaço Extracelular , Soroalbumina Bovina , Células Cultivadas , Espaço Extracelular/metabolismo , Trifosfato de Adenosina/metabolismo , Glucose
11.
Front Immunol ; 14: 1268756, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37915565

RESUMO

Nicotinamide phosphoribosyltransferase (NAMPT) plays a central role in mammalian cell metabolism by contributing to nicotinamide adenine dinucleotide biosynthesis. However, NAMPT activity is not limited to the intracellular compartment, as once secreted, the protein accomplishes diverse functions in the extracellular space. Extracellular NAMPT (eNAMPT, also called visfatin or pre-B-cell colony enhancing factor) has been shown to possess adipocytokine, pro-inflammatory, and pro-angiogenic activities. Numerous studies have reported the association between elevated levels of circulating eNAMPT and various inflammatory and metabolic disorders such as obesity, diabetes, atherosclerosis, arthritis, inflammatory bowel disease, lung injury and cancer. In this review, we summarize the current state of knowledge on eNAMPT biology, proposed roles in disease pathogenesis, and its potential as a disease biomarker. We also briefly discuss the emerging therapeutic approaches for eNAMPT inhibition.


Assuntos
Diabetes Mellitus , Neoplasias , Animais , Humanos , Nicotinamida Fosforribosiltransferase/metabolismo , Espaço Extracelular/metabolismo , Biomarcadores , Mamíferos/metabolismo
12.
Eur J Cell Biol ; 102(4): 151372, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37972445

RESUMO

Extracellular vesicles are small particles involved in intercellular signaling. They are produced by virtually all cell types, transport biological molecules, and are released into the extracellular space. Studies on extracellular vesicles have become more numerous in recent years, leading to promising research on their potential impact on health and disease. Despite significant progress in understanding the bioactivity of extracellular vesicles, most in vitro and in vivo studies overlook their transport through the extracellular matrix in tissues. The interaction or free diffusion of extracellular vesicles in their environment can provide valuable insights into their efficacy and function. Therefore, understanding the factors that influence the transport of extracellular vesicles in the extracellular matrix is essential for the development of new therapeutic approaches that involve the use of these extracellular vesicles. This review discusses the importance of the interaction between extracellular vesicles and the extracellular matrix and the different factors that influence their diffusion. In addition, we evaluate their role in tissue homeostasis, pathophysiology, and potential clinical applications. Understanding the complex interaction between extracellular vesicles and the extracellular matrix is critical in order to develop effective strategies to target specific cells and tissues in a wide range of clinical applications.


Assuntos
Vesículas Extracelulares , Vesículas Extracelulares/metabolismo , Matriz Extracelular/metabolismo , Espaço Extracelular , Homeostase
13.
Nat Commun ; 14(1): 6411, 2023 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-37828018

RESUMO

Progress in neuroscience research hinges on technical advances in visualizing living brain tissue with high fidelity and facility. Current neuroanatomical imaging approaches either require tissue fixation (electron microscopy), do not have cellular resolution (magnetic resonance imaging) or only give a fragmented view (fluorescence microscopy). Here, we show how regular light microscopy together with fluorescence labeling of the interstitial fluid in the extracellular space provide comprehensive optical access in real-time to the anatomical complexity and dynamics of living brain tissue at submicron scale. Using several common fluorescence microscopy modalities (confocal, light-sheet and 2-photon microscopy) in mouse organotypic and acute brain slices and the intact mouse brain in vivo, we demonstrate the value of this straightforward 'shadow imaging' approach by revealing neurons, microglia, tumor cells and blood capillaries together with their complete anatomical tissue contexts. In addition, we provide quantifications of perivascular spaces and the volume fraction of the extracellular space of brain tissue in vivo.


Assuntos
Encéfalo , Neurônios , Camundongos , Animais , Encéfalo/diagnóstico por imagem , Microscopia de Fluorescência/métodos , Espaço Extracelular , Cabeça
14.
Philos Trans R Soc Lond B Biol Sci ; 378(1890): 20220248, 2023 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-37778388

RESUMO

Citrullination is a post-translational modification catalysed by peptidyl arginine deiminase (PAD) enzymes, and dysregulation of protein citrullination is involved in various pathological disorders. During the past decade, a panel of citrullination inhibitors has been developed, while small molecules activating citrullination have rarely been reported so far. In this study, we screened citrullination activator using an antibody against citrullinated histone H3 (cit-H3), and a natural compound demethoxycurcumin (DMC) significantly activated citrullination. The requirement of PAD2 for DMC-activated citrullination was confirmed by a loss of function assay. Notably, DMC directly engaged with PAD2, and showed binding selectivity among PAD family enzymes. Point mutation assay indicated that residue E352 is essential for DMC targeting PAD2. Consistently, DMC induced typical phenotypes of cells with dysregulation of PAD2 activity, including citrullination-associated cell apoptosis and DNA damage. Overall, our study not only presents a strategy for rationally screening citrullination activators, but also provides a chemical approach for activating protein citrullination. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.


Assuntos
Citrulinação , Histonas , Desiminases de Arginina em Proteínas/genética , Desiminases de Arginina em Proteínas/metabolismo , Histonas/metabolismo , Processamento de Proteína Pós-Traducional , Espaço Extracelular , Hidrolases/genética , Hidrolases/metabolismo
15.
Int J Mol Sci ; 24(17)2023 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-37686050

RESUMO

Extracellular vesicles (EVs) are membrane vesicles released by cells into the extracellular space. EVs mediate cell-to-cell communication through local and systemic transportation of biomolecules such as DNA, RNA, transcription factors, cytokines, chemokines, enzymes, lipids, and organelles within the human body. EVs gained a particular interest from cancer biology scientists because of their role in the modulation of the tumor microenvironment through delivering bioactive molecules. In this respect, EVs represent an attractive therapeutic target and a means for drug delivery. The advantages of EVs include their biocompatibility, small size, and low immunogenicity. However, there are several limitations that restrict the widespread use of EVs in therapy, namely, their low specificity and payload capacity. Thus, in order to enhance the therapeutic efficacy and delivery specificity, the surface and composition of extracellular vesicles should be modified accordingly. In this review, we describe various approaches to engineering EVs, and further discuss their advantages and disadvantages to promote the application of EVs in clinical practice.


Assuntos
Vesículas Extracelulares , Humanos , Espaço Extracelular , Biologia , Comunicação Celular , Citocinas
16.
Biosensors (Basel) ; 13(9)2023 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-37754086

RESUMO

High-multiplex detection of protein biomarkers across tissue regions has been an attractive spatial biology approach due to significant advantages over traditional immunohistochemistry (IHC) methods. Different from most methods, spatial multiplex in situ tagging (MIST) transfers the spatial protein expression information to an ultrahigh-density, large-scale MIST array. This technique has been optimized to reach single-cell resolution by adoption of smaller array units and 30% 8-arm PEG polymer as transfer medium. Tissue cell nuclei stained with lamin B have been clearly visualized on the MIST arrays and are colocalized with detection of nine mouse brain markers. Pseudocells defined at 10 µm in size have been used to fully profile tissue regions including cells and the intercellular space. We showcased the versatility of our technology by successfully detecting 20 marker proteins in kidney samples with the addition of five minutes atop the duration of standard immunohistochemistry protocols. Spatial MIST is amenable to iterative staining and detection on the same tissue samples. When 25 proteins were co-detected on 1 mouse brain section for each round and 5 rounds were executed, an ultrahigh multiplexity of 125 proteins was obtained for each pseudocell. With its unique abilities, this single-cell spatial MIST technology has the potential to become an important method in advanced diagnosis of complex diseases.


Assuntos
Núcleo Celular , Neoplasias Cutâneas , Animais , Camundongos , Exobiologia , Espaço Extracelular , Rim , Polímeros
17.
Commun Biol ; 6(1): 961, 2023 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-37735577

RESUMO

Most Gram-negative bacteria synthesize osmo-regulated periplasmic glucans (OPG) in the periplasm or extracellular space. Pathogenicity of many pathogens is lost by knocking out opgG, an OPG-related gene indispensable for OPG synthesis. However, the biochemical functions of OpgG and OpgD, a paralog of OpgG, have not been elucidated. In this study, structural and functional analyses of OpgG and OpgD from Escherichia coli revealed that these proteins are ß-1,2-glucanases with remarkably different activity from each other, establishing a new glycoside hydrolase family, GH186. Furthermore, a reaction mechanism with an unprecedentedly long proton transfer pathway among glycoside hydrolase families is proposed for OpgD. The conformation of the region that forms the reaction pathway differs noticeably between OpgG and OpgD, which explains the observed low activity of OpgG. The findings enhance our understanding of OPG biosynthesis and provide insights into functional diversity for this novel enzyme family.


Assuntos
Glicosídeo Hidrolases , Proteínas Periplásmicas , Glicosídeo Hidrolases/genética , Escherichia coli/genética , Metabolismo dos Carboidratos , Espaço Extracelular , Glucanos
19.
Plant Signal Behav ; 18(1): 2245995, 2023 12 31.
Artigo em Inglês | MEDLINE | ID: mdl-37573516

RESUMO

In addition to rhizobia, many types of co-existent bacteria are found in leguminous root nodules, but their habitats are unclear. To investigate this phenomenon, we labeled Bradyrhizobium diazoefficiens USDA122 and Bradyrhizobium sp. SSBR45 with Discosoma sp. red fluorescent protein (DsRed) or enhanced green fluorescent protein (eGFP). USDA122 enhances soybean growth by forming effective root nodules, but SSBR45 does not form any nodules. Using low-magnification laser scanning confocal microscopy, we found that infected cells in the central zone of soybean nodules appeared to be occupied by USDA122. Notably, high-magnification microscopy after co-inoculation of non-fluorescent USDA122 and fluorescence-labeled SSBR45 also revealed that SSBR45 inhabits the intercellular spaces of healthy nodules. More unexpectedly, co-inoculation of eGFP-labeled USDA122 and DsRed-labeled SSBR45 (and vice versa) revealed the presence of USDA122 bacteria in both the symbiosomes of infected cells and in the apoplasts of healthy nodules. We then next inspected nodules formed after a mixed inoculation of differently-labeled USDA122, without SSBR45, and confirmed the inhabitation of the both populations of USDA122 in the intercellular spaces. In contrast, infected cells were occupied by single-labeled USDA122. We also observed Mesorhizobium loti in the intercellular spaces of active wild-type nodules of Lotus japonicus using transmission electron microscopy. Compatible intercellular rhizobia have been described during nodule formation of several legume species and in some mutants, but our evidence suggests that this type of colonization may occur much more commonly in leguminous root nodules.


Assuntos
Espaço Extracelular , Fabaceae , Rhizobium , Nódulos Radiculares de Plantas , Rhizobium/fisiologia , Espaço Extracelular/microbiologia , Nódulos Radiculares de Plantas/microbiologia , Nódulos Radiculares de Plantas/ultraestrutura , Proteínas de Fluorescência Verde/metabolismo , Lotus/microbiologia , Fabaceae/microbiologia , Microscopia Eletrônica de Transmissão , Simbiose
20.
Sci Rep ; 13(1): 13931, 2023 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-37626167

RESUMO

Plant-derived nanovesicles (NVs) and extracellular vesicles (EVs) are the next generation of nanocarrier platforms for biotherapeutics and drug delivery. EVs exist not only in the extracellular space, but also within the cell wall. Due to the limitations of existing isolation methods, the EVs extraction efficiency is low, and a large amount of plant material is wasted, which is of concern for rare and expensive medicinal plants. We proposed and validated a novel method for isolation of plant EVs by enzyme degradation of the plant cell wall to release the EVs. The released EVs can easily be collected. The new method was used for extraction of EVs from the roots of Morinda officinalis (MOEVs). For comparison, nanoparticles from the roots (MONVs) were extracted using the grinding method. The new method yielded a greater amount of MOEVs, and the vesicles had a smaller diameter compared to MONVs. Both MOEVs and MONVs were readily absorbed by endothelial cells without cytotoxic effect and promoted the expression of miR-155. The promotion of miR-155 by MOEVs was dose-dependent. More importantly, we found that MOEVs and MONVs were enriched toward bone tissue. These results support our hypothesis that EVs in plants could be efficiently extracted by enzymatic cell wall digestion and confirm the potential of MOEVs as therapeutic agents and drug carriers.


Assuntos
Vesículas Extracelulares , MicroRNAs , Células Endoteliais , Espaço Extracelular , Osso e Ossos
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