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2.
Urologiia ; (4): 14-17, 2020 Sep.
Artigo em Russo | MEDLINE | ID: mdl-32897008

RESUMO

OBJECTIVES: the study of the nature and severity of the reorganization of the structural elements of the bladder wall on experimental models of IC / BPS at the optical and ultrastructural levels. MATERIALS AND METHODS: The experimental model of IC / SBMP was created on 22 white New Zealand female rabbits weighing 1500-2000 g. The animals were divided into 2 groups: group 1 - 15 rabbits, which were introduced into the bladder wall urine taken from the animals bladder; Group 2 (control) - 7 animals that were injected into the bladder wall with a 0.9% NaCl solution. The biomaterial was examined by electron microscopy. The structural elements of the lamina propria of the bladder mucosa were evaluated. RESULTS: In the bladder mucosa of the experimental model, perivascular infiltration by inflammatory cells, pronounced edema of the lamina propria of the bladder mucosa, and the presence of numerous plasma cells having close contacts with macrophages, fibroblasts, and lymphocytes were revealed. In the nucleus of lymphocytes of a peripherally located supercondensed heterochromatin showed their apoptotic state. Edematous fluid was determined, penetrating mainly through the fenestra, located in the peripheral parts of the endothelial cells of capillaries and postcapillary venules. CONCLUSION: The study of the structural elements of the lamina propria of the bladder mucosa in the experiment by the method of electron microscopy made it possible to identify changes caused by the inflammatory process, both of an acute and productive nature. The results obtained showed that toxic damage to the lamina propria of the bladder mucosa causes apoptosis of fibroblasts of the lamina propria, leads to loosening of collagen fibers and, ultimately, to a decrease in the protective factors of the mucous membrane.


Assuntos
Cistite Intersticial , Animais , Elétrons , Células Endoteliais , Feminino , Microscopia Eletrônica , Membrana Mucosa , Coelhos
3.
Nat Commun ; 11(1): 4102, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32796823

RESUMO

Emerging evidence suggests that intestinal stromal cells (IntSCs) play essential roles in maintaining intestinal homeostasis. However, the extent of heterogeneity within the villi stromal compartment and how IntSCs regulate the structure and function of specialized intestinal lymphatic capillary called lacteal remain elusive. Here we show that selective hyperactivation or depletion of YAP/TAZ in PDGFRß+ IntSCs leads to lacteal sprouting or regression with junctional disintegration and impaired dietary fat uptake. Indeed, mechanical or osmotic stress regulates IntSC secretion of VEGF-C mediated by YAP/TAZ. Single-cell RNA sequencing delineated novel subtypes of villi fibroblasts that upregulate Vegfc upon YAP/TAZ activation. These populations of fibroblasts were distributed in proximity to lacteal, suggesting that they constitute a peri-lacteal microenvironment. Our findings demonstrate the heterogeneity of IntSCs and reveal that distinct subsets of villi fibroblasts regulate lacteal integrity through YAP/TAZ-induced VEGF-C secretion, providing new insights into the dynamic regulatory mechanisms behind lymphangiogenesis and lymphatic remodeling.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Fibroblastos/metabolismo , Mucosa Intestinal/metabolismo , Fatores de Transcrição/metabolismo , Fator C de Crescimento do Endotélio Vascular/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas de Ciclo Celular/genética , Células Cultivadas , Análise por Conglomerados , Ensaio de Imunoadsorção Enzimática , Fibroblastos/ultraestrutura , Citometria de Fluxo , Imunofluorescência , Hibridização in Situ Fluorescente , Mucosa Intestinal/ultraestrutura , Linfangiogênese/genética , Linfangiogênese/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica , Neovascularização Fisiológica/genética , Neovascularização Fisiológica/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética , Fator C de Crescimento do Endotélio Vascular/genética
4.
Nat Commun ; 11(1): 4107, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32796836

RESUMO

Foamy macrophages, which have prominent lipid droplets (LDs), are found in a variety of disease states. Toll-like receptor agonists drive triacylglycerol (TG)-rich LD development in macrophages. Here we explore the basis and significance of this process. Our findings indicate that LD development is the result of metabolic commitment to TG synthesis on a background of decreased fatty acid oxidation. TG synthesis is essential for optimal inflammatory macrophage activation as its inhibition, which prevents LD development, has marked effects on the production of inflammatory mediators, including IL-1ß, IL-6 and PGE2, and on phagocytic capacity. The failure of inflammatory macrophages to make PGE2 when TG-synthesis is inhibited is critical for this phenotype, as addition of exogenous PGE2 is able to reverse the anti-inflammatory effects of TG synthesis inhibition. These findings place LDs in a position of central importance in inflammatory macrophage activation.


Assuntos
Inflamação/metabolismo , Lipidômica/métodos , Triglicerídeos/metabolismo , Animais , Células Cultivadas , Citometria de Fluxo , Glucose/metabolismo , Glicerol/metabolismo , Células HEK293 , Humanos , Metabolismo dos Lipídeos/fisiologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Microscopia Eletrônica , Palmitatos/metabolismo , Análise de Sequência de RNA
5.
Nat Commun ; 11(1): 4121, 2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32807782

RESUMO

Vasoactive intestinal polypeptide receptor (VIP1R) is a widely expressed class B G protein-coupled receptor and a drug target for the treatment of neuronal, metabolic, and inflammatory diseases. However, our understanding of its mechanism of action and the potential of drug discovery targeting this receptor is limited by the lack of structural information of VIP1R. Here we report a cryo-electron microscopy structure of human VIP1R bound to PACAP27 and Gs heterotrimer, whose complex assembly is stabilized by a NanoBiT tethering strategy. Comparison with other class B GPCR structures reveals that PACAP27 engages VIP1R with its N-terminus inserting into the ligand binding pocket at the transmembrane bundle of the receptor, which subsequently couples to the G protein in a receptor-specific manner. This structure has provided insights into the molecular basis of PACAP27 binding and VIP receptor activation. The methodology of the NanoBiT tethering may help to provide structural information of unstable complexes.


Assuntos
Microscopia Crioeletrônica/métodos , Proteínas de Ligação ao GTP/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/metabolismo , Difusão Dinâmica da Luz , Humanos , Microscopia Eletrônica , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo
6.
ACS Infect Dis ; 6(9): 2319-2336, 2020 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-32786280

RESUMO

In December 2019, a novel beta (ß) coronavirus eventually named SARS-CoV-2 emerged in Wuhan, Hubei province, China, causing an outbreak of severe and even fatal pneumonia in humans. The virus has spread very rapidly to many countries across the world, resulting in the World Health Organization (WHO) to declare a pandemic on March 11, 2020. Clinically, the diagnosis of this unprecedented illness, called coronavirus disease-2019 (COVID-19), becomes difficult because it shares many symptoms with other respiratory pathogens, including influenza and parainfluenza viruses. Therefore, laboratory diagnosis is crucial for the clinical management of patients and the implementation of disease control strategies to contain SARS-CoV-2 at clinical and population level. Here, we summarize the main clinical and imaging findings of COVID-19 patients and discuss the advances, features, advantages, and limitations of different laboratory methods used for SARS-CoV-2 diagnosis.


Assuntos
Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , Infecções por Coronavirus/virologia , Humanos , Microscopia Eletrônica , Pandemias , Pneumonia Viral/virologia , Reação em Cadeia da Polimerase/métodos , Análise de Sequência , Testes Sorológicos/métodos , Manejo de Espécimes/métodos
7.
EBioMedicine ; 59: 102951, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32818801

RESUMO

BACKGROUND: . The occurrence of trans-placental transmission of severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) infection remains highly debated. Placental positivity for SARS-CoV-2 has been reported in selected cases, but infection or virus-associated disease of fetal tissues or newborns remains to be demonstrated. METHODS: We screened for SARS-CoV-2 spike (S) protein expression placentas from 101 women who delivered between February 7 and May 15, 2020, including 15 tested positive for SARS-CoV-2 RNA, 34 tested negative, and 52 not evaluated as they did not meet testing criteria (32), or delivered before COVID-19 pandemic declaration (20). Immunostain for SARS-CoV-2 nucleocapsid (N) was performed in the placentas of all COVID-19 positive women. One placenta resulted positive for the SARS-CoV-2 S and N proteins, which was further studied by RNA-in situ hybridization and RT-PCR for S transcripts, and by electron microscopy. A comprehensive immunohistochemical and immunofluorescence analysis of the placental inflammatory infiltrate completed the investigations. FINDINGS: SARS-CoV-2 S and N proteins were strongly expressed in the placenta of a COVID-19 pregnant woman whose newborn tested positive for viral RNA and developed COVID-19 pneumonia soon after birth. SARS-CoV-2 antigens, RNA and/or particles morphologically consistent with coronavirus were identified in villous syncytiotrophoblast, endothelial cells, fibroblasts, in maternal macrophages, and in Hofbauer cells and fetal intravascular mononuclear cells. The placenta intervillous inflammatory infiltrate consisted of neutrophils and monocyte-macrophages expressing activation markers. Absence of villitis was associated with an increase in the number of Hofbauer cells, which expressed PD-L1. Scattered neutrophil extracellular traps (NETs) were identified by immunofluorescence. INTERPRETATION: We provide first-time evidence for maternal-fetal transmission of SARS-CoV-2, likely propagated by circulating virus-infected fetal mononuclear cells. Placenta infection was associated with recruitment of maternal inflammatory cells in the intervillous space, without villitis. PD-L1 expression in syncytiotrophoblast and Hofbaeur cells, together with limited production of NETs, may have prevented immune cell-driven placental damage, ensuring sufficient maternal-fetus nutrient exchanges.


Assuntos
Infecções por Coronavirus/transmissão , Placenta/virologia , Pneumonia Viral/transmissão , Adulto , Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Armadilhas Extracelulares/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Recém-Nascido , Macrófagos/virologia , Microscopia Eletrônica , Nasofaringe/virologia , Proteínas do Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/metabolismo , Pandemias , Placenta/citologia , Placenta/patologia , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Gravidez , RNA Viral/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo
8.
Nat Commun ; 11(1): 3711, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32709891

RESUMO

The skeletal muscle T-tubule is a specialized membrane domain essential for coordinated muscle contraction. However, in the absence of genetically tractable systems the mechanisms involved in T-tubule formation are unknown. Here, we use the optically transparent and genetically tractable zebrafish system to probe T-tubule development in vivo. By combining live imaging of transgenic markers with three-dimensional electron microscopy, we derive a four-dimensional quantitative model for T-tubule formation. To elucidate the mechanisms involved in T-tubule formation in vivo, we develop a quantitative screen for proteins that associate with and modulate early T-tubule formation, including an overexpression screen of the entire zebrafish Rab protein family. We propose an endocytic capture model involving firstly, formation of dynamic endocytic tubules at transient nucleation sites on the sarcolemma, secondly, stabilization by myofibrils/sarcoplasmic reticulum and finally, delivery of membrane from the recycling endosome and Golgi complex.


Assuntos
Contração Muscular/fisiologia , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/ultraestrutura , Sarcolema/fisiologia , Sarcolema/ultraestrutura , Animais , Canais de Cálcio/metabolismo , Canais de Cálcio/ultraestrutura , Canais de Cálcio Tipo L/metabolismo , Proteínas de Transporte/metabolismo , Biologia do Desenvolvimento , Complexo de Golgi/metabolismo , Masculino , Microscopia Eletrônica , Proteínas Musculares/química , Músculo Esquelético/química , Miofibrilas/metabolismo , Sarcolema/química , Retículo Sarcoplasmático/metabolismo , Peixe-Zebra
9.
Nat Commun ; 11(1): 3722, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32709902

RESUMO

Human movement occurs through contraction of the basic unit of the muscle cell, the sarcomere. Sarcomeres have long been considered to be arranged end-to-end in series along the length of the muscle into tube-like myofibrils with many individual, parallel myofibrils comprising the bulk of the muscle cell volume. Here, we demonstrate that striated muscle cells form a continuous myofibrillar matrix linked together by frequently branching sarcomeres. We find that all muscle cells contain highly connected myofibrillar networks though the frequency of sarcomere branching goes down from early to late postnatal development and is higher in slow-twitch than fast-twitch mature muscles. Moreover, we show that the myofibrillar matrix is united across the entire width of the muscle cell both at birth and in mature muscle. We propose that striated muscle force is generated by a singular, mesh-like myofibrillar network rather than many individual, parallel myofibrils.


Assuntos
Fenômenos Mecânicos , Músculo Esquelético/fisiologia , Miofibrilas/fisiologia , Sarcômeros/fisiologia , Animais , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica , Contração Muscular/fisiologia , Desenvolvimento Muscular , Músculo Esquelético/citologia , Miofibrilas/patologia , Sarcômeros/patologia
10.
Nat Commun ; 11(1): 3464, 2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32651375

RESUMO

DOCK (dedicator of cytokinesis) proteins are multidomain guanine nucleotide exchange factors (GEFs) for RHO GTPases that regulate intracellular actin dynamics. DOCK proteins share catalytic (DOCKDHR2) and membrane-associated (DOCKDHR1) domains. The structurally-related DOCK1 and DOCK2 GEFs are specific for RAC, and require ELMO (engulfment and cell motility) proteins for function. The N-terminal RAS-binding domain (RBD) of ELMO (ELMORBD) interacts with RHOG to modulate DOCK1/2 activity. Here, we determine the cryo-EM structures of DOCK2-ELMO1 alone, and as a ternary complex with RAC1, together with the crystal structure of a RHOG-ELMO2RBD complex. The binary DOCK2-ELMO1 complex adopts a closed, auto-inhibited conformation. Relief of auto-inhibition to an active, open state, due to a conformational change of the ELMO1 subunit, exposes binding sites for RAC1 on DOCK2DHR2, and RHOG and BAI GPCRs on ELMO1. Our structure explains how up-stream effectors, including DOCK2 and ELMO1 phosphorylation, destabilise the auto-inhibited state to promote an active GEF.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Calorimetria , Proteínas Ativadoras de GTPase/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Células HEK293 , Células HeLa , Humanos , Immunoblotting , Cinética , Microscopia Eletrônica , Fosforilação , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo
11.
Nat Commun ; 11(1): 3440, 2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32651387

RESUMO

In recent years, exploration of the brain extracellular space (ECS) has made remarkable progress, including nanoscopic characterizations. However, whether ECS precise conformation is altered during brain pathology remains unknown. Here we study the nanoscale organization of pathological ECS in adult mice under degenerative conditions. Using electron microscopy in cryofixed tissue and single nanotube tracking in live brain slices combined with super-resolution imaging analysis, we find enlarged ECS dimensions and increased nanoscale diffusion after α-synuclein-induced neurodegeneration. These animals display a degraded hyaluronan matrix in areas close to reactive microglia. Furthermore, experimental hyaluronan depletion in vivo reduces dopaminergic cell loss and α-synuclein load, induces microgliosis and increases ECS diffusivity, highlighting hyaluronan as diffusional barrier and local tissue organizer. These findings demonstrate the interplay of ECS, extracellular matrix and glia in pathology, unraveling ECS features relevant for the α-synuclein propagation hypothesis and suggesting matrix manipulation as a disease-modifying strategy.


Assuntos
Encéfalo/metabolismo , Espaço Extracelular/metabolismo , Ácido Hialurônico/metabolismo , Sinucleinopatias/metabolismo , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Microglia/ultraestrutura , Microscopia Eletrônica , Doença de Parkinson/metabolismo , Espectroscopia de Luz Próxima ao Infravermelho
12.
J Fr Ophtalmol ; 43(7): 642-652, 2020 Sep.
Artigo em Francês | MEDLINE | ID: mdl-32631687

RESUMO

The COVID-19 pandemic has dramatically changed our daily lives as ophthalmologists. This general review firstly provides a better understanding of the virus responsible for the pandemic: the SARS-CoV-2, and the clinical manifestations of the COVID-19 disease. The second part is detailing the pathophysiology, clinical signs and challenges of ocular involvement, which seems rare and not functionally severe, but which may be a potential source of contamination. Finally, we discuss the preventive measures that need to be implemented in our daily practice to avoid any viral dissemination.


Assuntos
Betacoronavirus , Infecções por Coronavirus/complicações , Infecções Oculares Virais/virologia , Pneumonia Viral/complicações , Betacoronavirus/genética , Betacoronavirus/ultraestrutura , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/fisiopatologia , Infecções por Coronavirus/prevenção & controle , Técnicas de Diagnóstico Oftalmológico , Olho/virologia , Infecções Oculares Virais/fisiopatologia , Infecções Oculares Virais/prevenção & controle , Genoma Viral , Humanos , Microscopia Eletrônica , Pandemias/prevenção & controle , Pneumonia Viral/epidemiologia , Pneumonia Viral/fisiopatologia , Pneumonia Viral/prevenção & controle , Integração Viral
15.
Nat Commun ; 11(1): 3298, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620747

RESUMO

Communication between organelles is essential for their cellular homeostasis. Neurodegeneration reflects the declining ability of neurons to maintain cellular homeostasis over a lifetime, where the endolysosomal pathway plays a prominent role by regulating protein and lipid sorting and degradation. Here we report that TMEM16K, an endoplasmic reticulum lipid scramblase causative for spinocerebellar ataxia (SCAR10), is an interorganelle regulator of the endolysosomal pathway. We identify endosomal transport as a major functional cluster of TMEM16K in proximity biotinylation proteomics analyses. TMEM16K forms contact sites with endosomes, reconstituting split-GFP with the small GTPase RAB7. Our study further implicates TMEM16K lipid scrambling activity in endosomal sorting at these sites. Loss of TMEM16K function led to impaired endosomal retrograde transport and neuromuscular function, one of the symptoms of SCAR10. Thus, TMEM16K-containing ER-endosome contact sites represent clinically relevant platforms for regulating endosomal sorting.


Assuntos
Anoctaminas/metabolismo , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Lisossomos/metabolismo , Animais , Anoctaminas/genética , Transporte Biológico , Células COS , Linhagem Celular Tumoral , Células Cultivadas , Chlorocebus aethiops , Retículo Endoplasmático/ultraestrutura , Endossomos/ultraestrutura , Células HEK293 , Humanos , Metabolismo dos Lipídeos , Lisossomos/ultraestrutura , Camundongos Knockout , Microscopia Eletrônica , Mutação , Transporte Proteico , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/metabolismo
16.
Proc Natl Acad Sci U S A ; 117(31): 18780-18787, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32699144

RESUMO

Macular telangiectasia type 2 (MacTel), a late-onset macular degeneration, has been linked to a loss in the retina of Müller glial cells and the amino acid serine, synthesized by the Müller cells. The disease is confined mainly to a central retinal region called the MacTel zone. We have used electron microscopic connectomics techniques, optimized for disease analysis, to study the retina from a 48-y-old woman suffering from MacTel. The major observations made were specific changes in mitochondrial structure within and outside the MacTel zone that were present in all retinal cell types. We also identified an abrupt boundary of the MacTel zone that coincides with the loss of Müller cells and macular pigment. Since Müller cells synthesize retinal serine, we propose that a deficiency of serine, required for mitochondrial maintenance, causes mitochondrial changes that underlie MacTel development.


Assuntos
Conectoma/métodos , Retina , Doenças Retinianas , Feminino , Humanos , Degeneração Macular/diagnóstico por imagem , Degeneração Macular/patologia , Microscopia Eletrônica , Pessoa de Meia-Idade , Retina/citologia , Retina/diagnóstico por imagem , Retina/patologia , Doenças Retinianas/diagnóstico por imagem , Doenças Retinianas/patologia
17.
Parasitol Res ; 119(8): 2667-2678, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32627078

RESUMO

Coccidian parasites possess complex life cycles involving asexual proliferation followed by sexual development leading to the production of oocysts. Coccidian oocysts are persistent stages which are secreted by the feces and transmitted from host to host guaranteeing life cycle progression and disease transmission. The robust bilayered oocyst wall is formed from the contents of two organelles, the wall-forming bodies type I and II (WFBI, WFBII), located exclusively in the macrogametocyte. Eimeria nieschulzi has been used as a model parasite to study and follow gametocyte and oocyst development. In this study, the gametocyte and oocyst wall formation of E. nieschulzi was analyzed by electron microscopy and immuno-histology. A monoclonal antibody raised against the macrogametocytes of E. nieschulzi identified a tyrosine-rich glycoprotein (EnGAM82) located in WFBII. Correlative light and electron microscopy was used to examine the vesicle-specific localization and spatial distribution of GAM82-proteins during macrogametocyte maturation by this monoclonal antibody. In early and mid-stages, the GAM82-protein is ubiquitously distributed in WFBII. Few hours later, the protein is arranged in subvesicular structures. It was possible to show that the substructure of WFBII and the spatial distribution of GAM82-proteins probably represent pre-synthesized cross-linked materials prior to the inner oocyst wall formation. Dityrosine-cross-linked gametocyte proteins can also be confirmed and visualized by fluorescence microscopy (UV light, autofluorescence of WFBII).


Assuntos
Eimeria/citologia , Eimeria/ultraestrutura , Animais , Eimeria/crescimento & desenvolvimento , Glicoproteínas/química , Glicoproteínas/metabolismo , Estágios do Ciclo de Vida , Microscopia Eletrônica , Microscopia de Fluorescência , Oocistos/citologia , Oocistos/crescimento & desenvolvimento , Oocistos/metabolismo , Oocistos/ultraestrutura , Organelas/metabolismo , Organelas/ultraestrutura , Proteínas de Protozoários/metabolismo , Tirosina/análogos & derivados , Tirosina/química
18.
Nucleic Acids Res ; 48(14): 7623-7639, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32644123

RESUMO

RNA therapeutics are a promising strategy to treat genetic diseases caused by the overexpression or aberrant splicing of a specific protein. The field has seen major strides in the clinical efficacy of this class of molecules, largely due to chemical modifications and delivery strategies that improve nuclease resistance and enhance cell penetration. However, a major obstacle in the development of RNA therapeutics continues to be the imprecise, difficult, and often problematic nature of most methods used to measure cell penetration. Here, we review these methods and clearly distinguish between those that measure total cellular uptake of RNA therapeutics, which includes both productive and non-productive uptake, and those that measure cytosolic/nuclear penetration, which represents only productive uptake. We critically analyze the benefits and drawbacks of each method. Finally, we use key examples to illustrate how, despite rigorous experimentation and proper controls, our understanding of the mechanism of gymnotic uptake of RNA therapeutics remains limited by the methods commonly used to analyze RNA delivery.


Assuntos
RNA/metabolismo , RNA/uso terapêutico , Aptâmeros de Nucleotídeos/uso terapêutico , Núcleo Celular/metabolismo , Citosol/metabolismo , Doenças Genéticas Inatas/tratamento farmacológico , Técnicas Genéticas , Humanos , MicroRNAs/uso terapêutico , Microscopia Eletrônica , Oligonucleotídeos Antissenso/uso terapêutico , RNA/química , RNA/farmacocinética , RNA Interferente Pequeno/uso terapêutico , Espectrometria de Fluorescência
20.
Medicine (Baltimore) ; 99(28): e21101, 2020 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-32664131

RESUMO

INTRODUCTION: Amyloidosis and fibrillary glomerulonephritis (FGN) share similar electron microscopic signatures including random arrangement of fibrils. However, distinction between the 2 can often be made using Congo Red staining. PATIENT CONCERNS: Here we describe a unique case of FGN, which stained positive for Congo Red, as well as DnaJ heat shock protein family (Hsp40) member B9 which is more specific for FGN. The patient presented with acute kidney injury and severe proteinuria. DIAGNOSIS: Congophilic FGN. INTERVENTIONS: Six-month course of mycophenolate mofetil and prednisone. OUTCOMES: complete resolution of acute kidney injury and proteinuria TAKE HOME LESSONS:: To our knowledge, this is the first reported case of successful treatment of this rare condition using mycophenolate mofetil and prednisone.


Assuntos
Glomerulonefrite/terapia , Glomérulos Renais/ultraestrutura , Biópsia , Diagnóstico Diferencial , Inibidores Enzimáticos/uso terapêutico , Glomerulonefrite/diagnóstico , Glomerulonefrite/tratamento farmacológico , Glucocorticoides/uso terapêutico , Humanos , Masculino , Microscopia Eletrônica , Pessoa de Meia-Idade , Ácido Micofenólico/uso terapêutico , Prednisona/uso terapêutico
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