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1.
Invest Ophthalmol Vis Sci ; 63(1): 2, 2022 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-34978559

RESUMO

Purpose: Our studies in mouse eye lenses demonstrate that ephrin-A5 and EphA2 are needed for normal epithelial cells and lens transparency. We sought to determine whether EphA2 and ephrin-A5 are important for lens morphometrics, nucleus formation, and refractive index. Methods: We performed tissue morphometric measurements, electron microscopy, Western blots, and interferometric measurements using an X-ray synchrotron beam source to measure the gradient of refractive index (GRIN) to compare mouse lenses with genetic disruption of EphA2 or ephrin-A5. Results: Morphometric analysis revealed that although there is no change in the overall lens volume, there is a change in lens shape in both EphA2-/- lenses and ephrin-A5-/- lenses. Surprisingly, EphA2-/- lenses had small and soft lens nuclei different from hard lens nuclei of control lenses. SEM images revealed changes in cell morphology of EphA2-/- fiber cells close to the center of the lens. Inner EphA2-/- lens fibers had more pronounced tongue-and-groove interdigitations and formed globular membrane morphology only in the deepest layers of the lens nucleus. We did not observe nuclear defects in ephrin-A5-/- lenses. There was an overall decrease in magnitude of refractive index across EphA2-/- lenses, which is most pronounced in the nucleus. Conclusions: This work reveals that Eph-ephrin signaling plays a role in fiber cell maturation, nuclear compaction, and lens shape. Loss of EphA2 disrupts the nuclear compaction resulting in a small lens nucleus. Our data suggest that Eph-ephrin signaling may be required for fiber cell membrane reorganization and compaction and for establishing a normal GRIN.


Assuntos
Núcleo do Cristalino/crescimento & desenvolvimento , Receptor EphA2/fisiologia , Refração Ocular/fisiologia , Animais , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Efrina-A5/fisiologia , Técnicas de Genotipagem , Interferometria , Núcleo do Cristalino/metabolismo , Núcleo do Cristalino/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Varredura , Forma das Organelas/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/fisiologia , Raios X
2.
Invest Ophthalmol Vis Sci ; 63(1): 4, 2022 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-34982146

RESUMO

Purpose: Netarsudil, a Rho kinase inhibitor with norepinephrine transport inhibitory effect, lowers intraocular pressure, however, its effect on axon damage remains to be elucidated. The aim of the current study was to investigate the effect of netarsudil on TNF-induced axon loss and to examine whether it affects phosphorylated-AMP-activated kinase (p-AMPK) and autophagy in the optic nerve. Methods: Intravitreal administration of TNF or TNF with netarsudil was carried out on rats and quantification of axon number was determined. Electron microscopy determined autophagosome numbers. Localization of p-AMPK expression was examined by immunohistochemistry. The changes in p62, LC3-II, and p-AMPK levels were estimated in the optic nerve by immunoblot analysis. The effect of an AMPK activator A769662 or an AMPK inhibitor dorsomorphin on axon number was evaluated. Results: Morphometric analysis revealed apparent protection by netarsudil against TNF-induced axon degeneration. Netarsudil increased autophagosome numbers inside axons. Netarsudil treatment significantly upregulated optic nerve LC3-II levels in both the TNF-treated eyes and the control eyes. Increased p62 protein level induced by TNF was significantly ameliorated by netarsudil. The netarsudil administration alone lessened p62 levels. Netarsudil significantly upregulated the optic nerve p-AMPK levels. A769662 exhibited obvious axonal protection against TNF-induced damage. A769662 treatment upregulated LC3-II levels and the increment of p62 level induced by TNF was significantly ameliorated by A769662. Immunohistochemical analysis revealed that p-AMPK is present in axons. Netarsudil-mediated axonal protection was significantly suppressed by dorsomorphin administration. Conclusions: Netarsudil upregulated p-AMPK and autophagy. Netarsudil-mediated axonal protection may be associated with upregulated p-AMPK.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Autofagia/fisiologia , Axônios/efeitos dos fármacos , Benzoatos/farmacologia , Degeneração Neural/prevenção & controle , Nervo Óptico/efeitos dos fármacos , Fator de Necrose Tumoral alfa/toxicidade , beta-Alanina/análogos & derivados , Quinases Associadas a rho/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Animais , Axônios/enzimologia , Axônios/patologia , Compostos de Bifenilo/farmacologia , Inibidores Enzimáticos/farmacologia , Imuno-Histoquímica , Injeções Intravítreas , Masculino , Microscopia Eletrônica , Proteínas Associadas aos Microtúbulos/metabolismo , Degeneração Neural/enzimologia , Nervo Óptico/ultraestrutura , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Pironas/farmacologia , Ratos , Ratos Wistar , Proteína Sequestossoma-1/metabolismo , Tiofenos/farmacologia , beta-Alanina/farmacologia
3.
Meat Sci ; 184: 108690, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34656007

RESUMO

This study aimed to evaluate the effects of different cooking time (2, 4, and 6 h) and temperature (50, 60, 70, 80, and 90 °C) on physical, textual, and structural properties of longissimus lumborum muscle of yak, and to explore the thermal denaturation process of intramuscular collagen by using a new tool (collagen hybridizing peptide staining, CHP staining). The results showed that tenderness was affected by the interaction of cooking time and temperature and the changes in moisture and collagen composition. In comparison with cooking time, temperature had more obvious effects on cooking loss, moisture content and redness. Scanning electron microscopy showed that as the temperature increased, intramuscular connective tissue gradually degraded, and muscle fibers became more compact. CHP staining showed that the collagen in the perimysium first denatured at 50 °C, and more and more collagen denatured and degraded as the temperature increased.


Assuntos
Tecido Conjuntivo/química , Culinária/métodos , Carne Vermelha/análise , Animais , Bovinos , Colágeno/química , Tecido Conjuntivo/ultraestrutura , Microscopia Eletrônica de Varredura , Músculo Esquelético , Temperatura
4.
J Cell Biol ; 221(2)2022 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-34878519

RESUMO

The neuronal axon is packed with cytoskeletal filaments, membranes, and organelles, many of which move between the cell body and axon tip. Here, we used cryo-electron tomography to survey the internal components of mammalian sensory axons. We determined the polarity of the axonal microtubules (MTs) by combining subtomogram classification and visual inspection, finding MT plus and minus ends are structurally similar. Subtomogram averaging of globular densities in the MT lumen suggests they have a defined structure, which is surprising given they likely contain the disordered protein MAP6. We found the endoplasmic reticulum in axons is tethered to MTs through multiple short linkers. We surveyed membrane-bound cargos and describe unexpected internal features such as granules and broken membranes. In addition, we detected proteinaceous compartments, including numerous virus-like capsid particles. Our observations outline novel features of axonal cargos and MTs, providing a platform for identification of their constituents.


Assuntos
Axônios/ultraestrutura , Compartimento Celular , Microscopia Crioeletrônica , Espaço Intracelular/metabolismo , Mamíferos/metabolismo , Microtúbulos/ultraestrutura , Tomografia , Animais , Axônios/metabolismo , Capsídeo/metabolismo , Capsídeo/ultraestrutura , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Drosophila melanogaster/metabolismo , Drosophila melanogaster/ultraestrutura , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Gânglios Espinais/metabolismo , Microtúbulos/metabolismo , Análise Multivariada , Proteínas do Tecido Nervoso/metabolismo
5.
J Cutan Pathol ; 49(1): 17-28, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34272741

RESUMO

BACKGROUND: The abundance of publications of COVID-19-induced chilblains has resulted in a confusing situation. METHODS: This is a prospective single-institution study from 15 March to 13 May 2020. Thirty-two patients received PCR nasopharyngeal swabs. Of these, 28 patients had a thoracic CT-scan, 31 patients had blood and urine examinations, 24 patients had skin biopsies including immunohistochemical and direct immunofluorescence studies, and four patients had electron microscopy. RESULTS: COVID-19-induced chilblains are clinically and histopathologically identical to chilblains from other causes. Although intravascular thrombi are sometimes observed, no patient had a systemic coagulopathy or severe clinical course. The exhaustive clinical, radiological, and laboratory work-up in this study ruled-out other primary and secondary causes. Electron microscopy revealed rare, probable viral particles whose core and spikes measured from 120 to 133 nm within endothelium and eccrine glands in two cases. CONCLUSION: This study provides further clinicopathologic evidence of COVID-19-related chilblains. Negative PCR and antibody tests do not rule-out infection. Chilblains represent a good prognosis, occurring later in the disease course. No systemic coagulopathy was identified in any patient. Patients presenting with acral lesions should be isolated, and chilblains should be distinguished from thrombotic lesions (livedo racemosa, retiform purpura, or ischemic acral necrosis).


Assuntos
COVID-19/complicações , COVID-19/diagnóstico , Pérnio/etiologia , Pérnio/patologia , Dedos do Pé/patologia , Adolescente , Adulto , Idoso , Biópsia/métodos , COVID-19/metabolismo , COVID-19/virologia , Pérnio/diagnóstico , Pérnio/virologia , Criança , Diagnóstico Diferencial , Glândulas Écrinas/patologia , Glândulas Écrinas/ultraestrutura , Glândulas Écrinas/virologia , Endotélio/patologia , Endotélio/ultraestrutura , Endotélio/virologia , Feminino , Humanos , Livedo Reticular/patologia , Masculino , Microscopia Eletrônica/métodos , Pessoa de Meia-Idade , Prognóstico , Estudos Prospectivos , Púrpura/patologia , SARS-CoV-2/genética , Pele/patologia , Dedos do Pé/virologia , Adulto Jovem
6.
Int Immunopharmacol ; 102: 108424, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34915409

RESUMO

SARS-CoV2 mutants B.1.1.7, B.1.351, and P.1 contain a key mutation N501Y. B.1.135 and P.1 lineages have another mutation, E484K. Here, we decode the effect of these two mutations on the host receptor, ACE2, and neutralizing antibody (B38) recognition. The N501Y RBD mutant binds to ACE2 with higher affinity due to improved π-π stacking and π-cation interactions. The higher binding affinity of the E484K mutant is caused due to the formation of additional hydrogen bond and salt-bridge interactions with ACE2. Both the mutants bind to the B38 antibody with reduced affinity due to the loss of several hydrogen-bonding interactions. The insights obtained from the study are crucial to interpret the increased transmissibility and reduced neutralization efficacy of rapidly emerging SARS-CoV2 VOCs.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Anticorpos Neutralizantes/metabolismo , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/genética , Enzima de Conversão de Angiotensina 2/ultraestrutura , Afinidade de Anticorpos/genética , Sítios de Ligação/genética , Cristalografia por Raios X , Humanos , Mutação , SARS-CoV-2/genética , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/ultraestrutura , Internalização do Vírus
7.
J Cell Biol ; 221(1)2022 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-34749397

RESUMO

ADP-ribosylation factors (Arfs) are small GTPases regulating membrane traffic in the secretory pathway. They are closely related and appear to have overlapping functions, regulators, and effectors. The functional specificity of individual Arfs and the extent of redundancy are still largely unknown. We addressed these questions by CRISPR/Cas9-mediated genomic deletion of the human class I (Arf1/3) and class II (Arf4/5) Arfs, either individually or in combination. Most knockout cell lines were viable with slight growth defects only when lacking Arf1 or Arf4. However, Arf1+4 and Arf4+5 could not be deleted simultaneously. Class I Arfs are nonessential, and Arf4 alone is sufficient for viability. Upon Arf1 deletion, the Golgi was enlarged, and recruitment of vesicle coats decreased, confirming a major role of Arf1 in vesicle formation at the Golgi. Knockout of Arf4 caused secretion of ER-resident proteins, indicating specific defects in coatomer-dependent ER protein retrieval by KDEL receptors. The knockout cell lines will be useful tools to study other Arf-dependent processes.


Assuntos
Fatores de Ribosilação do ADP/metabolismo , Técnicas de Inativação de Genes , Complexo de Golgi/metabolismo , Forma Celular , Retículo Endoplasmático/metabolismo , Deleção de Genes , Complexo de Golgi/ultraestrutura , Células HeLa , Humanos
8.
Biochim Biophys Acta Biomembr ; 1864(1): 183749, 2022 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-34506795

RESUMO

Gangliosides induced a smelting process in nanostructured amyloid fibril-like films throughout the surface properties contributed by glycosphingolipids when mixed with 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC)/Aß(1-40) amyloid peptide. We observed a dynamical smelting process when pre-formed amyloid/phospholipid mixture is laterally mixed with gangliosides. This particular environment, gangliosides/phospholipid/Aß(1-40) peptide mixed interfaces, showed complex miscibility behavior depending on gangliosides content. At 0% of ganglioside covered surface respect to POPC, Aß(1-40) peptide forms fibril-like structure. In between 5 and 15% of gangliosides, the fibrils dissolve into irregular domains and they disappear when the proportion of gangliosides reach the 20%. The amyloid interfacial dissolving effect of gangliosides is taken place at lateral pressure equivalent to the organization of biological membranes. Domains formed at the interface are clearly evidenced by Brewster Angle Microscopy and Atomic Force Microscopy when the films are transferred onto a mica support. The domains are thioflavin T (ThT) positive when observed by fluorescence microscopy. We postulated that the smelting process of amyloids fibrils-like structure at the membrane surface provoked by gangliosides is a direct result of a new interfacial environment imposed by the complex glycosphingolipids. We add experimental evidence, for the first time, how a change in the lipid environment (increase in ganglioside proportion) induces a rapid loss of the asymmetric structure of amyloid fibrils by a simple modification of the membrane condition (a more physiological situation).


Assuntos
Peptídeos beta-Amiloides/química , Gangliosídeos/química , Glicoesfingolipídeos/química , Lipídeos de Membrana/química , Nanoestruturas/química , Fragmentos de Peptídeos/química , Amiloide/química , Peptídeos beta-Amiloides/ultraestrutura , Microscopia de Força Atômica , Nanoestruturas/ultraestrutura , Fragmentos de Peptídeos/ultraestrutura , Fosfatidilcolinas/química , Propriedades de Superfície
9.
Biochim Biophys Acta Biomembr ; 1864(1): 183781, 2022 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-34555419

RESUMO

Surface-active amphiphiles find applications in a wide range of areas of industry such as agrochemicals, personal care, and pharmaceuticals. In many of these applications, interaction with cell membranes is a key factor for achieving their purpose. How do amphiphiles interact with lipid membranes? What are their bases for membrane specificity? Which biophysical properties of membranes are susceptible to modulation by amphiphilic membrane-effectors? What aspects of this interaction are important for performing their function? In our work on membrane biophysics over the years, questions like these have arisen and we now share some of our findings and discuss them in this review. This topic was approached focusing on the membrane properties and their alterations rather than on the amphiphile structure requirements for their interaction. Here, we do not aim to provide a comprehensive list of the modes of action of amphiphiles of biological interest but to help in understanding them.


Assuntos
Membrana Celular/química , Lipídeos de Membrana/química , Tensoativos/química , Biofísica , Membrana Celular/ultraestrutura
10.
Biochim Biophys Acta Biomembr ; 1864(1): 183791, 2022 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-34624277

RESUMO

Cell membranes provide an environment that is essential to the functions of membrane proteins. Cell membranes are mainly composed of proteins and highly diverse phospholipids. The influence of diverse lipid compositions of native cell membranes on the dynamics of the embedded membrane proteins has not been examined. Here we employ solid-state NMR to investigate the dynamics of E. coli Aquaporin Z (AqpZ) in its native inner cell membranes, and reveal the influence of diverse lipid compositions on the dynamics of AqpZ by comparing it in native cell membranes to that in POPC/POPG bilayers. We demonstrate that the dynamic rigidity of AqpZ generally conserves in both native cell membranes and POPC/POPG bilayers, due to its tightly packed tetrameric structure. In the gel and the liquid crystal phases of lipids, our experimental results show that AqpZ is more dynamic in native cell membranes than that in POPC/POPG bilayers. In addition, we observe that phase transitions of lipids in native membranes are less sensitive to temperature variations compared with that in POPC/POPG bilayers, which results in that the dynamics of AqpZ is less affected by the phase transitions of lipids in native cell membranes than that in POPC/POPG bilayers. This study provides new insights into the dynamics of membrane proteins in native cell membranes.


Assuntos
Aquaporinas/química , Membrana Celular/química , Proteínas de Escherichia coli/química , Proteínas de Membrana/química , Fosfolipídeos/química , Aquaporinas/genética , Aquaporinas/ultraestrutura , Membrana Celular/genética , Membrana Celular/ultraestrutura , Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/ultraestrutura , Proteínas de Membrana/ultraestrutura , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Fosfolipídeos/genética
11.
FASEB J ; 36(1): e22067, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34914140

RESUMO

The objective of the current study was to examine the drug-induced effects of the EP2 agonist, omidenapag (OMD), on human corneal stroma, two- and three-dimensional (2D and 3D) cultures of human corneal stroma fibroblasts (HCSFs). The drug-induced effects on 2D monolayers and 3D spheroids were characterized by examining the ultrastructures by scanning electron microscope (SEM), transendothelial electrical resistance (TEER) measurements, and fluorescein isothiocyanate (FITC)-dextran permeability. The physical properties of 3D spheroids with respect to size and stiffness were also examined. In addition, the gene expressions of extracellular matrix (ECM) molecules, including collagen (COL) 1, 4, and 6, and fibronectin (FN), a tissue inhibitor of metalloproteinase (TIMP) 1-4, matrix metalloproteinase (MMP) 2, 9, and 14, aquaporin1 (AQP1), and several endoplasmic reticulum (ER) stress-related factors were evaluated. In the 2D HCSFs, OMD induced (1) a significant increase in ECM deposits, as evidenced by SEM, the mRNA expression of COL4 and FN, and (2) a decrease in TEER values and a concentration-dependent increase in FITC-dextran permeability. In the case of 3D spheroids, OMD had no effect on size but a substantial increase in stiffness was observed. Furthermore, such OMD-induced effects on stiffness were dramatically modulated by the osmotic pressure of the system. In contrast to the above 2D cultures, among the ECM molecules and the modulators of 3D spheroids, namely, TIMPS and MMPs, the down-regulation of COL1, TIMP1 and 2 and the up-regulation of MMP9 were observed. Interestingly, such diversity in terms of OMD-induced gene expressions between 2D and 3D cultures was also recognized in AQP1 (2D; no significant change, 3D; significant up-regulation) and ER stress-related genes. The findings presented herein suggest that the EP2 agonist, OMD, alters the physical stiffness of 3D spheroids obtained from human corneal stroma fibroblasts and this alteration is dependent on the osmotic pressures. 2D and 3D cell cultures may be useful for evaluating the drug induced effects of OMD toward human corneal stroma.


Assuntos
Córnea/metabolismo , Fibroblastos/metabolismo , Pressão Osmótica/efeitos dos fármacos , Receptores de Prostaglandina E Subtipo EP2 , Esferoides Celulares/metabolismo , Córnea/ultraestrutura , Estresse do Retículo Endoplasmático , Matriz Extracelular/metabolismo , Matriz Extracelular/ultraestrutura , Proteínas do Olho/metabolismo , Feminino , Fibroblastos/ultraestrutura , Humanos , Masculino , Receptores de Prostaglandina E Subtipo EP2/agonistas , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Esferoides Celulares/ultraestrutura
12.
J Exp Med ; 219(1)2022 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-34783859

RESUMO

Inflammasome proteins play an important role in many diseases of high unmet need, making them attractive drug targets. However, drug discovery for inflammasome proteins has been challenging in part due to the difficulty in solving high-resolution structures using cryo-EM or crystallography. Recent advances in the structural biology of NLRP3 and NLRP1 have provided the first set of data that proves a promise for structure-based drug design for this important family of targets.


Assuntos
Descoberta de Drogas/métodos , Inflamassomos/metabolismo , Complexos Multiproteicos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas NLR/metabolismo , Animais , Microscopia Crioeletrônica , Sistemas de Liberação de Medicamentos/métodos , Humanos , Inflamassomos/antagonistas & inibidores , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/ultraestrutura , Proteína 3 que Contém Domínio de Pirina da Família NLR/química , Proteína 3 que Contém Domínio de Pirina da Família NLR/ultraestrutura , Proteínas NLR/química , Proteínas NLR/ultraestrutura , Preparações Farmacêuticas/administração & dosagem , Conformação Proteica , Multimerização Proteica , Transdução de Sinais/efeitos dos fármacos
13.
J Cell Biol ; 221(1)2022 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-34787650

RESUMO

Proper cilia formation in multiciliated cells (MCCs) is necessary for appropriate embryonic development and homeostasis. Multicilia share many structural characteristics with monocilia and primary cilia, but there are still significant gaps in our understanding of the regulation of multiciliogenesis. Using the Xenopus embryo, we show that CEP97, which is known as a negative regulator of primary cilia formation, interacts with dual specificity tyrosine phosphorylation regulated kinase 1A (Dyrk1a) to modulate multiciliogenesis. We show that Dyrk1a phosphorylates CEP97, which in turn promotes the recruitment of Polo-like kinase 1 (Plk1), which is a critical regulator of MCC maturation that functions to enhance centriole disengagement in cooperation with the enzyme Separase. Knockdown of either CEP97 or Dyrk1a disrupts cilia formation and centriole disengagement in MCCs, but this defect is rescued by overexpression of Separase. Thus, our study reveals that Dyrk1a and CEP97 coordinate with Plk1 to promote Separase function to properly form multicilia in vertebrate MCCs.


Assuntos
Centríolos/metabolismo , Cílios/metabolismo , Organogênese , Proteínas Tirosina Quinases/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Movimento Celular , Embrião não Mamífero/metabolismo , Embrião não Mamífero/ultraestrutura , Humanos , Fosforilação , Ligação Proteica , Proteínas Tirosina Quinases/química , Proteínas Proto-Oncogênicas/metabolismo , Especificidade por Substrato , Xenopus , Proteínas de Xenopus/química
14.
J Cell Biol ; 221(1)2022 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-34817533

RESUMO

The key endosomal regulators Rab5, EEA1, and APPL1 are frequently applied in fluorescence microscopy to mark early endosomes, whereas Rab7 is used as a marker for late endosomes and lysosomes. However, endogenous levels of these proteins localize poorly in immuno-EM, and systematic studies on their native ultrastructural distributions are lacking. To address this gap, we here present a quantitative, on-section correlative light and electron microscopy (CLEM) approach. Using the sensitivity of fluorescence microscopy, we label hundreds of organelles that are subsequently visualized by EM and classified by ultrastructure. We show that Rab5 predominantly marks small, endocytic vesicles and early endosomes. EEA1 colocalizes with Rab5 on early endosomes, but unexpectedly also labels Rab5-negative late endosomes, which are positive for PI(3)P but lack Rab7. APPL1 is restricted to small Rab5-positive, tubulo-vesicular profiles. Rab7 primarily labels late endosomes and lysosomes. These data increase our understanding of the structural-functional organization of the endosomal system and introduce quantitative CLEM as a sensitive alternative for immuno-EM.


Assuntos
Endossomos/ultraestrutura , Microscopia Eletrônica , Proteínas de Transporte Vesicular/ultraestrutura , Antígenos/metabolismo , Linhagem Celular Tumoral , Endossomos/metabolismo , Imunofluorescência , Humanos , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Proteínas de Transporte Vesicular/metabolismo
15.
Parasitol Int ; 86: 102472, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34597759

RESUMO

Kudoa ocellatus n. sp. was found in the musculature of Astronotus ocelattus (Agassiz, 1831) from the Arari River on Marajó Island in Pará, Brazil. The new species forms pseudocysts in the epaxial and hypaxial musculature composed of various spores that are pseudoquadrate in the apical view. In the lateral view, the spores were triangular or pyramidal. In the lateral view, the spores were 46 ± 0.11 µm (4.5-4.8) in length and 6.6 ± 0.3 µm (6.2-7.2) in width, with four pyriform polar capsules of equal size that measured 2.0 ± 0.16 µm (1.8-2.2) in length and 1.5 ± 0.18 µm (1.3-1.8) in width. Based on the partial (1418 bps) sequence of the SSU rDNA gene, Kudoa ocellatus n. sp. was distinct from all the other Kudoa species deposited in GenBank. The phylogenetic Bayesian Inference and P distance placed the new species together with the other Kudoa species that parasitize freshwater Amazonian fish. The morphological evidence, together with the SSU rDNA gene sequence, supported the description of Kudoa ocellatus n. sp., a distinct new species of the genus, which parasitizes a freshwater Amazonian cichlid.


Assuntos
Ciclídeos , Doenças dos Peixes/epidemiologia , Myxozoa/classificação , Doenças Parasitárias em Animais/epidemiologia , Animais , Brasil/epidemiologia , Doenças dos Peixes/parasitologia , Interações Hospedeiro-Parasita , Myxozoa/genética , Myxozoa/ultraestrutura , Doenças Parasitárias em Animais/parasitologia , Prevalência
16.
Biochim Biophys Acta Mol Basis Dis ; 1868(1): 166279, 2022 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-34600082

RESUMO

The tumor stroma plays a pivotal role in colon cancer genesis and progression. It was observed that collagen fibers in the extracellular matrix (ECM) of cancer stroma, undergo a strong remodeling. These fibrous proteins result more aligned and compact than in physiological conditions, creating a microenvironment that favors cancer development. In this work, micro-FTIR spectroscopy was applied to investigate the chemical modifications in the tumor stroma. Using Fuzzy C-means clustering, mean spectra from diseased and normal stroma were compared and collagen was found to be responsible for the main differences between them. Specifically, the modified absorptions at 1203, 1238, 1284 cm-1 and 1338 cm-1 wavenumbers, were related to the amide III band and CH2 bending of side chains. These signals are sensitive to the interactions between the α-chains in the triple helices of collagen structure. This provided robust chemical evidence that in cancer ECM, collagen fibers are more parallelized, stiff and ordered than in normal tissue. Principal Component Analysis (PCA) applied to the spectra from malignant and normal stroma confirmed these findings. Using LDA (Linear Discriminant Analysis) classification, the absorptions 1203, 1238, 1284 and 1338 cm-1 were examined as spectral biomarkers, obtaining quite promising results. The use of a PCA-LDA prediction model on samples with moderate tumor degree further showed that the stroma chemical modifications are more indicative of malignancy compared to the epithelium. These preliminary findings have shown that micro-FTIR spectroscopy, focused on collagen signals, could become a promising tool for colon cancer diagnosis.


Assuntos
Carcinogênese/genética , Carcinoma/diagnóstico , Colágeno/química , Neoplasias do Colo/diagnóstico , Espectroscopia de Infravermelho com Transformada de Fourier , Carcinoma/química , Carcinoma/patologia , Colágeno/ultraestrutura , Colo/química , Colo/patologia , Neoplasias do Colo/química , Neoplasias do Colo/patologia , Epitélio/química , Epitélio/patologia , Matriz Extracelular/química , Matriz Extracelular/patologia , Humanos , Análise de Componente Principal , Microambiente Tumoral/genética
17.
Biochim Biophys Acta Mol Cell Res ; 1869(1): 119161, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34655689

RESUMO

Membraneless organelles have emerged during the evolution of eukaryotic cells as intracellular domains in which multiple proteins organize into complex structures to perform specialized functions without the need of a lipid bilayer compartment. Here we describe the perinuclear space of eukaryotic cells as a highly organized network of cytoskeletal filaments that facilitates assembly of biomolecular condensates. Using bioinformatic analyses, we show that the perinuclear proteome is enriched in intrinsic disorder with several proteins predicted to undergo liquid-liquid phase separation. We also analyze immunofluorescence and transmission electron microscopy images showing the association between the nucleus and other organelles, such as mitochondria and lysosomes, or the labeling of specific proteins within the perinuclear region of cells. Altogether our data support the existence of a perinuclear dense sub-micron region formed by a well-organized three-dimensional network of structural and signaling proteins, including several proteins containing intrinsically disordered regions with phase behavior. This network of filamentous cytoskeletal proteins extends a few micrometers from the nucleus, contributes to local crowding, and organizes the movement of molecular complexes within the perinuclear space. Our findings take a key step towards understanding how membraneless regions within eukaryotic cells can serve as hubs for biomolecular condensates assembly, in particular the perinuclear space. Finally, evaluation of the disease context of the perinuclear proteins revealed that alterations in their expression can lead to several pathological conditions, and neurological disorders and cancer are among the most frequent.


Assuntos
Citoesqueleto de Actina/metabolismo , Membrana Nuclear/metabolismo , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/ultraestrutura , Animais , Células Cultivadas , Embrião de Galinha , Proteínas Intrinsicamente Desordenadas/metabolismo , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Microscopia Eletrônica de Transmissão/métodos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Membrana Nuclear/ultraestrutura , Proteoma/genética , Proteoma/metabolismo , Peixe-Zebra
18.
Parasitol Int ; 86: 102468, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34520840

RESUMO

Parastrigea brasiliana (Szidat, 1928) Dubois, 1964, was described from (Cochlearius cochlearius) in South America. The taxonomy of this species has been unstable due that it was described as a member of Strigea Abildgaard, 1790. However, the same author one year later transferred it to Apharyngostrigea Ciurea, 1927 and since then, it has been alternatively placed in the genus Apharyngostrigea or Parastrigea Szidat, 1928 from Strigeidae. In the current research, specimens identified as P. brasiliana were collected from type host in southeastern Mexico. We sequenced three molecular markers: the internal transcribed spacers ITS1 and ITS2 including the 5.8S gene (ITS region), the D1-D3 domains of the large subunit (LSU) from nuclear DNA and cytochrome c oxidase subunit I (cox 1) from mitochondrial DNA. These sequences were aligned with other sequences available in the GenBank dataset from Strigeidae. Maximum likelihood and Bayesian analyses inferred with three molecular markers consistently showed that P. brasiliana is not closely related to other members of the genus Parastrigea and are placed in a reciprocal monophyletic clade inside Apharyngostrigea, with very low genetic divergence, varying from 0 to 0.09% for the ITS, from 0 to 0.08% for the LSU and from 0.21 to 0.43% for cox 1. Consequently, we proposed to reallocate it to A. brasiliana. The phylogenetic analyses obtained are key and very useful for re-evaluate the morphology of A. brasiliana because this species share morphological characters with the genera Parastrigea (concentration of vitelline follicles distributed in two lateral expansions on the forebody) and Apharyngostrigea (absence of pharynx). Finally, the current record of A. brasiliana expands its distribution range in four countries, namely, the USA, Mexico, Venezuela and Brazil, in the Neotropical region.


Assuntos
Doenças das Aves/parasitologia , Aves , Trematódeos , Infecções por Trematódeos/veterinária , Animais , DNA de Helmintos/análise , DNA Mitocondrial/análise , Proteínas de Helminto/análise , México , Microscopia Eletrônica de Varredura/veterinária , Trematódeos/anatomia & histologia , Trematódeos/classificação , Trematódeos/genética , Trematódeos/ultraestrutura , Infecções por Trematódeos/parasitologia
19.
Dermatol Surg ; 48(1): 120-125, 2022 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-34904578

RESUMO

BACKGROUND: Topical medications play a large role in the management of cutaneous diseases, but penetration is limited. Device-assisted drug delivery using mechanical destruction, lasers, and other energy-based modalities can increase penetration and absorption through creation of transcutaneous channels. OBJECTIVE: To examine real-time, in vivo cutaneous changes in response to various devices used to improve topical drug delivery through optical coherence tomography (OCT) imaging. METHODS AND MATERIALS: Treatment was performed with 8 medical devices, including mechanical destruction, lasers, and other energy-based modalities. Optical coherence tomography was used for real-time, noninvasive, in vivo imaging. RESULTS: Using OCT, microneedling and radiofrequency microneedling demonstrated no cutaneous channels. Both low-energy, low-density, fractional nonablative lasers produced transient channels, which closed within hours. The fractional nonablative 1,927-nm thulium fiber and 1,550-nm erbium fiber lasers created channels with epidermal debris within, which were still closing at 24 hours. The fractional thermomechanical ablative device and the fractional ablative CO2 laser produced channels that were still open at 24 hours. CO2 laser channels had thick rims of coagulated tissue and remained open for longer. CONCLUSION: Demonstrable differences among the devices were seen, and only some can produce observable channels, the characteristics of which vary with each technology.


Assuntos
Sistemas de Liberação de Medicamentos/instrumentação , Lasers , Absorção Cutânea/efeitos da radiação , Pele/diagnóstico por imagem , Administração Cutânea , Humanos , Pele/metabolismo , Pele/ultraestrutura , Tomografia de Coerência Óptica
20.
Proc Natl Acad Sci U S A ; 118(51)2021 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-34911759

RESUMO

Chiral asymmetry is important in a wide variety of disciplines and occurs across length scales. While several natural chiral biomolecules exist only with single handedness, they can produce complex hierarchical structures with opposite chiralities. Understanding how the handedness is transferred from molecular to the macroscopic scales is far from trivial. An intriguing example is the transfer of the handedness of helicoidal organizations of cellulose microfibrils in plant cell walls. These cellulose helicoids produce structural colors if their dimension is comparable to the wavelength of visible light. All previously reported examples of a helicoidal structure in plants are left-handed except, remarkably, in the Pollia condensata fruit; both left- and right-handed helicoidal cell walls are found in neighboring cells of the same tissue. By simultaneously studying optical and mechanical responses of cells with different handednesses, we propose that the chirality of helicoids results from differences in cell wall composition. In detail, here we showed statistical substantiation of three different observations: 1) light reflected from right-handed cells is red shifted compared to light reflected from left-handed cells, 2) right-handed cells occur more rarely than left-handed ones, and 3) right-handed cells are located mainly in regions corresponding to interlocular divisions. Finally, 4) right-handed cells have an average lower elastic modulus compared to left-handed cells of the same color. Our findings, combined with mechanical simulation, suggest that the different chiralities of helicoids in the cell wall may result from different chemical composition, which strengthens previous hypotheses that hemicellulose might mediate the rotations of cellulose microfibrils.


Assuntos
Parede Celular/química , Commelinaceae/química , Frutas/química , Parede Celular/ultraestrutura , Celulose/química , Cor , Módulo de Elasticidade , Microfibrilas/química , Polissacarídeos/química
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