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1.
Microsyst Nanoeng ; 10: 35, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38482463

RESUMO

Droplet microfluidics has emerged as a critical component of several high-throughput single-cell analysis techniques in biomedical research and diagnostics. Despite significant progress in the development of individual assays, multiparametric optical sensing of droplets and their encapsulated contents has been challenging. The current approaches, most commonly involving microscopy-based high-speed imaging of droplets, are technically complex and require expensive instrumentation, limiting their widespread adoption. To address these limitations, we developed the OptiDrop platform; this platform is a novel optofluidic setup that leverages the principles of flow cytometry. Our platform enables on-chip detection of the scatter and multiple fluorescence signals from the microfluidic droplets and their contents using optical fibers. The highly customizable on-chip optical fiber-based signal detection system enables simplified, miniaturized, low-cost, multiparametric sensing of optical signals with high sensitivity and single-cell resolution within each droplet. To demonstrate the ability of the OptiDrop platform, we conducted a differential expression analysis of the major histocompatibility complex (MHC) protein in response to IFNγ stimulation. Our results showed the platform's ability to sensitively detect cell surface biomarkers using fluorescently labeled antibodies. Thus, the OptiDrop platform combines the versatility of flow cytometry with the power of droplet microfluidics to provide wide-ranging, scalable optical sensing solutions for research and diagnostics.

2.
J Biosci ; 492024.
Artigo em Inglês | MEDLINE | ID: mdl-38287676

RESUMO

Oculocutaneous albinism (OCA) is characterized by reduced melanin biosynthesis affecting the retina, thus impairing visual function. The disease pathology of OCA is poorly understood at the cellular level due to unavailability of suitable biological model systems. This study aimed to develop a disease-specific in vitro model for OCA type 1A, the most severe form caused by TYR (tyrosinase) gene mutations, using retinal pigment epithelium (RPE) differentiated from patient-derived human-induced pluripotent stem cells (hiPSCs). A comparative study between healthy and OCA1A RPE cells revealed that while healthy RPE cells exhibited timely onest of pigmentation during differentiation, OCA1A RPE cells failed to pigment even after an extended culture period. This observation was validated by ultrastructural studies using electron microscopy, hinting at melanosome-specific defects. Immunocytochemistry demonstrated abnormal expression patterns of melanogenesis-specific protein markers in OCA1A RPE cells, indicating reduced or absence of melanin synthesis. Next, a quantitative assay was performed to confirm the absence of melanin production in OCA1A RPE cells. Tyrosinase assay showed no activity in OCA1A compared with healthy RPE, suggesting non-functionality of TYR, further corroborated by western blot analysis showing complete absence of the protein. Gene expression by RNA sequencing of healthy and OCA1A RPE cells uncovered differential gene expression associated with lens development, visual perception, transmembrane transporter activity, and key signaling pathways. This disease-in-a-dish model of OCA1A provides an excellent platform to understand disease mechanism, identify potential therapeutic targets, and facilitate gene therapy or gene correction.


Assuntos
Albinismo Oculocutâneo , Células-Tronco Pluripotentes Induzidas , Humanos , Melaninas/genética , Melaninas/metabolismo , Monofenol Mono-Oxigenase/genética , Monofenol Mono-Oxigenase/química , Monofenol Mono-Oxigenase/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Albinismo Oculocutâneo/genética , Albinismo Oculocutâneo/terapia
3.
EBioMedicine ; 50: 260-273, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31727601

RESUMO

BACKGROUND: Plethora of efforts fails to yield a single drug to reverse the pathogenesis of Parkinson's disease (PD) and related α-synucleopathies. METHODS: Using chemical biology, we identified a small molecule inhibitor of c-abl kinase, PD180970 that could potentially clear the toxic protein aggregates. Genetic, molecular, cell biological and immunological assays were performed to understand the mechanism of action. In vivo preclinical disease model of PD was used to assess its neuroprotection efficacy. FINDINGS: In this report, we show the ability of a small molecule inhibitor of tyrosine kinases, PD180970, to induce autophagy (cell lines and mice midbrain) in an mTOR-independent manner and ameliorate the α-synuclein mediated toxicity. PD180970 also exerts anti-neuroinflammatory potential by inhibiting the release of proinflammatory cytokines such as IL-6 (interleukin-6) and MCP-1 (monocyte chemoattractant protein-1) through reduction of TLR-4 (toll like receptor-4) mediated NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation. In vivo studies show that PD180970 is neuroprotective by degrading the toxic protein oligomers through induction of autophagy and subsiding the microglial activation. INTERPRETATION: These protective mechanisms ensure the negation of Parkinson's disease related motor impairments. FUND: This work was supported by Wellcome Trust/DBT India Alliance Intermediate Fellowship (500159-Z-09-Z), DST-SERB grant (EMR/2015/001946), DBT (BT/INF/22/SP27679/2018) and JNCASR intramural funds to RM, and SERB, DST (SR/SO/HS/0121/2012) to PAA, and DST-SERB (SB/YS/LS-215/2013) to JPC and BIRAC funding to ETA C-CAMP.


Assuntos
Doenças Neurodegenerativas/etiologia , Doenças Neurodegenerativas/metabolismo , Fármacos Neuroprotetores/farmacologia , Agregação Patológica de Proteínas/metabolismo , Animais , Biomarcadores , Linhagem Celular , Citocinas/metabolismo , Modelos Animais de Doenças , Humanos , Imuno-Histoquímica , Interleucina-6/metabolismo , Lipopolissacarídeos/metabolismo , Macroautofagia , Masculino , Camundongos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/fisiopatologia , Neurônios/metabolismo , Fármacos Neuroprotetores/uso terapêutico , Estresse Oxidativo , Agregados Proteicos/efeitos dos fármacos , Agregação Patológica de Proteínas/tratamento farmacológico , Piridonas/farmacologia , Piridonas/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , alfa-Sinucleína/metabolismo
4.
Nat Commun ; 10(1): 89, 2019 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-30626868

RESUMO

The importance of gut microbiota in human health and pathophysiology is undisputable. Despite the abundance of metagenomics data, the functional dynamics of gut microbiota in human health and disease remain elusive. Urolithin A (UroA), a major microbial metabolite derived from polyphenolics of berries and pomegranate fruits displays anti-inflammatory, anti-oxidative, and anti-ageing activities. Here, we show that UroA and its potent synthetic analogue (UAS03) significantly enhance gut barrier function and inhibit unwarranted inflammation. We demonstrate that UroA and UAS03 exert their barrier functions through activation of aryl hydrocarbon receptor (AhR)- nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent pathways to upregulate epithelial tight junction proteins. Importantly, treatment with these compounds attenuated colitis in pre-clinical models by remedying barrier dysfunction in addition to anti-inflammatory activities. Cumulatively, the results highlight how microbial metabolites provide two-pronged beneficial activities at gut epithelium by enhancing barrier functions and reducing inflammation to protect from colonic diseases.


Assuntos
Cumarínicos/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , Proteínas de Junções Íntimas/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células CACO-2 , Cumarínicos/química , Células Epiteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células HT29 , Humanos , Mucosa Intestinal/metabolismo , Macrófagos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 2 Relacionado a NF-E2/genética , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Organismos Livres de Patógenos Específicos , Proteínas de Junções Íntimas/genética
5.
J Biol Chem ; 282(8): 5625-32, 2007 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-17182610

RESUMO

Gephyrin is a bifunctional modular protein that, in neurons, clusters glycine receptors and gamma-aminobutyric acid, type A receptors in the postsynaptic membrane of inhibitory synapses. By x-ray crystallography and cross-linking, the N-terminal G-domain of gephyrin has been shown to form trimers and the C-terminal E-domain dimers, respectively. Gephyrin therefore has been proposed to form a hexagonal submembranous lattice onto which inhibitory receptors are anchored. Here, crystal structure-based substitutions at oligomerization interfaces revealed that both G-domain trimerization and E-domain dimerization are essential for the formation of higher order gephyrin oligomers and postsynaptic gephyrin clusters. Insertion of the alternatively spliced C5' cassette into the G-domain inhibited clustering by interfering with trimerization, and mutation of the glycine receptor beta-subunit binding region prevented the localization of the clusters at synaptic sites. Together our findings show that domain interactions mediate gephyrin scaffold formation.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Complexos Multiproteicos/metabolismo , Sinapses/metabolismo , Animais , Proteínas de Transporte/genética , Linhagem Celular , Humanos , Proteínas de Membrana/genética , Complexos Multiproteicos/genética , Neurônios/metabolismo , Ligação Proteica/fisiologia , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína/fisiologia , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Receptores de Glicina/metabolismo , Sinapses/genética , Xenopus
6.
EMBO J ; 23(13): 2510-9, 2004 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-15201864

RESUMO

Gephyrin is a bi-functional modular protein involved in molybdenum cofactor biosynthesis and in postsynaptic clustering of inhibitory glycine receptors (GlyRs). Here, we show that full-length gephyrin is a trimer and that its proteolysis in vitro causes the spontaneous dimerization of its C-terminal region (gephyrin-E), which binds a GlyR beta-subunit-derived peptide with high and low affinity. The crystal structure of the tetra-domain gephyrin-E in complex with the beta-peptide bound to domain IV indicates how membrane-embedded GlyRs may interact with subsynaptic gephyrin. In vitro, trimeric full-length gephyrin forms a network upon lowering the pH, and this process can be reversed to produce stable full-length dimeric gephyrin. Our data suggest a mechanism by which induced conformational transitions of trimeric gephyrin may generate a reversible postsynaptic scaffold for GlyR recruitment, which allows for dynamic receptor movement in and out of postsynaptic GlyR clusters, and thus for synaptic plasticity.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Receptores de Glicina/metabolismo , Animais , Sítios de Ligação , Proteínas de Transporte/química , Proteínas de Transporte/efeitos dos fármacos , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/ultraestrutura , Cromatografia em Gel , Coenzimas/metabolismo , Cristalografia por Raios X , Dimerização , Concentração de Íons de Hidrogênio , Hidrólise , Espectrometria de Massas , Proteínas de Membrana/química , Proteínas de Membrana/efeitos dos fármacos , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/ultraestrutura , Metaloproteínas/metabolismo , Modelos Químicos , Modelos Moleculares , Cofatores de Molibdênio , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/metabolismo , Pteridinas/metabolismo , Ratos , Receptores de Glicina/química , Receptores de Glicina/genética , Soluções , Sulfatos/química , Ressonância de Plasmônio de Superfície , Sinapses/metabolismo , Tripsina/farmacologia
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