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1.
J Gene Med ; 26(4): e3683, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38571451

RESUMO

BACKGROUND: Acute pancreatitis (AP) is a potentially lethal acute disease highly involved in coagulation disorders. Pyroptosis has been reported to exacerbate coagulation disorders, yet this implication has not been illustrated completely in AP. METHODS: RNA sequencing data of peripheral blood of AP patients were downloaded from the Gene Expression Omnibus database. Gene set variation analysis and single sample gene set enrichment analysis were used to calculate the enrichment score of coagulation-related signatures and pyroptosis. Spearman and Pearson correlation analysis was used for correlation analysis. Peripheral blood samples and related clinical parameters were collected from patients with AP and healthy individuals. A severe AP (SAP) model of mice was established using caerulein and lipopolysaccharide. Enzyme-linked immunosorbent assay, chemiluminescence immunoassay and immunohistochemical analysis were employed to detect the level of coagulation indicators and pyroptosis markers in serum and pancreas tissues. Additionally, we evaluated the effect of pyroptosis inhibition and NLRC4 silence on the function of human umbilical vein endothelial cells (HUVECs). RESULTS: Coagulation disorders were significantly positively correlated to the severity of AP, and they could be a predictor for AP severity. Further analyses indicated that six genes-DOCK9, GATA3, FCER1G, NLRC4, C1QB and C1QC-may be involved in coagulation disorders of AP. Among them, NLRC4 was positively related to pyroptosis that had a positive association with most coagulation-related signatures. Data from patients showed that NLRC4 and other pyroptosis markers, including IL-1ß, IL-18, caspase1 and GSDMD, were significant correlation to AP severity. In addition, NLRC4 was positively associated with coagulation indicators in AP patients. Data from mice showed that NLRC4 was increased in the pancreas tissues of SAP mice. Treatment with a pyroptosis inhibitor effectively alleviated SAP and coagulation disorders in mice. Finally, inhibiting pyroptosis or silencing NLRC4 could relieve endothelial dysfunction in HUVECs. CONCLUSIONS: NLRC4-mediated pyroptosis damages the function of endothelial cells and thereby exacerbates coagulation disorders of AP. Inhibiting pyroptosis could improve coagulation function and alleviate AP.


Assuntos
Transtornos da Coagulação Sanguínea , Pancreatite , Animais , Humanos , Camundongos , Doença Aguda , Transtornos da Coagulação Sanguínea/genética , Transtornos da Coagulação Sanguínea/complicações , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Pancreatite/genética , Piroptose
2.
Nat Commun ; 15(1): 1599, 2024 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-38383552

RESUMO

Lipids play crucial roles in many biological processes. Mapping spatial distributions and examining the metabolic dynamics of different lipid subtypes in cells and tissues are critical to better understanding their roles in aging and diseases. Commonly used imaging methods (such as mass spectrometry-based, fluorescence labeling, conventional optical imaging) can disrupt the native environment of cells/tissues, have limited spatial or spectral resolution, or cannot distinguish different lipid subtypes. Here we present a hyperspectral imaging platform that integrates a Penalized Reference Matching algorithm with Stimulated Raman Scattering (PRM-SRS) microscopy. Using this platform, we visualize and identify high density lipoprotein particles in human kidney, a high cholesterol to phosphatidylethanolamine ratio inside granule cells of mouse hippocampus, and subcellular distributions of sphingosine and cardiolipin in human brain. Our PRM-SRS displays unique advantages of enhanced chemical specificity, subcellular resolution, and fast data processing in distinguishing lipid subtypes in different organs and species.


Assuntos
Microscopia , Microscopia Óptica não Linear , Animais , Camundongos , Humanos , Microscopia Óptica não Linear/métodos , Análise Espectral Raman/métodos , Lipídeos
3.
bioRxiv ; 2024 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-38370804

RESUMO

Fluorescent biosensors revolutionized biomedical science by enabling the direct measurement of signaling activities in living cells, yet the current technology is limited in resolution and dimensionality. Here, we introduce highly sensitive chemigenetic kinase activity biosensors that combine the genetically encodable self-labeling protein tag HaloTag7 with bright far-red-emitting synthetic fluorophores. This technology enables five-color biosensor multiplexing, 4D activity imaging, and functional super-resolution imaging via stimulated emission depletion (STED) microscopy.

4.
Anal Methods ; 16(10): 1489-1495, 2024 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-38369952

RESUMO

High fluorescence background poses a substantial challenge to surface-enhanced Raman scattering (SERS), thereby limiting its broader applicability across diverse domains. In this work, silver nanoparticle (Ag NP)-loaded graphene oxide aerogel nanomaterials (GO-Ag ANM) were prepared for sensitive SERS detection of fluorescent explosive 2,4,8,10-tetranitrobenzo-1,3a,6,6a-tetraazapentaenopyridine (BPTAP) by a fluorescence quenching strategy. By harnessing the fluorescence quenching properties of graphene and the localized surface plasmon resonance of silver nanoparticles, the synthesized aerogels exhibited effective fluorescence quenching and Raman enhancement capabilities when employed for BPTAP analysis with 532 nm laser excitation. Significantly, precise control over the loading quantity of silver nanoparticles (Ag NPs) resulted in the remarkable sensitivity of the surface-enhanced Raman scattering (SERS) effect. This method allowed for the detection of fluorescent explosive BPTAP at an extraordinarily low concentration of 1 × 10-7 M. Furthermore, the approach also demonstrated excellent detection capabilities for the dyes R6G, CV, and RhB. This study offers valuable insights for the sensitive detection of fluorescent molecules.

5.
Curr Protoc ; 4(1): e970, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38270527

RESUMO

Raman microscopy is a vibrational imaging technology that can detect molecular chemical bond vibrational signals. Since this signal is originated from almost every vibrational mode of molecules with different vibrational energy levels, it provides spatiotemporal distribution of various molecules in living organisms without the need for any labeling. The limitations of low signal strength in Raman microscopy have been effectively addressed by incorporating a stimulated emission process, leading to the development of stimulated Raman scattering (SRS) microscopy. Furthermore, the issue of low spatial resolution has been resolved through the application of computational techniques, specifically image deconvolution. In this article, we present a comprehensive guide to super-resolution SRS microscopy using an Adam-based pointillism deconvolution (A-PoD) algorithm, complemented by a user-friendly graphical user interface (GUI). We delve into the crucial parameters and conditions necessary for achieving super-resolved images through SRS imaging. Additionally, we provide a step-by-step walkthrough of the preprocessing steps and the use of GUI-supported A-PoD. This complete package offers a user-friendly platform for super-resolution SRS microscopy, enhancing the versatility and applicability of this advanced microscopy technique to reveal nanoscopic multimolecular nature. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Super-resolution stimulated Raman scattering microscopy with graphical user interface-supported A-PoD Support Protocol: Deuterium labeling on cells with heavy water for metabolic imaging.


Assuntos
Microscopia , Microscopia Óptica não Linear , Algoritmos , Óxido de Deutério , Rotulagem de Produtos
6.
J Biophotonics ; 17(2): e202300344, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38010367

RESUMO

This study reports on the first use of the optical Kerr effect (OKE) in breast cancer tissue. This proposed optical biopsy method utilizes a Femtosecond Optical Kerr Gate to detect changes in dielectric relaxation and conductivity created by a cancerous infection. Here, the temporal behavior of the OKE is tracked in normal and cancerous samples of human and mouse breast. These tissues display a double peaked temporal structure and its decay rate changes depending on the tissue's infection status. The decay of the secondary peak, attributed to ultrafast plasma response, indicates that the tissue's conductivity has doubled once infected. A slower molecular contribution to the Kerr effect can also be observed in healthy tissues. These findings suggest two possible biomarkers for the use of OKE in optical biopsy. Both markers arise from alterations in the infected tissue's cellular structure, which changes the rate at which electronic and molecular processes occur.


Assuntos
Neoplasias da Mama , Humanos , Animais , Camundongos , Feminino , Neoplasias da Mama/diagnóstico por imagem , Biópsia
7.
bioRxiv ; 2024 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-37425829

RESUMO

Primary tumors with similar mutational profiles can progress to vastly different outcomes where transcriptional state, rather than mutational profile, predicts prognosis. A key challenge is to understand how distinct tumor cell states are induced and maintained. In triple negative breast cancer cells, invasive behaviors and aggressive transcriptional signatures linked to poor patient prognosis can emerge in response to contact with collagen type I. Herein, collagen-induced migration heterogeneity within a TNBC cell line was leveraged to identify transcriptional programs associated with invasive versus non-invasive phenotypes and implicate molecular switches. Phenotype-guided sequencing revealed that invasive cells upregulate iron uptake and utilization machinery, anapleurotic TCA cycle genes, actin polymerization promoters, and a distinct signature of Rho GTPase activity and contractility regulating genes. The non-invasive cell state is characterized by actin and iron sequestration modules along with glycolysis gene expression. These unique tumor cell states are evident in patient tumors and predict divergent outcomes for TNBC patients. Glucose tracing confirmed that non-invasive cells are more glycolytic than invasive cells, and functional studies in cell lines and PDO models demonstrated a causal relationship between phenotype and metabolic state. Mechanistically, the OXPHOS dependent invasive state resulted from transient HO-1 upregulation triggered by contact with dense collagen that reduced heme levels and mitochondrial chelatable iron levels. This induced expression of low cytoplasmic iron response genes regulated by ACO1/IRP1. Knockdown or inhibition of HO-1, ACO1/IRP1, MRCK, or OXPHOS abrogated invasion. These findings support an emerging theory that heme and iron flux serve as important regulators of TNBC aggressiveness.

8.
J Am Chem Soc ; 145(47): 25664-25672, 2023 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-37921495

RESUMO

Polymeric spherulites are typically formed by melt crystallization: spherulitic growth in solution is rare and requires complex polymers and dilute solutions. Here, we report the mild and unique formation of luminescent spherulites at room temperature via the simple molecule benzene-1,4-dithiol (BDT). Specifically, BDT polymerized into oligomers (PBDT) via disulfide bonds and assembled into uniform supramolecular nanoparticles in aqueous buffer; these nanoparticles were then dissolved back into PBDT in a good solvent (i.e., dimethylformamide) and underwent chain elongation to form spherulites (rPBDT) in 10 min. The spherulite geometry was modulated by changing the PBDT concentration and reaction time. Due to the step-growth polymerization and reorganization of PBDT, these spherulites not only exhibited robust structure but also showed broad clusterization-triggered emission. The biocompatibility and efficient cellular uptake of the spherulites further underscore their value as traceable drug carriers. This system provides a new pathway for designing versatile superstructures with value for hierarchical assembly of small molecules into a complicated biological system.


Assuntos
Nanopartículas , Polímeros , Cristalização , Polímeros/química , Congelamento
9.
Biomed Opt Express ; 14(11): 5764-5780, 2023 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-38021123

RESUMO

Triple negative breast cancer (TNBC) is a highly aggressive form of cancer. Detecting TNBC early is crucial for improving disease prognosis and optimizing treatment. Unfortunately, conventional imaging techniques fall short in providing a comprehensive differentiation of TNBC subtypes due to their limited sensitivity and inability to capture subcellular details. In this study, we present a multimodal imaging platform that integrates heavy water (D2O)-probed stimulated Raman scattering (DO-SRS), two-photon fluorescence (TPF), and second harmonic generation (SHG) imaging. This platform allows us to directly visualize and quantify the metabolic activities of TNBC subtypes at a subcellular level. By utilizing DO-SRS imaging, we were able to identify distinct levels of de novo lipogenesis, protein synthesis, cytochrome c metabolic heterogeneity, and lipid unsaturation rates in various TNBC subtype tissues. Simultaneously, TPF imaging provided spatial distribution mapping of NAD[P]H and flavin signals in TNBC tissues, revealing a high redox ratio and significant lipid turnover rate in TNBC BL2 (HCC1806) samples. Furthermore, SHG imaging enabled us to observe diverse orientations of collagen fibers in TNBC tissues, with higher anisotropy at the tissue boundary compared to the center. Our multimodal imaging platform offers a highly sensitive and subcellular approach to characterizing not only TNBC, but also other tissue subtypes and cancers.

11.
Light Sci Appl ; 12(1): 234, 2023 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-37714845

RESUMO

A new approach for early detection of multiple cancers is presented by integrating SERS spectroscopy of serum molecular fingerprints and machine learning.

12.
Acc Mater Res ; 4(9): 726-728, 2023 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-37766942
13.
ACS Appl Mater Interfaces ; 15(36): 42293-42303, 2023 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-37651748

RESUMO

Colorimetric biosensors based on gold nanoparticle (AuNP) aggregation are often challenged by matrix interference in biofluids, poor specificity, and limited utility with clinical samples. Here, we propose a peptide-driven nanoscale disassembly approach, where AuNP aggregates induced by electrostatic attractions are dissociated in response to proteolytic cleavage. Initially, citrate-coated AuNPs were assembled via a short cationic peptide (RRK) and characterized by experiments and simulations. The dissociation peptides were then used to reversibly dissociate the AuNP aggregates as a function of target protease detection, i.e., main protease (Mpro), a biomarker for severe acute respiratory syndrome coronavirus 2. The dissociation propensity depends on peptide length, hydrophilicity, charge, and ligand architecture. Finally, our dissociation strategy provides a rapid and distinct optical signal through Mpro cleavage with a detection limit of 12.3 nM in saliva. Our dissociation peptide effectively dissociates plasmonic assemblies in diverse matrices including 100% human saliva, urine, plasma, and seawater, as well as other types of plasmonic nanoparticles such as silver. Our peptide-enabled dissociation platform provides a simple, matrix-insensitive, and versatile method for protease sensing.


Assuntos
COVID-19 , Nanopartículas Metálicas , Humanos , Ouro , Peptídeos , Peptídeo Hidrolases
14.
GEN Biotechnol ; 2(3): 247-261, 2023 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-37363411

RESUMO

Studies have shown that brain lipid metabolism is associated with biological aging and influenced by dietary and genetic manipulations; however, the underlying mechanisms are elusive. High-resolution imaging techniques propose a novel and potent approach to understanding lipid metabolic dynamics in situ. Applying deuterium water (D2O) probing with stimulated Raman scattering (DO-SRS) microscopy, we revealed that lipid metabolic activity in Drosophila brain decreased with aging in a sex-dependent manner. Female flies showed an earlier occurrence of lipid turnover decrease than males. Dietary restriction (DR) and downregulation of insulin/IGF-1 signaling (IIS) pathway, two scenarios for lifespan extension, led to significant enhancements of brain lipid turnover in old flies. Combining SRS imaging with deuterated bioorthogonal probes (deuterated glucose and deuterated acetate), we discovered that, under DR treatment and downregulation of IIS pathway, brain metabolism shifted to use acetate as a major carbon source for lipid synthesis. For the first time, our study directly visualizes and quantifies spatiotemporal alterations of lipid turnover in Drosophila brain at the single organelle (lipid droplet) level. Our study not only demonstrates a new approach for studying brain lipid metabolic activity in situ but also illuminates the interconnection of aging, dietary, and genetic manipulations on brain lipid metabolic regulation.

15.
Res Sq ; 2023 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-37214848

RESUMO

The Optical Kerr Effect was demonstrated for the first time as a new optical biopsy method to detect normal and grades of cancer of human breast tissues. The technique works by temporally tracking the various electronic and molecular processes that give rise to the nonlinear index of refraction (n2). The rate at which these processes populate and dissipate varies depending on the internal properties of the sample. It is shown here that in tissues, the variances in the ultrafast plasma Kerr responses that relates to the dielectric relaxation can be used as a biomarker for cancer. The relaxation of this response changes significantly between healthy and different grades of triple negative breast cancer tissues. This change can be attributed to a doubling or tripling of the tissue's conductivity depending on the cancer grade.

16.
J Vis Exp ; (195)2023 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-37246865

RESUMO

Essential aromatic amino acids (AAAs) are building blocks for synthesizing new biomasses in cells and sustaining normal biological functions. For example, an abundant supply of AAAs is important for cancer cells to maintain their rapid growth and division. With this, there is a rising demand for a highly specific, noninvasive imaging approach with minimal sample preparation to directly visualize how cells harness AAAs for their metabolism in situ. Here, we develop an optical imaging platform that combines deuterium oxide (D2O) probing with stimulated Raman scattering (DO-SRS) and integrates DO-SRS with two-photon excitation fluorescence (2PEF) into a single microscope to directly visualize the metabolic activities of HeLa cells under AAA regulation. Collectively, the DO-SRS platform provides high spatial resolution and specificity of newly synthesized proteins and lipids in single HeLa cell units. In addition, the 2PEF modality can detect autofluorescence signals of nicotinamide adenine dinucleotide (NADH) and Flavin in a label-free manner. The imaging system described here is compatible with both in vitro and in vivo models, which is flexible for various experiments. The general workflow of this protocol includes cell culture, culture media preparation, cell synchronization, cell fixation, and sample imaging with DO-SRS and 2PEF modalities.


Assuntos
Aminoácidos Aromáticos , Microscopia , Humanos , Células HeLa , Microscopia/métodos , Proteínas/metabolismo , Aminoácidos/metabolismo
17.
ACS Sens ; 8(4): 1733-1741, 2023 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-36950737

RESUMO

The surface-enhanced Raman scattering (SERS) technique is a promising method for the detection of explosives such as 2,4,6-trinitrotoluene (TNT) and 3-nitro-1,2,4-triazol-5-one (NTO) because of its high sensitivity to trace substances. However, most SERS detection processes are often nonautomated as well as exhibit low efficiency and toxic exposure, which often poses potential danger to operators. Herein, we propose the integration of SERS with digital microfluidics (SERS-DMF) for automated, high-throughput, and high-sensitivity detection of explosives. First, we carefully designed a DMF chip comprising 40 drive electrodes and 8 storage electrodes to achieve a high-throughput process. And different concentrations of target molecules, silver nanoparticles (Ag NPs), and salts were loaded into the DMF chip. Then, the droplet aggregation, incubation, and detection processes were automatically controlled using the SERS-DMF platform. In addition, Ag NPs were efficiently aggregated by screening different types and concentrations of salts, resulting in "hotspots" and the SERS effect. With the help of the SERS-DMF platform, two explosive samples were automatically detected with high throughput and high sensitivity. The detection limits of TNT and NTO were 10-7 and 10-8 M, respectively. In addition, compared with nonautomatic operations, the SERS-DMF platform exhibited better reproducibility and higher efficiency for the detection of explosives. The proposed SERS-DMF thus has considerable potential as an analytical technique for detecting hazardous substances.


Assuntos
Substâncias Explosivas , Nanopartículas Metálicas , Trinitrotolueno , Microfluídica , Sais , Reprodutibilidade dos Testes , Prata
18.
Nat Methods ; 20(3): 448-458, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36797410

RESUMO

Stimulated Raman scattering (SRS) offers the ability to image metabolic dynamics with high signal-to-noise ratio. However, its spatial resolution is limited by the numerical aperture of the imaging objective and the scattering cross-section of molecules. To achieve super-resolved SRS imaging, we developed a deconvolution algorithm, adaptive moment estimation (Adam) optimization-based pointillism deconvolution (A-PoD) and demonstrated a spatial resolution of lower than 59 nm on the membrane of a single lipid droplet (LD). We applied A-PoD to spatially correlated multiphoton fluorescence imaging and deuterium oxide (D2O)-probed SRS (DO-SRS) imaging from diverse samples to compare nanoscopic distributions of proteins and lipids in cells and subcellular organelles. We successfully differentiated newly synthesized lipids in LDs using A-PoD-coupled DO-SRS. The A-PoD-enhanced DO-SRS imaging method was also applied to reveal metabolic changes in brain samples from Drosophila on different diets. This new approach allows us to quantitatively measure the nanoscopic colocalization of biomolecules and metabolic dynamics in organelles.


Assuntos
Microscopia , Análise Espectral Raman , Microscopia/métodos , Análise Espectral Raman/métodos , Proteínas/metabolismo , Lipídeos
19.
Cell Mol Neurobiol ; 43(5): 2219-2241, 2023 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-36571634

RESUMO

Traumatic brain injury (TBI) can lead to neurodegenerative diseases such as Alzheimer's disease (AD) through mechanisms that remain incompletely characterized. Similar to AD, TBI models present with cellular metabolic alterations and modulated cleavage of amyloid precursor protein (APP). Specifically, AD and TBI tissues display increases in amyloid-ß as well as its precursor, the APP C-terminal fragment of 99 a.a. (C99). Our recent data in cell models of AD indicate that C99, due to its affinity for cholesterol, induces the formation of transient lipid raft domains in the ER known as mitochondria-associated endoplasmic reticulum (ER) membranes ("MAM" domains). The formation of these domains recruits and activates specific lipid metabolic enzymes that regulate cellular cholesterol trafficking and sphingolipid turnover. Increased C99 levels in AD cell models promote MAM formation and significantly modulate cellular lipid homeostasis. Here, these phenotypes were recapitulated in the controlled cortical impact (CCI) model of TBI in adult mice. Specifically, the injured cortex and hippocampus displayed significant increases in C99 and MAM activity, as measured by phospholipid synthesis, sphingomyelinase activity and cholesterol turnover. In addition, our cell type-specific lipidomics analyses revealed significant changes in microglial lipid composition that are consistent with the observed alterations in MAM-resident enzymes. Altogether, we propose that alterations in the regulation of MAM and relevant lipid metabolic pathways could contribute to the epidemiological connection between TBI and AD.


Assuntos
Doença de Alzheimer , Lesões Encefálicas Traumáticas , Camundongos , Animais , Doença de Alzheimer/metabolismo , Mitocôndrias/metabolismo , Regulação para Cima , Retículo Endoplasmático/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Lesões Encefálicas Traumáticas/metabolismo , Lipídeos
20.
Biophys Rep (N Y) ; 2(1): 100043, 2022 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-36425084

RESUMO

Microtubules are self-assembling biological nanotubes made of the protein tubulin that are essential for cell motility, cell architecture, cell division, and intracellular trafficking. They demonstrate unique mechanical properties of high resilience and stiffness due to their quasi-crystalline helical structure. It has been theorized that this hollow molecular nanostructure may function like a quantum wire where optical transitions can take place, and photoinduced changes in microtubule architecture may be mediated via changes in disulfide or peptide bonds or stimulated by photoexcitation of tryptophan, tyrosine, or phenylalanine groups, resulting in subtle protein structural changes owing to alterations in aromatic flexibility. Here, we measured the Raman spectra of a microtubule and its constituent protein tubulin both in dry powdered form and in aqueous solution to determine if molecular bond vibrations show potential Fano resonances, which are indicative of quantum coupling between discrete phonon vibrational states and continuous excitonic many-body spectra. The key findings of this work are that we observed the Raman spectra of tubulin and microtubules and found line shapes characteristic of Fano resonances attributed to aromatic amino acids and disulfide bonds.

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