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Cell mass and chemical composition are important aggregate cellular properties that are especially relevant to physiological processes, such as growth control and tissue homeostasis. Despite their importance, it has been difficult to measure these features quantitatively at the individual cell level in intact tissue. Here, we introduce normalized Raman imaging (NoRI), a stimulated Raman scattering (SRS) microscopy method that provides the local concentrations of protein, lipid, and water from live or fixed tissue samples with high spatial resolution. Using NoRI, we demonstrate that protein, lipid, and water concentrations at the single cell are maintained in a tight range in cells under the same physiological conditions and are altered in different physiological states, such as cell cycle stages, attachment to substrates of different stiffness, or by entering senescence. In animal tissues, protein and lipid concentration varies with cell types, yet an unexpected cell-to-cell heterogeneity was found in cerebellar Purkinje cells. The protein and lipid concentration profile provides means to quantitatively compare disease-related pathology, as demonstrated using models of Alzheimer's disease. This demonstration shows that NoRI is a broadly applicable technique for probing the biological regulation of protein mass, lipid mass, and water mass for studies of cellular and tissue growth, homeostasis, and disease.
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Microscopía Óptica no Lineal , Espectrometría Raman , Metabolismo de los Lípidos , Lípidos , Microscopía/métodos , Proteínas , Espectrometría Raman/métodosRESUMEN
Labeling the genome and envelope of a virus with multicolor quantum dots (QDs) simultaneously enables real-time monitoring of viral uncoating and genome release, contributing to our understanding of virus infection mechanisms. However, current labeling techniques require genetic modification, which alters the virus's composition and infectivity. To address this, we utilized the CRISPR/Cas13 system and a bioorthogonal metabolic method to label the Japanese encephalitis virus (JEV) genome and envelopes with different-colored QDs in situ. This technique allows one-step two-color labeling of the viral envelope and intraviral genome with QDs harnessing virus infection. In combination with single-virus tracking, we visualized JEV uncoating and genome release in real time near the endoplasmic reticulum of live cells. This labeling strategy allows for real-time visualization of uncoating and genome release at the single-virus level, and it is expected to advance the study of other viral infection mechanisms.
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Puntos Cuánticos , Virosis , Virus , Humanos , Envoltura Viral/metabolismo , Proteínas del Envoltorio ViralRESUMEN
All protein-directed syntheses of metal nanoclusters (NCs) and nanoparticles (NPs) have attracted considerable attention because protein scaffolds provide a unique metal coordination environment and can adjust the shape and morphology of NCs and NPs. However, the detailed formation mechanisms of NCs or NPs directed by protein templates remain unclear. In this study, by taking advantage of the ferritin nanocage as a biotemplate to monitor the growth of Fe-O NCs as a function of time, we synthesized a series of iron NCs with different sizes and shapes and subsequently solved their corresponding three-dimensional atomic-scale structures by X-ray protein crystallography and cryo-electron microscopy. The time-dependent structure analyses revealed the growth process of these Fe-O NCs with the 4-fold channel of ferritin as nucleation sites. To our knowledge, the newly biosynthesized Fe35O23Glu12 represents the largest Fe-O NCs with a definite atomic structure. This study contributes to our understanding of the formation mechanism of iron NCs and provides an effective method for metal NC synthesis.
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Ferritinas , Tamaño de la Partícula , Ferritinas/química , Nanopartículas del Metal/química , Hierro/química , Modelos Moleculares , Cristalografía por Rayos X , Compuestos Férricos/químicaRESUMEN
The architecture of cell culture, two-dimensional (2D) versus three-dimensional (3D), significantly impacts various cellular factors, including cell-cell interactions, nutrient and oxygen gradients, metabolic activity, and gene expression profiles. This can result in different cellular responses during cancer drug treatment, with 3D-cultured cells often exhibiting higher resistance to chemotherapeutic drugs. While various genetic and proteomic analyses have been employed to investigate the underlying mechanisms of this increased resistance, complementary techniques that provide experimental evidence of spatial molecular profiling data are limited. Stimulated Raman scattering (SRS) microscopy has demonstrated its capability to measure both intracellular drug uptake and growth inhibition. In this work, we applied three-band (C-D, C-H, and fingerprint regions) SRS imaging to 2D and 3D cell cultures and performed a comparative analysis of drug uptake and response with the goal of understanding whether the difference in drug uptake explains the drug resistance in 3D culture compared to 2D. Our investigations revealed that despite similar intracellular drug levels in 2D and 3D A549 cells during lapatinib treatment, the growth of 3D spheroids was less impacted, supporting an enhanced drug tolerance in the 3D microenvironment. We further elucidated drug penetration patterns and the resulting heterogeneous cellular responses across different spheroid layers. Additionally, we investigated the role of the extracellular matrix in modulating drug delivery and cell response and discovered that limited drug penetration in 3D could also contribute to lower drug response. Our study provides valuable insights into the intricate mechanisms of increased drug resistance in 3D tumor models during cancer drug treatments.
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Epidermal growth factor receptor (EGFR) is a transmembrane protein commonly targeted by tyrosine kinase inhibitors (TKIs) as a front-line therapy for patients with many cancers including nonsmall cell lung cancer (NSCLC). Effective treatment requires efficient intracellular drug uptake and target binding. However, despite the recent success in the development of new TKI drugs, the mechanisms of uptake for many TKIs are still poorly understood due to the difficulty in imaging and measuring nonfluorescent drug molecules at a subcellular resolution. It has previously been shown that weakly basic TKI drugs are sequestered in lysosomes. Leveraging this property, we apply hyperspectral stimulated Raman scattering imaging to directly visualize and quantify two Food and Drug Administration-approved EGFR inhibitor drugs (lapatinib and afatinib) inside living cells and the changes in their cellular uptake upon the addition of organic cation transporter inhibitors. These single-cell quantitative measurements provide new insight into the role of membrane transporters in the uptake of TKI drugs in living cells.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Mutación , Receptores ErbB/metabolismo , Resistencia a AntineoplásicosRESUMEN
The ongoing Coronavirus Disease 2019 (COVID-19) pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) threatens global public health and economy unprecedentedly, requiring accelerating development of prophylactic and therapeutic interventions. Molecular understanding of neutralizing antibodies (NAbs) would greatly help advance the development of monoclonal antibody (mAb) therapy, as well as the design of next generation recombinant vaccines. Here, we applied H2L2 transgenic mice encoding the human immunoglobulin variable regions, together with a state-of-the-art antibody discovery platform to immunize and isolate NAbs. From a large panel of isolated antibodies, 25 antibodies showed potent neutralizing activities at sub-nanomolar levels by engaging the spike receptor-binding domain (RBD). Importantly, one human NAb, termed PR1077, from the H2L2 platform and 2 humanized NAb, including PR953 and PR961, were further characterized and subjected for subsequent structural analysis. High-resolution X-ray crystallography structures unveiled novel epitopes on the receptor-binding motif (RBM) for PR1077 and PR953, which directly compete with human angiotensin-converting enzyme 2 (hACE2) for binding, and a novel non-blocking epitope on the neighboring site near RBM for PR961. Moreover, we further tested the antiviral efficiency of PR1077 in the Ad5-hACE2 transduction mouse model of COVID-19. A single injection provided potent protection against SARS-CoV-2 infection in either prophylactic or treatment groups. Taken together, these results shed light on the development of mAb-related therapeutic interventions for COVID-19.
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Anticuerpos Neutralizantes/inmunología , COVID-19/virología , SARS-CoV-2/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Neutralizantes/ultraestructura , Anticuerpos Antivirales/inmunología , COVID-19/epidemiología , COVID-19/inmunología , COVID-19/metabolismo , Epítopos/inmunología , Humanos , Ratones , Ratones Transgénicos , Pruebas de Neutralización , Pandemias , Unión Proteica , Dominios Proteicos , Receptores Virales/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunologíaRESUMEN
BACKGROUND: In patients with hypertrophic cardiomyopathy (HCM), ischemic myocardial fibrosis assessed by late gadolinium enhancement (I-LGE) using cardiovascular magnetic resonance (CMR) have been reported. However, the clinical significance of I-LGE has not been completely understood. We aim to evaluate the I-LGE differ phenotypically from HCM without LGE or nonischemic myocardial fibrosis assessed by late gadolinium enhancement (NI-LGE) in the left ventricle (LV). METHODS: The patients with HCM whom was underwent CMR were enrolled, using cine cardiac magnetic resonance to evaluate LV function and LGE to detect the myocardial fibrosis. Three groups were assorted: 1) HCM without LGE; 2) HCM with LGE involved the subendocardial layer was defined as I-LGE; 3) HCM with LGE not involved the subendocardial layer was defined as NI-LGE. RESULTS: We enrolled 122 patients with HCM in the present study. LGE was detected in 58 of 122 (48%) patients with HCM, and 22 (18%) of patients reported I-LGE. HCM with I-LGE had increased higher left ventricular mass index (LVMI) (P < 0.0001) than HCM with NI-LGE or without LGE. In addition, HCM with I-LGE had a larger LV end- systolic volume (P = 0.045), lower LV ejection fraction (LVEF) (P = 0.026), higher LV myocardial mass (P < 0.001) and thicker LV wall (P < 0.001) more than HCM without LGE alone. The I-LGE were significantly associated with LVEF (OR: 0.961; P = 0.016), LV mass (OR: 1.028; P < 0.001), and maximal end-diastolic LVWT (OR: 1.567; P < 0.001). On multivariate analysis, LVEF (OR: 0.948; P = 0.013) and maximal end-diastolic LVWT (OR: 1.548; P = 0.001) were associated with higher risk for I-LGE compared to HCM without LGE. Noticeably, the maximal end-diastolic LVWT (OR: 1.316; P = 0.011) was the only associated with NI-LGE compared to HCM without LGE. CONCLUSIONS: I-LGE is not uncommon in patients with HCM. HCM with I-LGE was associated with significant LV hypertrophy, extensive LGE and poor LV ejection fraction. We should consider focal ischemic myocardial fibrosis when applying LGE to risk stratification for HCM.
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Cardiomiopatía Hipertrófica , Medios de Contraste , Humanos , Gadolinio , Imagen por Resonancia Cinemagnética , Cardiomiopatía Hipertrófica/diagnóstico , Miocardio/patología , Fibrosis , Espectroscopía de Resonancia MagnéticaRESUMEN
N-Isopropylbenzylamine (N-ipb), a chain isomer of methamphetamine (METH) with similar physical properties, has been used as a substitute for METH in seized drug samples. However, the abuse potential of N-ipb remains unclear. Therefore, this study aimed to evaluate the abuse potential of N-ipb in comparison to METH, by using conditioned place preference (CPP), locomotor sensitization and intravenous self-administration tests. The results showed that N-ipb at a dose of 3 mg·kg-1 significantly induced CPP in mice, which was comparable to the effect of METH at 1 mg·kg-1 . Either acute or repeated N-ipb injections (1 or 3 mg·kg-1 ) failed to raise the locomotor activity. However, acute treatment with 10 mg·kg-1 N-ipb elevated the locomotor activity compared with saline, while chronic injection of 10 mg·kg-1 N-ipb induced a delayed and attenuated sensitization compared with 1 mg·kg-1 METH. Rats could acquire N-ipb self-administration at a dose of 1 mg·kg-1 ·infusion-1 , and a typical inverted U-shaped dose-response curve was obtained for N-ipb. The mean dose of N-ipb that maintained the maximum response was greater than that of METH, indicating that N-ipb is less potent for reinforcement than METH. In the economic behavioural analysis, comparison of essential values derived from the demand elasticity revealed that N-ipb is less efficacy as a reinforcer than METH. The present data demonstrate that N-ipb functions as a reinforcer and has a potential for abuse. However, the potency of psychomotor stimulation and the reinforcing effectiveness of N-ipb are lower than those of METH.
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Aminas , Estimulantes del Sistema Nervioso Central , Metanfetamina , Ratones , Ratas , Animales , Estimulantes del Sistema Nervioso Central/farmacología , Roedores , Actividad Motora , Metanfetamina/farmacologíaRESUMEN
A planar conjugated ligand functionalized with bithiophene and its Ru(II), Os(II), and Ir(III) complexes have been constructed as single-molecule platform for synergistic photodynamic, photothermal, and chemotherapy. The complexes have significant two-photon absorption at 808â nm and remarkable singlet oxygen and superoxide anion production in aqueous solution and cells when exposed to 808â nm infrared irradiation. The most potent Ru(II) complex Ru7 enters tumor cells via the rare macropinocytosis, locates in both nuclei and mitochondria, and regulates DNA-related chemotherapeutic mechanisms intranuclearly including DNA topoisomerase and RNA polymerase inhibition and their synergistic effects with photoactivated apoptosis, ferroptosis and DNA cleavage. Ru7 exhibits high efficacy in vivo for malignant melanoma and cisplatin-resistant non-small cell lung cancer tumors, with a 100 % survival rate of mice, low toxicity to normal cells and low residual rate. Such an infrared two-photon activatable metal complex may contribute to a new generation of single-molecule-based integrated diagnosis and treatment platform to address drug resistance in clinical practice and phototherapy for large, deeply located solid tumors.
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Antineoplásicos , Complejos de Coordinación , Rayos Infrarrojos , Fotones , Tiofenos , Complejos de Coordinación/química , Complejos de Coordinación/farmacología , Complejos de Coordinación/síntesis química , Animales , Humanos , Antineoplásicos/farmacología , Antineoplásicos/química , Tiofenos/química , Tiofenos/farmacología , Ratones , Fotoquimioterapia , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/farmacología , Fármacos Fotosensibilizantes/síntesis química , Rutenio/química , Rutenio/farmacología , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Terapia Fototérmica , Iridio/química , Estructura Molecular , Apoptosis/efectos de los fármacosRESUMEN
Although neuronal Toll-like receptors (TLRs) (e.g., TLR2, TLR3, and TLR7) have been implicated in itch sensation, the roles of keratinocyte TLRs in chronic itch are elusive. Herein, we evaluated the roles of keratinocyte TLR2 and TLR7 in chronic itch under dry skin and psoriasis conditions, which was induced by either acetone-ether-water treatment or 5% imiquimod cream in mice, respectively. We found that TLR2 and TLR7 signaling were significantly upregulated in dry skin and psoriatic skin in mice. Chronic itch and epidermal hyperplasia induced by dry skin or psoriasis were comparably reduced in TLR2 and TLR7 knockout mice. In the dry skin model, the enhanced messenger RNA (mRNA) expression levels of pruritic CXCL1/2, IL-31, IL-33, ST2, IL-6, IL-17A, TNF-α, and IFN-γ were inhibited in TLR2-/- mice, while CXCL2, IL-31, and IL-6 were inhibited in TLR7-/- mice. In psoriasis model, the enhanced mRNA expression levels of pruritic CXCL1/2, IL-31, IL-33, ST2, IL-6, and TNF-α were inhibited in TLR2-/- mice, while CXCL1/2, IL-31, IL-33, ST2, IL-6, IL-17A, and TNF-α were inhibited in TLR7-/- mice. Incubation with Staphylococcus aureus (S. aureus) peptidoglycan (PGN-SA) (a TLR2 agonist), imiquimod (a TLR7 agonist), and miR142-3p (a putative TLR7 agonist) were sufficient to upregulate the expression of pruritic cytokines or chemokines in cultured keratinocyte HaCaT cells. Finally, pharmacological blockade of C-X-C Motif Chemokine Receptor 1/2 and high mobility group box protein 1 dose-dependently attenuated acute and chronic itch in mice. Together, these results indicate that keratinocyte TLR2 and TLR7 signaling pathways are distinctly involved in the pathogenesis of chronic itch.
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Quimiocinas , Citocinas , Prurito , Psoriasis , Receptor Toll-Like 2 , Receptor Toll-Like 7 , Animales , Ratones , Citocinas/metabolismo , Imiquimod/efectos adversos , Proteína 1 Similar al Receptor de Interleucina-1 , Interleucina-17 , Interleucina-33 , Interleucina-6 , Queratinocitos/metabolismo , Psoriasis/tratamiento farmacológico , ARN Mensajero , Receptor Toll-Like 2/genética , Receptor Toll-Like 7/genética , Factor de Necrosis Tumoral alfa/efectos adversos , Modelos Animales de Enfermedad , Ratones Noqueados , Células HaCaT , HumanosRESUMEN
Intratumoral heterogeneity is a substantial cause of drug resistance development during chemotherapy or other drug treatments for cancer. Therefore, monitoring and measuring cell exposure and response to drugs at the single-cell level are crucial. Previous research suggested that the single-cell growth rate can be used to investigate drug-cell interactions. However, currently established methods for quantifying single-cell growth are limited to isolated or monolayer cells. Here, we introduce a technique that accurately measures both 2D and 3D cell growth rates using label-free ratiometric stimulated Raman scattering (SRS) microscopy. We use deuterated amino acids, leucine, isoleucine, and valine, as tracers and measure the C-D SRS signal from deuterium-labeled proteins and the C-H SRS signal from unlabeled proteins simultaneously to determine the cell growth rate at the single-cell level. The technique offers single-cell level drug sensitivity measurement with a shorter turnaround time (within 12 h) than most traditional assays. The submicrometer resolution of the imaging technique allows us to examine the effects of chemotherapeutic drugs, including kinase inhibitors, mitotic inhibitors, and topoisomerase II inhibitors, on both the cell growth rate and morphology. The capability of quantifying 3D cell growth rates provides insight into a deeper understanding of the cell-drug interaction in the actual tumor environment.
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Microscopía , Proteínas , Microscopía/métodos , Proteínas/metabolismo , Aminoácidos , Proliferación Celular , Espectrometría RamanRESUMEN
Particles in biopharmaceutical products present high risks due to their detrimental impacts on product quality and safety. Identification and quantification of particles in drug products are important to understand particle formation mechanisms, which can help develop control strategies for particle formation during the formulation development and manufacturing process. However, existing analytical techniques such as microflow imaging and light obscuration measurement lack the sensitivity and resolution to detect particles with sizes smaller than 2 µm. More importantly, these techniques are not able to provide chemical information to determine particle composition. In this work, we overcome these challenges by applying the stimulated Raman scattering (SRS) microscopy technique to monitor the C-H Raman stretching modes of the proteinaceous particles and silicone oil droplets formed in the prefilled syringe barrel. By comparing the relative signal intensity and spectral features of each component, most particles can be classified as protein-silicone oil aggregates. We further show that morphological features are poor indicators of particle composition. Our method has the capability to quantify aggregation in protein therapeutics with chemical and spatial information in a label-free manner, potentially allowing high throughput screening or investigation of aggregation mechanisms.
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Agregado de Proteínas , Aceites de Silicona , Aceites de Silicona/química , Espectrometría Raman , Proteínas/química , Microscopía , Tamaño de la PartículaRESUMEN
BACKGROUND/AIM: Metformin is a drug for treating type 2 diabetes mellitus (T2DM). Recently, metformin has been shown to reduce the risks of asthma-associated outcomes and asthma deterioration, thereby holding promise as a superior medicine for diabetic patients with asthma. However, the mechanism by which metformin reduces diabetic asthma is yet to be clarified. This study aimed at ascertaining the downstream molecules underlying the effect of metformin on the activation of mast cells (MCs) and airway reactivity in a concomitant diabetic and asthmatic rat model. METHODS: A T2DM model was induced utilizing a high-fat diet and streptozotocin. Then, 10% ovalbumin was utilized to stimulate asthma-like pathology in the T2DM rats. RBL-2H3 cells were induced by anti-dinitrophenyl-specific immunoglobulin E for constructing an in vitro model. Luciferase assay and RNA immunoprecipitation (IP) assay were conducted to identify the interaction between microRNA-152-3p (miR-152-3p) and DNA methyltransferase 1 (DNMT1), while chromatin IP to identify the binding of DNMT1 to insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF-1R) promoters. The effects of metformin on both pathological changes in vivo and biological behaviors of cells were evaluated. Using gain- and loss-of-function approaches, we assessed the role of the two interactions in the metformin-induced effect. RESULTS: It was suggested that metformin could impede the MC activation and airway resistance in the concomitant diabetic and asthmatic rats. Additionally, metformin downregulated IR and IGF-1R through DNMT1-dependent methylation to repress MC activation and airway resistance. DNMT1 was testified to be a target gene of miR-152-3p. Furthermore, miR-152-3p-induced silencing of DNMT1 was blocked by metformin, hence restraining MC activation and airway resistance. CONCLUSION: The findings cumulatively demonstrate that metformin downregulates IR/IGF-1R to block MC activation and airway resistance via impairing the binding affinity between miR-152-3p and DNMT1.
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Asma , Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Metformina , MicroARNs , Ratas , Animales , MicroARNs/genética , MicroARNs/metabolismo , Metilación , Diabetes Mellitus Tipo 2/metabolismo , Metformina/farmacología , Receptor de Insulina/metabolismo , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Mastocitos/metabolismo , Asma/tratamiento farmacológico , Asma/genéticaRESUMEN
OBJECTIVE: To identify the additional diagnostic value of CNV-seq over conventional karyotyping on the part of chromosomal abnormalities in prenatal diagnosis. METHOD: This was a systematic review conducted in accordance with PRISMA criteria. In order to clarify related research, PubMed, Web of Science databases (including Core Collection, BIOSIS Previews, MEDLINE, and so on), The Cochrane Library and Wiley Online Library were searched with the terms: "prenatal diagnosis," "CNV-seq," "karyotyping," published from January 2010 to May 2022. No language restrictions. RenMan 5.4 was used for the meta-analysis. RESULTS: Eight studies were included in this systemic review and meta-analysis, including 11 091 pregnant women with high-risk pregnancy factors or with structurally abnormal fetus under ultrasound. CNV-seq detected a 2% (95% CI, -0% to 4%) additional chromosomal anomalies over conventional karyotyping in the six series. A 4% (95% CI, 3%-6%) pooled mean incremental yield of pathogenic CNVs by CNV-seq over karyotyping was observed, with a 1%-16% range. CONCLUSION: CNV-seq, applied in prenatal diagnosis, may detect more chromosomal abnormalities when compared with karyotyping. With the advantages of wide coverage, high throughput, high resolution, no culture, good compatibility, and adjustable sequencing depth, CNV-seq has high application value in prenatal diagnosis.
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Aberraciones Cromosómicas , Trastornos de los Cromosomas , Embarazo , Femenino , Humanos , Cariotipificación , Diagnóstico Prenatal , Trastornos de los Cromosomas/diagnóstico , Embarazo de Alto Riesgo , Variaciones en el Número de Copia de ADNRESUMEN
Bone is difficult to image using traditional histopathological methods, leading to challenges in intraoperative pathological evaluation that is critical in guiding surgical treatment, particularly in orthopedic oncology. In this study, we demonstrate that a multimodal quantitative imaging approach that combines stimulated Raman scattering (SRS) microscopy, two-photon fluorescence (TPF) microscopy, and second-harmonic generation (SHG) microscopy can provide useful diagnostic information regarding intact bone tissue fragments from surgical excision or biopsy specimens. We imaged bone samples from 17 patient cases and performed quantitative chemical and morphological analyses of both mineral and organic components of bone. Our main findings show that carbonate content combined with morphometric analysis of bone organic matrix can separate several major classes of bone cancer-associated diagnostic categories with an average accuracy of 92%. This proof-of-principle study demonstrates that quantitative multimodal imaging and machine learning-based analysis of bony tissue can provide crucial diagnostic information for guiding clinical decisions in orthopedic oncology. Moreover, the general methodology of morphological and chemical imaging combined with machine learning can be readily extended to other tissue types for tissue diagnosis in intraoperative and other clinical settings.
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Microscopía , Espectrometría Raman , Huesos/diagnóstico por imagen , Humanos , Imagen Multimodal , Fotones , Espectrometría Raman/métodosRESUMEN
Mammalian cell display technology uses eukaryotic protein expression system to display proteins on cell surfaces and has become an important method in biological research. Although mammalian cell display technology has many advantages and development potential, certain attributes of the displayed protein remain uncharacterized, such as whether the displayed proteins re-enter the cell and how displayed proteins move into the cell. Here, we present the endocytosis mechanism, motility behavior, and transport kinetics of displayed proteins determined using HaloTag as the displayed protein and quantum dot-based single-particle tracking. The displayed protein enters the cell through clathrin-mediated endocytosis and is transported through the cell in three stages, which is dependent on microfilaments and microtubules. The dynamic information obtained in this study provides answers to questions about endocytosis and postendocytosis transport of displayed proteins and, therefore, is expected to facilitate the development of surface display technology.
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Puntos Cuánticos , Citoesqueleto de Actina , Animales , Membrana Celular , Endocitosis , MamíferosRESUMEN
To explore the mechanism regarding the regulation of spinal cord ischemia (SCI) in rats by mild hypothermia. A SCI rat model was established through aorta occlusion, and in some cases, the rats were intervened with mild hypothermia, after which motor function, microglia activation, and M1/M2 polarization in rats were measured. Also, the expression of inflammatory cytokines (IL-1ß, IL-6 and TNF-α) and neuronal apoptosis were examined. Lipopolysaccharide (LPS)-induced M1 microglia and IL-4-induced M2 microglia were intrathecally injected into rats to evaluate the effect of microglial polarization on SCI. In in vitro experiments, primary microglial cells were treated under hypothermic condition, in which M1/M2 polarization and microglia apoptosis, the levels of iNOS, CD86, CD206, Arg-1 and inflammatory cytokines were assessed. Western blot analysis detected the activation of the TLR4/NF-κB pathway to investigate the role of this pathway in M1/M2 polarization. SCI treatment impaired motor function, induced higher M1 microglia proportion, and increased the levels of pro-inflammatory cytokines in rats, and mild hypothermic treatment attenuated these trends. Moreover, injection of M1 microglia increased M1 microglia proportion and increased the levels of pro-inflammatory cytokines, while injection of M2 microglia induced the reverse results, i.e. decreased M1 microglia proportion and reduced pro-inflammatory cytokine levels. In LPS-induced microglial cells, mild hypothermia treatment increased M2 microglia proportion and decreased pro-inflammatory cytokine levels, relative to normothermia. Mild hypothermia inactivated the TLR4/NF-κB pathway in LPS-treated microglia. TLR4 overexpression reversed the function of mild hypothermia in LPS-stimulated microglia, and under normal condition, TLR4/NF-κB pathway suppressed microglial M2 polarization. Mild hypothermia inhibits TLR4/NF-κB pathway and promotes microglial M2 polarization, thus attenuating SCI-induced injury and inflammation.
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Hipotermia , Traumatismos de la Médula Espinal , Isquemia de la Médula Espinal , Animales , Hipotermia/metabolismo , Microglía/metabolismo , Parálisis/metabolismo , Ratas , Traumatismos de la Médula Espinal/complicaciones , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/terapia , Isquemia de la Médula Espinal/terapiaRESUMEN
An Ag-catalysed three-component reaction of alkynyl aryl ketones bearing an ortho-methoxy group, element selenium, and arylboronic acid, providing a facile route to selenofunctionalized chromone products has been developed. This protocol features high efficiency and high regioselectivity, and the use of selenium powder as the selenium source. Mechanistic experiments indicated that the combined oxidative effect of (bis(trifluoroacetoxy)iodo)benzene and oxygen in the air pushes the catalytic redox cycle of the Ag catalyst and the phenylselenium trifluoroacetate formed in situ is the key intermediate of the PIFA-mediated 6-endo-electrophilic cyclization and selenofunctionalization reaction of alkynyl aryl ketones.
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Cetonas , Selenio , Ácidos Borónicos , Ciclización , PlataRESUMEN
OBJECTIVE: Atherosclerosis (AS) is an inflammatory disease and the formation of atherosclerotic plaque plays a critical role in AS progression. We aimed to investigate the effect of long non-coding RNA (lncRNA) activated by DNA damage (NORAD)/microRNA-495-3p (miR-495-3p)/Krüppel-like factor 5 (KLF5) axis on atherosclerotic plaque formation. METHODS: The ApoE-/- mice were fed a high-fat diet to construct AS mouse models and the modeled mice were treated with altered NORAD, miR-495-3p or KLF5. NORAD, miR-495-3p and KLF5 expression in mouse aorta tissues were evaluated, and the levels of inflammatory factors, oxidative stress factors, endothelial function indices and blood lipid in mice were all determined. The atherosclerotic plaque area, lipid deposition area, collagen fibers and CD68 expression in mouse aorta tissues were assessed. The regulatory relation between NORAD and miR-495-3p, and the target relation between miR-495-3p and KLF5 were confirmed. RESULTS: NORAD and KLF5 were increased whereas miR-495-3p was decreased in atherosclerotic mouse aortas. Inhibited NORAD or elevated miR-495-3p suppressed inflammation, oxidative stress, endothelial dysfunction, blood lipid level, atherosclerotic plaque area, collagen fibers and CD68 expression in atherosclerotic mouse aortas. Effects of elevated miR-495-3p on atherosclerotic mice could be reversed by up-regulation of KLF5. NORAD served as a sponge of miR-495-3p and miR-495-3p directly targeted KLF5. CONCLUSION: Silenced NORAD elevated miR-495-3p to suppress atherosclerotic plaque formation via reducing KLF5. Findings in our research may be helpful for exploring molecular mechanisms of AS.
Asunto(s)
Apolipoproteínas E/genética , Aterosclerosis/genética , Factores de Transcripción de Tipo Kruppel/genética , MicroARNs/genética , ARN Largo no Codificante/genética , Animales , Aterosclerosis/patología , Daño del ADN/genética , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/genética , Humanos , Inflamación/genética , Inflamación/patología , Ratones , Ratones Noqueados , Placa Aterosclerótica/genética , Placa Aterosclerótica/patologíaRESUMEN
PURPOSE: The aim of this study was to identify trends and focuses in the field of Fuchs endothelial corneal dystrophy (FECD) research. METHODS: A bibliometric analysis based on the Web of Science Core Collection was conducted. All publications related to FECD from 2001 to 2020 were extracted and analyzed. VOSviewer v.1.6.17 was used to construct a visualization map and evaluate the trends and focuses in FECD research. RESULTS: A total of 1,041 publications were extracted. The rate of global publications has steadily increased. The United States produced the highest number of publications (461), the highest number of citations (18,757), and the highest H index (69). Melles GRJ published the highest number of papers (60), and Price FW had the highest number of citations (4,154) in the FECD research field. The highest number of publications came from the journal Cornea (279). Keywords were classified into four clusters: (1) corneal transplantation surgery, (2) surgical techniques and instruments, (3) corneal parameter measurement, and (4) genetic and molecular pathomechanisms. The average appearing years (AAYs) of the keywords were evaluated. Recently appearing keywords included "Tcf4 gene" (AAY of 2018.3), "ctg18.1" (AAY of 2017.2), "trinucleotide repeat expansion" (AAY of 2018.3), "rock inhibitor" (AAY of 2017.4), and "descemetorhexis" (AAY of 2017.4). CONCLUSIONS: The United States has a dominant position in FECD research. Although corneal transplantation surgery has been the most mainstream area of FECD research field for a long time, gene mutations such as the TCF4 CTG trinucleotide repeat expansion, nonsurgical interventions such as rho-associated kinase inhibitors, and newer surgical methods such as descemetorhexis without endothelial keratoplasty are potential research hotspots.