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RNA-Seq analysis revealed the molecular mechanisms of photobiomodulation effect on human fibroblasts
摘要： Background The Photobiomodulation (PBM) effect has been applied to various clinical therapy for a long time. However, the mechanism related to the PBM effect in terms of wavelengths has been lack of in-depth study, except that ultraviolet radiation has attracted much attention due to its strong cell killing effect. Purpose To clarify the principle behind PBM and the main mechanism of improvement. Methods To carry on this study, we created light equipment using three LED chips, which emit 390 nm ultraviolet radiation, 415 nm blue light and 660 nm red light, respectively. We choose human fibroblasts (HF) to be irradiated by three different wavelengths for PBM test. In this study, we used cell counting kit (CCK-8) test to show the cell proliferation roughly and reported on a systematic RNA sequencing (RNA-seq) analysis at transcriptional expression levels from HF, which accepted PBM of different wavelengths of light. Results We found that 415 nm blue light inhibited cell proliferation and 660 nm red light stimulated cell proliferation while 390 nm ultraviolet radiation has little influence on cell proliferation. Furthermore, RNA-seq results showed that CSF1R, PPP3CC, ITGAL, ITGAM, IL2RB and several other differentially expressed genes (DEGs) are involved in the cell proliferation. Relative DEGs values for matrix metalloproteinases (MMPs) gene family has shown a great difference in blue and red light radiation especially on MMP25, MMP9, MMP21 and MMP13. Conclusion Taken together, the results provide a valuable resource to describe the variation of HFs under PBM of different light at gene level.
关键词： MMPs,RNA-Seq,Human fibroblasts(HF),Photobiomodulation (PBM),LED
更新于2025-09-23 15:19:57
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Optofluidic real-time cell sorter for longitudinal CTC studies in mouse models of cancer
摘要： Circulating tumor cells (CTCs) play a fundamental role in cancer progression. However, limited blood volume and the rarity of CTCs in the bloodstream preclude longitudinal, in-depth studies of these cells using existing liquid biopsy techniques. Here, we present an optofluidic system that continuously collects fluorescently labeled CTCs from a genetically engineered mouse model (GEMM) for several hours per day over multiple days or weeks. The system is based on a microfluidic cell sorting chip connected serially to an unanesthetized mouse via an implanted arteriovenous shunt. Pneumatically controlled microfluidic valves capture CTCs as they flow through the device, and CTC-depleted blood is returned back to the mouse via the shunt. To demonstrate the utility of our system, we profile CTCs isolated longitudinally from animals over 4 days of treatment with the BET inhibitor JQ1 using single-cell RNA sequencing (scRNA-Seq) and show that our approach eliminates potential biases driven by intermouse heterogeneity that can occur when CTCs are collected across different mice. The CTC isolation and sorting technology presented here provides a research tool to help reveal details of how CTCs evolve over time, allowing studies to credential changes in CTCs as biomarkers of drug response and facilitating future studies to understand the role of CTCs in metastasis.
关键词： single-cell RNA-Seq,metastasis,circulating tumor cells,GEMM,microfluidic
更新于2025-09-19 17:15:36
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Integrated Analysis of Transcriptomic and Metabolomic Data Reveals the Mechanism by Which LED Light Irradiation Extends the Postharvest Quality of Pak-choi (Brassica campestris L. ssp. chinensis (L.) Makino var. communis Tsen et Lee)
摘要： Low-intensity (10 μmol m?2 s?1) white LED (light-emitting diode) light effectively delayed senescence and maintained the quality of postharvest pakchoi during storage at 20 °C. To investigate the mechanism of LED treatment in maintaining the quality of pakchoi, metabolite profiles reported previously were complemented by transcriptomic profiling to provide greater information. A total of 7761 differentially expressed genes (DEGs) were identified in response to the LED irradiation of pak-choi during postharvest storage. Several pathways were markedly induced by LED irradiation, with photosynthesis being the most notable. More specifically, porphyrin and chlorophyll metabolism and glucosinolate biosynthesis were significantly induced by LED irradiation, which is consistent with metabolomics reported previously. Additionally, chlorophyllide a, chlorophyll, as well as total glucosinolate content was positively induced by LED irradiation. Overall, LED irradiation delayed the senescence of postharvest pak-choi mainly by activating photosynthesis, inducting glucosinolate biosynthesis, and inhibiting the down-regulation of porphyrin and chlorophyll metabolism pathways. The present study provides new insights into the effect and the underlying mechanism of LED irradiation on delaying the senescence of pak-choi. LED irradiation represents a useful approach for extending the shelf life of pak-choi.
关键词： postharvest quality,Pak-choi,LED irradiation,RNA-seq,metabolomics
更新于2025-09-19 17:13:59
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Transcriptome Profile Alteration with Cadmium Selenide/Zinc Sulfide Quantum Dots in Saccharomyces cerevisiae
摘要： Quantum Dots (QDs) are becoming more prevalent in products used in our daily lives, such as TVs and laptops, due to their unique and tunable optical properties. The possibility of using QDs as fluorescent probes in applications, such as medical imaging, has been a topic of interest for some time, but their potential toxicity and long-term effects on the environment are not well understood. In the present study, we investigated the effects of yellow CdSe/ZnS-QDs on Saccharomyces cerevisiae. We utilized growth assays, RNA-seq, reactive oxygen species (ROS) detection assays, and cell wall stability experiments to investigate the potential toxic effects of CdSe/ZnS-QDs. We found CdSe/ZnS-QDs had no negative effects on cell viability; however, cell wall-compromised cells showed more sensitivity in the presence of 10 μg/mL CdSe/ZnS-QDs compared to non-treated cells. In CdSe/ZnS-treated and non-treated cells, no significant change in superoxide was detected, but according to our transcriptomic analysis, thousands of genes in CdSe/ZnS-treated cells became differentially expressed. Four significantly differentiated genes found, including FAF1, SDA1, DAN1, and TIR1, were validated by consistent results with RT-qPCR assays. Our transcriptome analysis led us to conclude that exposure of CdSe/ZnS-QDs on yeast significantly affected genes implicated in multiple cellular processes.
关键词： yeast,QDs,gene expression,toxicity,CdSe/ZnS,RNA-seq
更新于2025-09-16 10:30:52
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A protocol for laser microdissection (LMD) followed by transcriptome analysis of plant reproductive tissue in phylogenetically distant angiosperms
摘要： Background: Plant development is controlled by the action of many, often connected gene regulatory networks. Differential gene expression controlled by internal and external cues is a major driver of growth and time specific differentiation in plants. Transcriptome analysis is the state-of-the-art method to detect spatio-temporal changes in gene expression during development. Monitoring changes in gene expression at early stages or in small plant organs and tissues requires an accurate technique of tissue isolation, which subsequently results in RNA of sufficient quality and quantity. Laser-microdissection enables such accurate dissection and collection of desired tissue from sectioned material at a microscopic level for RNA extraction and subsequent downstream analyses, such as transcriptome, proteome, genome or miRNA. Results: A protocol for laser-microdissection, RNA extraction and RNA-seq was optimized and verified for three distant angiosperm species: Arabidopsis thaliana (Brassicaceae), Oryza sativa (Poaceae) and Eschscholzia californica (Papaveraceae). Previously published protocols were improved in processing speed by reducing the vacuum intensity and incubation time during tissue fixation and incubation time and cryoprotection and by applying adhesive tape. The sample preparation and sectioning of complex and heterogenous flowers produced adequate histological quality and subsequent RNA extraction from micro-dissected gynoecia reliably generated samples of sufficient quality and quantity on all species for RNA-seq. Expression analysis of growth stage specific A. thaliana and O. sativa transcriptomes showed distinct patterns of expression of chromatin remodelers on different time points of gynoecium morphogenesis from the initiation of development to post-meiotic stages. Conclusion: Here we describe a protocol for plant tissue preparation, cryoprotection, cryo-sectioning, laser microdissection and RNA sample preparation for Illumina sequencing of complex plant organs from three phyletically distant plant species. We are confident that this approach is widely applicable to other plant species to enable transcriptome analysis with high spatial resolution in non-model plant species. The protocol is rapid, produces high quality sections of complex organs and results in RNA of adequate quality well suited for RNA-seq approaches. We provide detailed description of each stage of sample preparation with the quality and quantity measurements as well as an analysis of generated transcriptomes.
关键词： Non-model species,Cryosectioning,Evo-devo,Development,RNA-seq,Laser microdissection (LMD)
更新于2025-09-12 10:27:22
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Gene expression profiling of single cells from archival tissue with laser-capture microdissection and Smart-3SEQ
摘要： RNA sequencing (RNA-seq) is a sensitive and accurate method for quantifying gene expression. Small samples or those whose RNA is degraded, such as formalin-fixed paraffin-embedded (FFPE) tissue, remain challenging to study with nonspecialized RNA-seq protocols. Here, we present a new method, Smart-3SEQ, that accurately quantifies transcript abundance even with small amounts of total RNA and effectively characterizes small samples extracted by laser-capture microdissection (LCM) from FFPE tissue. We also obtain distinct biological profiles from FFPE single cells, which have been impossible to study with previous RNA-seq protocols, and we use these data to identify possible new macrophage phenotypes associated with the tumor microenvironment. We propose Smart-3SEQ as a highly cost-effective method to enable large gene expression profiling experiments unconstrained by sample size and tissue availability. In particular, Smart-3SEQ’s compatibility with FFPE tissue unlocks an enormous number of archived clinical samples; combined with LCM it allows unprecedented studies of small cell populations and single cells isolated by their in situ context.
关键词： laser-capture microdissection,single-cell gene expression,RNA-seq,tumor microenvironment,FFPE tissue,Smart-3SEQ
更新于2025-09-11 14:15:04