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Label-Free Digital Holo-tomographic Microscopy Reveals Virus-Induced Cytopathic Effects in Live Cells
摘要： Cytopathic effects (CPEs) are a hallmark of infections. CPEs are difficult to observe due to phototoxicity from classical light microscopy. We report distinct patterns of virus infections in live cells using digital holo-tomographic microscopy (DHTM). DHTM is label-free and records the phase shift of low-energy light passing through the specimen on a transparent surface with minimal perturbation. DHTM measures the refractive index (RI) and computes the refractive index gradient (RIG), unveiling optical heterogeneity in cells. We find that vaccinia virus (VACV), herpes simplex virus (HSV), and rhinovirus (RV) infections progressively and distinctly increased RIG. VACV infection, but not HSV and RV infections, induced oscillations of cell volume, while all three viruses altered cytoplasmic membrane dynamics and induced apoptotic features akin to those caused by the chemical compound staurosporine. In sum, we introduce DHTM for quantitative label-free microscopy in infection research and uncover virus type-specific changes and CPE in living cells with minimal interference.
关键词： tomography,virus infection,apoptosis,herpes simplex virus,live-cell microscopy,membrane blebbing,cell volume,refractive index,cell contraction,rhinovirus,label-free microscopy,vaccinia virus
更新于2025-09-09 09:28:46
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Role of C/EBP Homologous Protein in Retinal Ganglion Cell Death After Ischemia/Reperfusion Injury
摘要： To investigate the role of C/EBP homologous protein (CHOP), a proapoptotic protein, and the unfolded protein response (UPR) marker that is involved in endoplasmic reticulum (ER) stress-mediated apoptosis in mouse retinal ganglion cell (RGC) death following ischemia/reperfusion (I/R) injury.
关键词： apoptosis,retinal ischemia,CHOP,retinal ganglion cell
更新于2025-09-04 15:30:14
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Knockdown of FOXO6 inhibits high glucose–induced oxidative stress and apoptosis in retinal pigment epithelial cells
摘要： Oxidative stress and apoptosis in retinal pigment epithelium cells are involved in the pathogenesis of diabetic retinopathy (DR). Forkhead box class O 6 (FOXO6) is a member of the FOXO family that can regulate diabetes‐induced oxidative stress. However, the role of FOXO6 in DR has not been clarified. The aim of the present study was to investigate the effects of FOXO6 on high glucose (HG)‐induced oxidative stress and apoptosis in ARPE‐19 cells. The results showed that FOXO6 was overexpressed in clinical vitreous samples from DR patients and in HG‐induced ARPE‐19 cells. Knockdown of FOXO6 by small interfeing RNA targeting FOXO6 (si‐FOXO6) mitigated the HG‐induced the production of reactive oxygen species and malondialdehyde, as well as the inhibition of superoxide dismutase activity. Knockdown of FOXO6 reduced the rate of cell apoptosis in HG‐induced ARPE‐19 cells. The increase in bax expression and decrease in bcl‐2 expression caused by HG stimulation were reversed by si‐FOXO6 transfection. Furthermore, knockdown of FOXO6 enhanced the activation of Akt/Nrf2 pathway in HG‐stimulated ARPE‐19 cells. Taken together, suppression of FOXO6 protects ARPE‐19 cells from HG‐induced oxidative stress and apoptosis, which is in part mediated by the activation of Akt/Nrf2 pathway.
关键词： Akt/Nrf2 pathway,oxidative stress,retinal pigment epithelium cells,forkhead box class O 6,diabetic retinopathy,apoptosis
更新于2025-09-04 15:30:14
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Pyropheophorbide-α methyl ester-mediated photodynamic therapy induces apoptosis and inhibits LPS-induced inflammation in RAW264.7 macrophages
摘要： Backgroud: This study aimed to determine the effect of pyropheophorbide-α methyl ester (MPPa)-mediated photodynamic therapy (MPPa-PDT) on the apoptosis and inflammation of murine macrophage RAW264.7 cells. Methods: Uptake and subcellular localization of MPPa was detected by ?ow cytometry and confocal fluorescence microscope. Cell viability was assessed by CCK-8; ROS levels were assessed by DCFH-DA. Cell apoptosis was measured by ?ow cytometry and Hoechst 33342 staining, whereas mitochondrial membrane potential was detected by JC-1 staining. Secretion of tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) was determined using ELISA kits. Caspase-3, cleaved caspase-3, procaspase-9, cleaved caspase-9, PARP, cleaved PARP, Bcl-2, Bax, NF-κB p-p65, p-IKKα/β, and p-IκBα were measured by western blotting. Nuclear factor κB (NF-κB)-p65 nuclear translocation was observed by immunofluorescence. Results: MPPa -PDT influenced cell viability in a light dose-dependent manner. It induced ROS formation and RAW264.7 cell apoptosis. It also increased the expression of cleaved caspase-3, cleaved caspase-9, cleaved PARP and Bax, decreased the expression of Bcl-2. While TNF-α, IL-1β, and IL-6 increased in LPS group (model of inflammation), it deceased in LPS-MPPa-PDT group. NF-κB p-p65, p-IKKα/β, and p-IκBα had higher expression in LPS group while that reduced in LPS-MPPa-PDT group. Simultaneously, MPPa-PDT inhibited nuclear translocation of NF-κB-p65 caused by LPS. Conclusions: MPPa-PDT can induce apoptosis and attenuate inflammation in mouse RAW264.7 macrophages, thereby suggesting a promising therapy for atherosclerosis.
关键词： inflammation,atherosclerosis,MPPa-PDT,RAW264.7,apoptosis
更新于2025-09-04 15:30:14
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Reduced graphene oxide decorated with gold nanoparticle as signal amplification element on ultra-sensitive electrochemiluminescence determination of caspase-3 activity and apoptosis using peptide based biosensor
摘要： Introduction: Growing demands for ultrasensitive biosensing have led to the development of numerous signal amplification strategies. In this report, a novel electrochemiluminescence (ECL) method was developed for the detection and determination of caspase-3 activity based on reduced graphene oxide sheets decorated by gold nanoparticles as signal amplification element and horseradish peroxidase enzyme (HRP) as ECL intensity enhancing agent. Methods: The ECL intensity of the luminol was improved by using the streptavidin coated magnetic beads and HRP in the presence of hydrogen peroxide. The cleavage behavior of caspase-3 was characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques using biotinylated peptide (DEVD containing peptide) which was coated on reduced graphene oxide decorated with gold nanoparticle. The surface modification of graphene oxide was successfully confirmed by FTIR, UV-vis and x-ray spectroscopy. Results: ECL based biosensor showed that the linear dynamic range (LDR) and the lower limit of quantification (LLOQ) were 0.5-100 and 0.5 femtomolar (fM), respectively. Finally, the performance of the engineered peptide based biosensor was validated in the A549 cell line as real samples. Conclusion: The prepared peptide based biosensor could be considered as an excellent candidate for early detection of apoptosis, cell turnover, and cancer related diseases.
关键词： DEVD-peptide,Electrochemiluminescence,Apoptosis,Reduced graphene oxide,Caspase-3,Gold nanoparticle
更新于2025-09-04 15:30:14
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Evaluation of apoptotic cell death mechanisms induced by hypericin-mediated photodynamic therapy in colon cancer cells
摘要： Hypericin (HYP)-mediated photodynamic therapy (PDT) is a new alternative treatment strategy for colon cancer inducing various cell-death pathways. Apoptotic cell death is the desired cellular fate in cancer cells. Therefore, we investigated the apoptotic pathways by determining apoptosis-related proteins (survivin, caspase-3, caspase-9, Bcl-2, and Bax) at the mRNA level using real-time polymerase chain reaction (qPCR), and the percentage of survivin was determined by survivin ELISA in HT-29 and Caco-2 colon cancer cell lines. The downregulation of survivin was significant 16 h after PDT for both cells, while caspase-3 upregulation was apparent 24 h after PDT. Caspase-9 and caspase-3 upregulations were parallel to each other. There was no Bcl-2 expression in HT-29 cells, but we observed downregulation in Bcl-2 expression in Caco-2 cells after HYP photoactivation. The Bax expression downregulated significantly after 24 h incubation in HT-29 cells, while it was upregulated after 16 h incubation in Caco-2 cells. The present study provides evidence that HYP-induced alterations in apoptosis-related protein expression ended in different responses in HT-29 and Caco-2 colon cancer cells, and these may be used in the treatment of chemotherapy-resistant cancer types.
关键词： survivin,apoptosis,HT-29 cells,Caco-2 cells,PDT,Hypericin
更新于2025-09-04 15:30:14
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Nickel Nanowires Combined with Surface-Enhanced Raman Spectroscopy: Application in Label-Free Detection of Cytochrome c-Mediated Apoptosis
摘要： Intrinsic properties of nickel have enabled its wide applications as an effective catalyst. In this study, nickel nanowires (Ni NWs) as electron donors for oxidized cytochrome c (Cyt c) are investigated, which are NW diameter, temperature, and pH value-dependent. The reductive and magnetic properties facilitate the Ni NWs to rapidly and conveniently reduce Cyt c in complicated biological samples. Moreover, we find that the Ni NWs combined with resonance Raman spectroscopy have specificity towards Cyt c detection in real biological samples, which is successfully used to distinguish the redox state of the released Cyt c from isolated mitochondria in apoptotic Hela cells. Moreover, rapid label-free Cyt c quantification can be achieved by surface-enhanced Raman spectroscopy with a limit of detection range of 1 nM and long concentration linear (1nM?1μM). The proposed Ni NWs-based reduction approach will significantly simplify the traditional biological methods and has great potential in the application of Cyt c-related apoptotic studies.
关键词： nickel nanowires,surface-enhanced Raman spectroscopy,cytochrome c,label-free detection,apoptosis
更新于2025-09-04 15:30:14
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Mitochondrial Fission Is Required for Blue Light-Induced Apoptosis and Mitophagy in Retinal Neuronal R28 Cells
摘要： Light emitting diodes (LEDs) are widely used to provide illumination due to their low energy requirements and high brightness. However, the LED spectrum contains an intense blue light component which is phototoxic to the retina. Recently, it has been reported that blue light may directly impinge on mitochondrial function in retinal ganglion cells (RGCs). Mitochondria are high dynamic organelles that undergo frequent fission and fusion events. The aim of our study was to elucidate the role of mitochondrial dynamics in blue light-induced damage in retinal neuronal R28 cells. We found that exposure to blue light (450 nm, 1000 lx) for up to 12 h significantly up-regulated the expression of mitochondrial fission protein Drp1, while down-regulating the expression of mitochondrial fusion protein Mfn2 in cells. Mitochondrial fission was simultaneously stimulated by blue light irradiation. In addition, exposure to blue light increased the production of reactive oxygen species (ROS), disrupted mitochondrial membrane potential (MMP), and induced apoptosis in R28 cells. Notably, Drp1 inhibitor Mdivi-1 and Drp1 RNAi not only attenuated blue light-induced mitochondrial fission, but also alleviated blue light-induced ROS production, MMP disruption and apoptosis in cells. Compared with Mdivi-1 and Drp1 RNAi, the antioxidant N-acetyl-L-cysteine (NAC) only slightly inhibited mitochondrial fission, while significantly alleviating apoptosis after blue light exposure. Moreover, we examined markers for mitophagy, which is responsible for the clearance of dysfunctional mitochondria. It was found that blue light stimulated the conversion of LC3B-I to LC3B-II as well as the expression of PINK1 in R28 cells. Mdivi-1 or Drp1 RNAi efficiently inhibited the blue light-induced expression of PINK1 and co-localization of LC3 with mitochondria. Thus, our data suggest that mitochondrial fission is required for blue light-induced mitochondrial dysfunction and apoptosis in RGCs.
关键词： mitophagy,apoptosis,mitochondrial fission,blue light,retinal neuronal cells
更新于2025-09-04 15:30:14