A novel ESIPT fluorescent probe for early detection and assessment of ferroptosis-mediated acute kidney injury via peroxynitrite fluctuation.

BACKGROUND: Acute kidney injury (AKI) poses a severe risk to public health, mostly manifested by damage and death of renal tubular epithelial cells. However, routine blood examination, a conventional approach for clinical detection of AKI, is not available for identifying early-stage AKI. Plenty of reported methods were lack of early biomarkers and real time evaluation tools, which resulted in a vital challenge for early diagnosis of AKI. Therefore, developing novel probes for early detection and assessment of AKI is exceedingly crucial. RESULTS: Based on ESIPT mechanism, a new fluorescent probe (MEO-NO) with 2-(2'-hydroxyphenyl) benzothiazole (HBT) derivatives as fluorophore has been synthesized for dynamic imaging peroxynitrite (ONOO-) levels in ferroptosis-mediated AKI. Upon the addition of ONOO-, MEO-NO exhibited obvious fluorescence changes, a significant Stokes shift (130 nm) and rapid response (approximately 45 s), and featured exceptional sensitivity (LOD = 7.28 nM) as well as high selectivity from the competitive species at physiological pH. In addition, MEO-NO was conducive to the biological depth imaging ONOO- in cells, zebrafish, and mice. Importantly, MEO-NO could monitor ONOO- levels during sorafenib-induced ferroptosis and CP-induced AKI. With the assistance of MEO-NO, we successfully visualized and tracked ONOO- variations for early detection and assessment of ferroptosis-mediated AKI in cells, zebrafish and mice models. SIGNIFICANCE AND NOVELTY: Benefiting from the superior performance of MEO-NO, experimental results further demonstrated that the levels of ONOO- was overexpressed during ferroptosis-mediated AKI in cells, zebrafish, and mice models. The developed novel probe MEO-NO provided a strong visualization tool for imagining ONOO-, which might be a potential method for the prevention, diagnosis, and treatment of ferroptosis-mediated AKI.

YAP upregulates AMPKα1 to induce cancer cell senescence.

Yes-associated protein (YAP)-a major effector protein of the Hippo pathway- regulates cell proliferation, differentiation, apoptosis, and senescence. Amp-activated protein kinase (AMPK) is a key sensor that monitors cellular nutrient supply and energy status. Although YAP and AMPK are considered to regulate cellular senescence, it is still unclear whether AMPK is involved in YAP-regulated cellular senescence. Here, we found that YAP promoted AMPKα1 aggregation and localization around mitochondria by co-transfecting CFP-YAP and YFP-AMPKα1 plasmids. Subsequent live cell fluorescence resonance energy transfer (FRET) assay did not exhibit direct interaction between YAP and AMPKα1. FRET, Co-immunoprecipitation, and western blot experiments revealed that YAP directly bound to TEAD, enhancing the expression of AMPKα1 and p-AMPKα. Treatment with verteporfin inhibited YAP's binding to TEAD and reversed the elevated expression of AMPKα1 in the cells overexpressing CFP-YAP. Verteporfin also reduced the proportion of AMPKα1 puncta in the cells co-expressing CFP-YAP and YFP-AMPKα1. In addition, the AMPKα1 puncta were demonstrated to inhibit cell viability, autophagy, and proliferation, and ultimately promote cell senescence. In conclusion, YAP binds to TEAD to upregulate AMPKα1 and promotes the formation of AMPKα1 puncta around mitochondria under the condition of co-expression of CFP-YAP and YFP-AMPKα1, in which AMPKα1 puncta lead to cellular senescence.

S-acylation of a non-secreted peptide controls plant immunity via secreted-peptide signal activation.

Small peptides modulate multiple processes in plant cells, but their regulation by post-translational modification remains unclear. ROT4 (ROTUNDIFOLIA4) belongs to a family of Arabidopsis non-secreted small peptides, but knowledge on its molecular function and how it is regulated is limited. Here, we find that ROT4 is S-acylated in plant cells. S-acylation is an important form of protein lipidation, yet so far it has not been reported to regulate small peptides in plants. We show that this modification is essential for the plasma membrane association of ROT4. Overexpression of S-acylated ROT4 results in a dramatic increase in immune gene expression. S-acylation of ROT4 enhances its interaction with BSK5 (BRASSINOSTEROID-SIGNALING KINASE 5) to block the association between BSK5 and PEPR1 (PEP RECEPTOR1), a receptor kinase for secreted plant elicitor peptides (PEPs), thereby activating immune signaling. Phenotype analysis indicates that S-acylation is necessary for ROT4 functions in pathogen resistance, PEP response, and the regulation of development. Collectively, our work reveals an important role for S-acylation in the cross-talk of non-secreted and secreted peptide signaling in plant immunity.

Aurora kinase A-mediated phosphorylation triggers structural alteration of Rab1A to enhance ER complexity during mitosis.

Morphological rearrangement of the endoplasmic reticulum (ER) is critical for metazoan mitosis. Yet, how the ER is remodeled by the mitotic signaling remains unclear. Here, we report that mitotic Aurora kinase A (AURKA) employs a small GTPase, Rab1A, to direct ER remodeling. During mitosis, AURKA phosphorylates Rab1A at Thr75. Structural analysis demonstrates that Thr75 phosphorylation renders Rab1A in a constantly active state by preventing interaction with GDP-dissociation inhibitor (GDI). Activated Rab1A is retained on the ER and induces the oligomerization of ER-shaping protein RTNs and REEPs, eventually triggering an increase of ER complexity. In various models, from Caenorhabditis elegans and Drosophila to mammals, inhibition of Rab1AThr75 phosphorylation by genetic modifications disrupts ER remodeling. Thus, our study reveals an evolutionarily conserved mechanism explaining how mitotic kinase controls ER remodeling and uncovers a critical function of Rab GTPases in metaphase.

Suramin, an antiparasitic drug, stimulates adipocyte differentiation and promotes adipogenesis.

BACKGROUND: Previous studies demonstrated that mast cells with their degranulated component heparin are the major endogenous factors that stimulate preadipocyte differentiation and promote fascial adipogenesis, and this effect is related to the structure of heparin. Regarding the structural and physiological properties of the negatively charged polymers, hexasulfonated suramin, a centuries-old medicine that is still used for treating African trypanosomiasis and onchocerciasis, is assumed to be a heparin-related analog or heparinoid. This investigation aims to elucidate the influence of suramin on the adipogenesis. METHODS: To assess the influence exerted by suramin on adipogenic differentiation of primary white adipocytes in rats, this exploration was conducted both in vitro and in vivo. Moreover, it was attempted to explore the role played by the sulfonic acid groups present in suramin in mediating this adipogenic process. RESULTS: Suramin demonstrated a dose- and time-dependent propensity to stimulate the adipogenic differentiation of rat preadipocytes isolated from the superficial fascia tissue and from adult adipose tissue. This stimulation was concomitant with a notable upregulation in expression levels of pivotal adipogenic factors as the adipocyte differentiation process unfolded. Intraperitoneal injection of suramin into rats slightly increased adipogenesis in the superficial fascia and in the epididymal and inguinal fat depots. PPADS, NF023, and NF449 are suramin analogs respectively containing 2, 6, and 8 sulfonic acid groups, among which the last two moderately promoted lipid droplet formation and adipocyte differentiation. The number and position of sulfonate groups may be related to the adipogenic effect of suramin. CONCLUSIONS: Suramin emerges as a noteworthy pharmaceutical agent with the unique capability to significantly induce adipocyte differentiation, thereby fostering adipogenesis.

Tea consumption and risk of lung diseases: a two­sample Mendelian randomization study.

BACKGROUND: Numerous studies have reported the association between tea intake and lung diseases. However, the probable relationship between tea consumption on lung diseases still remain controversial and it is unclear whether these findings are due to reverse causality or confounding factor. METHODS: In order to systematically investigate the causal connection between tea intake on respiratory system disorders, we employed a two-sample Mendelian randomized (MR) study. Genetic instruments for tea intake were identified from a genome-wide association study (GWAS) involving 447,385 individuals. Data on lung diseases were collected from a variety of publicly available genome-wide association studies. The main method used for MR analysis is the inverse variance weighting (IVW) method. To ensure the accuracy of the findings, further sensitivity analysis was conducted. RESULTS: The IVW method in our MR analysis revealed no evidence to support a causal relationship between tea intake and lung diseases (IPF: OR = 0.997, 95% CI = 0.994-1.000, p = 0.065; Lung cancer: OR = 1.003, 95% CI = 0.998-1.008, P = 0.261; COPD: OR = 1.001, 95% CI = 0.993-1.006, p = 0.552; acute bronchitis: OR = 0.919, 95% CI = 0.536-1.576, p = 0.759; tuberculosis: OR = 1.002, 95% CI = 0.998-1.008, p = 0.301; pneumonia: OR = 0.789, 95% CI = 0.583-1.068, p = 0.125). The reliability of the results was further demonstrated by four additional MR analysis techniques and additional sensitivity testing. CONCLUSION: We found no evidence of a link between tea intake on lung diseases in our MR results based on genetic information.

Dependence of ghost on the incident light angle into dichroic mirror.

The dichroic mirror (DM) is a key component in microscope. We found a ghost in the reflection channel of a dual-channel fluorescence microscope and studied the relationship between the ghost and the incidence angle θ into the DM. The DM emission surface reflection generated ghost if the θ is not 45 ° . We analyzed the distance and intensity relationship between the ghost and the primary image, which is θ -dependent and was demonstrated by imaging live cells and a stage micrometer. The ghost can be eliminated by placing the DM between objective and tube lens, but not between tube lens and detector, ensuring that the incident light into the DM is approximately parallel. Furthermore, the transmitted light of the DM is shifted towards a longer wavelength with increasing θ . Collectively, microscopists must carefully optimize the θ when designing a microscope to avoid the ghost.

Superresolution live-cell imaging reveals that the localization of TMEM106B to filopodia in oligodendrocytes is compromised by the hypomyelination-related D252N mutation.

Hypomyelination leukodystrophies constitute a group of heritable white matter disorders exhibiting defective myelin development. Initially identified as a lysosomal protein, the TMEM106B D252N mutant has recently been associated with hypomyelination. However, how lysosomal TMEM106B facilitates myelination and how the D252N mutation disrupts that process are poorly understood. We used superresolution Hessian structured illumination microscopy (Hessian-SIM) and spinning disc-confocal structured illumination microscopy (SD-SIM) to find that the wild-type TMEM106B protein is targeted to the plasma membrane, filopodia, and lysosomes in human oligodendrocytes. The D252N mutation reduces the size of lysosomes in oligodendrocytes and compromises lysosome changes upon starvation stress. Most importantly, we detected reductions in the length and number of filopodia in cells expressing the D252N mutant. PLP1 is the most abundant myelin protein that almost entirely colocalizes with TMEM106B, and coexpressing PLP1 with the D252N mutant readily rescues the lysosome and filopodia phenotypes of cells. Therefore, interactions between TMEM106B and PLP1 on the plasma membrane are essential for filopodia formation and myelination in oligodendrocytes, which may be sustained by the delivery of these proteins from lysosomes via exocytosis.

Rapid, artifact-reduced, image reconstruction for super-resolution structured illumination microscopy.

Super-resolution structured illumination microscopy (SR-SIM) is finding increasing application in biomedical research due to its superior ability to visualize subcellular dynamics in living cells. However, during image reconstruction artifacts can be introduced and when coupled with time-consuming postprocessing procedures, limits this technique from becoming a routine imaging tool for biologists. To address these issues, an accelerated, artifact-reduced reconstruction algorithm termed joint space frequency reconstruction-based artifact reduction algorithm (JSFR-AR-SIM) was developed by integrating a high-speed reconstruction framework with a high-fidelity optimization approach designed to suppress the sidelobe artifact. Consequently, JSFR-AR-SIM produces high-quality, super-resolution images with minimal artifacts, and the reconstruction speed is increased. We anticipate this algorithm to facilitate SR-SIM becoming a routine tool in biomedical laboratories.

Experimental study of 131I-caerin 1.1 and 131I-c(RGD)2 for internal radiation therapy of esophageal cancer xenografts.

OBJECTIVE: The aim of this study was to analyze and compare the therapeutic effects of 131I-caerin 1.1 and 131I-c(RGD)2 on TE-1 esophageal cancer cell xenografts. METHODS: (1) The in vitro antitumor effects of the polypeptides caerin 1.1 and c(RGD)2 were verified by MTT and clonogenic assays. 131I-caerin 1.1 and 131I-c(RGD)2 were prepared by chloramine-T (Ch-T) direct labeling, and their basic properties were measured. The binding and elution of 131I-caerin 1.1, 131I-c(RGD)2, and Na131I (control group) in esophageal cancer TE-1 cells were studied through cell binding and elution assays. (2) The antiproliferative effect and cytotoxicity of 131I-caerin 1.1, 131I-c(RGD)2, Na131I, caerin 1.1 and c(RGD)2 on TE-1 cells were detected by Cell Counting Kit-8 (CCK-8) assay. (3) A nude mouse esophageal cancer (TE-1) xenograft model was established to study and compare the efficacy of 131I-caerin 1.1 and 131I-c(RGD)2 in internal radiation therapy for esophageal cancer. RESULTS: (1) Caerin 1.1 inhibited the in vitro proliferation of TE-1 cells in a concentration-dependent manner, with an IC50 of 13.00 µg/mL. The polypeptide c(RGD)2 had no evident inhibitory effect on the in vitro proliferation of TE-1 cells. Therefore, the antiproliferative effects of caerin 1.1 and c(RGD)2 on esophageal cancer cells were significantly different (P < 0.05). The clonogenic assay showed that the clonal proliferation of TE-1 cells decreased as the concentration of caerin 1.1 increased. Compared with the control group (drug concentration of 0 µg/mL), the caerin 1.1 group showed significantly lower clonal proliferation of TE-1 cells (P < 0.05). (2) The CCK-8 assay showed that 131I-caerin 1.1 inhibited the in vitro proliferation of TE-1 cells, while 131I-c(RGD)2 had no evident inhibitory effect on proliferation. The two polypeptides showed significantly different antiproliferative effects on esophageal cancer cells at higher concentrations (P < 0.05). Cell binding and elution assays showed that 131I-caerin 1.1 stably bound to TE-1 cells. The cell binding rate of 131I-caerin 1.1 was 15.8 % ± 1.09 % at 24 h and 6.95 % ± 0.22 % after 24 h of incubation and elution. The cell binding rate of 131I-c(RGD)2 was 0.06 % ± 0.02 % at 24 h and 0.23 % ± 0.11 % after 24 h of incubation and elution. (3) In the in vivo experiment, 3 days after the last treatment, the tumor sizes of the phosphate-buffered saline (PBS) group, caerin 1.1 group, c(RGD)2 group, 131I group, 131I-caerin 1.1 group, and 131I-c(RGD)2 group were 68.29 ± 2.67 mm3, 61.78 ± 3.58 mm3, 56.67 ± 5.65 mm3, 58.88 ± 1.71 mm3, 14.40 ± 1.38 mm3, and 60.14 ± 0.47 mm3, respectively. Compared with the other treatment groups, the 131I-caerin 1.1 group had significantly smaller tumor sizes (P < 0.001). After treatment, the tumors were isolated and weighed. The tumor weights in the PBS group, caerin 1.1 group, c(RGD)2 group, 131I group, 131I-caerin 1.1 group, and 131I-c(RGD)2 group were 39.50 ± 9.54 mg, 38.25 ± 5.38 mg, 38.35 ± 9.53 mg, 28.25 ± 8.50 mg, 9.50 ± 4.43 mg, and 34.75 ± 8.06 mg, respectively. The tumor weights in the 131I-caerin 1.1 group were significantly lighter than those in the other groups (P < 0.01). CONCLUSION: 131I-caerin 1.1 has tumor-targeting properties, is capable of targeted binding to TE-1 esophageal cancer cells, can be stably retained in tumor cells, and has an evident cytotoxic killing effect, while 131I-c(RGD)2 has no evident cytotoxic effect. 131I-caerin 1.1 better suppressed tumor cell proliferation and tumor growth than pure caerin 1.1, 131I-c(RGD)2, and pure c(RGD)2.

Quantitative structured illumination microscopy via a physical model-based background filtering algorithm reveals actin dynamics.

Despite the prevalence of superresolution (SR) microscopy, quantitative live-cell SR imaging that maintains the completeness of delicate structures and the linearity of fluorescence signals remains an uncharted territory. Structured illumination microscopy (SIM) is the ideal tool for live-cell SR imaging. However, it suffers from an out-of-focus background that leads to reconstruction artifacts. Previous post hoc background suppression methods are prone to human bias, fail at densely labeled structures, and are nonlinear. Here, we propose a physical model-based Background Filtering method for living cell SR imaging combined with the 2D-SIM reconstruction procedure (BF-SIM). BF-SIM helps preserve intricate and weak structures down to sub-70 nm resolution while maintaining signal linearity, which allows for the discovery of dynamic actin structures that, to the best of our knowledge, have not been previously monitored.

Correlative Escherichia coli Transcription Rate and Bubble Conformation Remodeled by NusA and NusG.

Transcription is highly regulated by a variety of transcription factors, among which NusA and NusG act contradictorily in Escherichia coli (E. coli) that NusA stabilizes a paused RNA polymerase (RNAP) and NusG suppresses it. The mechanism of the NusA and NusG regulations on RNAP transcription has been addressed, but their effect on the conformational changes of the transcription bubble correlated with transcription kinetics remains elusive. By using single-molecule magnetic trap, we identify a reduction in the transcription rate of ∼40% events by NusA. Although the rest ∼60% of transcription events exhibit unaffected transcription rates, a NusA-enhanced standard deviation of the transcription rate is observed. NusA remodeling also increases the extent of DNA unwinding in the transcription bubble by 1-2 base pairs, which can be reduced by NusG. The NusG remodeling is more significant on the RNAP molecules with reduced transcription rates rather than those without. Our results provide a quantitative view on the mechanisms of transcriptional regulation by NusA and NusG factors.

Regorafenib induces Bim-mediated intrinsic apoptosis by blocking AKT-mediated FOXO3a nuclear export.

Regorafenib (REGO) is a synthetic oral multi-kinase inhibitor with potent antitumor activity. In this study, we investigate the molecular mechanisms by which REGO induces apoptosis. REGO induced cytotoxicity, inhibited the proliferation and migration ability of cells, and induced nuclear condensation, and reactive oxygen species (ROS)-dependent apoptosis in cancer cells. REGO downregulated PI3K and p-AKT level, and prevented FOXO3a nuclear export. Most importantly, AKT agonist (SC79) not only inhibited REGO-induced FOXO3a nuclear localization and apoptosis but also restored the proliferation and migration ability of cancer cells, further demonstrating that REGO prevented FOXO3a nuclear export by deactivating PI3K/AKT. REGO treatment promotes Bim expression via the FOXO3a nuclear localization pathway following PI3K/AKT inactivation. REGO induced Bim upregulation and translocation into mitochondria as well as Bim-mediated Bax translocation into mitochondria. Fluorescence resonance energy transfer (FRET) analysis showed that REGO enhanced the binding of Bim to Bak/Bax. Knockdown of Bim, Bak and Bax respectively almost completely inhibited REGO-induced apoptosis, demonstrating the key role of Bim by directly activating Bax/Bak. Knockdown of Bax but not Bak inhibited REGO-induced Drp1 oligomerization in mitochondria. In conclusion, our data demonstrate that REGO promotes apoptosis via the PI3K/AKT/FOXO3a/Bim-mediated intrinsic pathway.

SVM classifier of cervical histopathology images based on texture and morphological features.

BACKGROUND: Cervical histopathology image classification is a crucial indicator in cervical biopsy results. OBJECTIVE: The objective of this study is to identify histopathology images of cervical cancer at an early stage by extracting texture and morphological features for the Support Vector Machine (SVM) classifier. METHODS: We extract three different texture features and one morphological feature of cervical histopathology images: first-order histogram, K-means clustering, Gray Level Co-occurrence Matrix (GLCM) and nucleus feature. The original dataset used in our experiment is obtained from 20 patients diagnosed with cervical cancer, including 135 whole slide images (WSIs). Given an entire WSI, the patches on its tissue region are extracted randomly. RESULTS: We finally obtain 3,000 patches, including 1,000 normal, 1,000 hysteromyoma and 1,000 cancer images. Among them, 80% of the entire data set is randomly selected as training set and the remaining 20% as test set. The accuracy of SVM classification using first-order histogram, K-means clustering, GLAM and nucleus feature for extracting features are respectively 87.4%, 90.6%, 91.6% and 93.5%. CONCLUSIONS: The classification accuracy of the SVM combining the four features is 96.8%, and the proposed nucleus feature plays a key role in the SVM classification of cervical histopathology images.

Bad is essential for Bcl-xL-enhanced Bax shuttling between mitochondria and cytosol.

Although Bcl-xL has been shown to retrotranslocate Bax from mitochondria to cytosol, other studies have found that Bcl-xL also stabilizes the mitochondrial localization of Bax. It is still unclear what causes the difference in Bcl-xL-regulated Bax localization. Bad, a BH3-only protein with a high affinity for Bcl-xL, may play an important role in Bcl-xL-regulated Bax shuttling. Here, we found that Bcl-xL enhanced both translocalization and retrotranslocation of mitochondrial Bax, as evidenced by quantitative co-localization, western blots and fluorescence loss in photobleaching (FLIP) analyses. Notably, Bad knockdown prevented Bcl-xL-mediated Bax retrotranslocation, indicating Bad was essential for this process. Quantitative fluorescence resonance energy transfer (FRET) imaging in living cells and co-immunoprecipitation analyses showed that the interaction of Bcl-xL with Bad was stronger than that with Bax. The Bad mimetic ABT-737 dissociated Bax from Bcl-xL on isolated mitochondria, suggesting that mitochondrial Bax was directly liberated to cytosol due to Bad binding to Bcl-xL. In addition, MK-2206, an Akt inhibitor, decreased Bad phosphorylation while increasing cytosolic Bax proportion. Our data firmly demonstrate a notion that Bad binds to mitochondrial Bcl-xL to release Bax, resulting in retrotranslocation of Bax to cytosol, and that the amount of Bad involved is regulated by Akt signaling.

Live-cell imaging analysis on the anti-apoptotic function of the Bcl-xL transmembrane carboxyl terminal domain.

The Transmembrane Carboxyl Terminal Domain (TMD) of some Bcl-2 family proteins has been demonstrated to play a key role in modulating apoptosis. We here ustilzed live-cell fluorescence imaging to evaluate how the Bcl-xL TMD (XT) regulate apoptosis. Cell viability assay revealed that XT had strong anti-apoptotic ability similarly to the full-length Bcl-xL. Fluorescence images of living cells co-expressing CFP-XT and Bad-YFP or YFP-Bax revealed that XT recruited Bad to mitochondria but prevented Bax translocation to mitochondria, and also significantly suppressed Bad/Bax-mediated apoptosis, indicating that XT prevents the pro-apoptotic function of Bad and Bax. Fluorescence Resonance Energy Transfer (FRET) analyses determined that XT directly interacted with Bad and Bax, and deletion of XT completely eliminated the mitochondrial localization and homo-oligomerization of Bcl-xL. Fluorescence images of living cells co-expressing CFP-XT and YFP-Bax revealed that XT significantly prevented mitochondrial Bax oligomerization, resulting in cytosolic Bax distribution. Collectively, XT is necessary for the mitochondrial localization and anti-apoptotic capacity of Bcl-xL, and XT, similarly to the full-length Bcl-xL, forms homo-oligomers on mitochondria to directly interact with Bad and Bax to inhibit their apoptotic functions.

Sirt3 activates autophagy to prevent DOX-induced senescence by inactivating PI3K/AKT/mTOR pathway in A549 cells.

Sirtuin 3 (Sirt3), a mitochondrial deacetylase, regulates mitochondrial redox homeostasis and autophagy and is involved in physiological and pathological processes such as aging, cellular metabolism, and tumorigenesis. We here investigate how Sirt3 regulates doxorubicin (DOX)-induced senescence in lung cancer A549 cells. Sirt3 greatly reduced DOX-induced upregulation of senescence marker proteins p53, p16, p21 and SA-ß-Gal activity as well as ROS levels. Notably, Sirt3 reversed DOX-induced autophagic flux blockage, as shown by increased p62 degradation and LC3II/LC3I ratio. Importantly, the autophagy inhibitors 3-methyladenine (3-MA) and chloroquine (CQ) partially abolished the antioxidant stress and antiaging effects of Sirt3, while the autophagy activator rapamycin (Rap) potentiated these effects of Sirt3, demonstrating that autophagy mediates the anti-aging effects of Sirt3. Additionally, Sirt3 inhibited the DOX-induced activation of the phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway, which in turn activated autophagy. The PI3K inhibitor LY294002 promoted the antioxidant stress and antiaging effects of Sirt3, while the AKT activator SC-79 reversed these effects of Sirt3. Taken together, Sirt3 counteracts DOX-induced senescence by improving autophagic flux.

Live-cell super-resolution imaging unconventional dynamics and assemblies of nuclear pore complexes.

Super-resolution microscopy has promoted the development of cell biology, but imaging proteins with low copy numbers in cellular structures remains challenging. The limited number of designated proteins within nuclear pore complexes (NPCs) impedes continuous observation in live cells, although they are often used as a standard for evaluating various SR methods. To address this issue, we tagged POM121 with Halo-SiR and imaged it using structured illumination microscopy with sparse deconvolution (Sparse-SIM). Remarkably, POM121-SiR exhibited more than six-fold fluorescence intensity and four-fold enhanced contrast compared to the same protein labeled with tandem-linked mCherry, while showing negligible photo-bleaching during SR imaging for 200 frames. Using this technique, we discovered various types of NPCs, including ring-like and cluster-like structures, and observed dynamic remodeling along with the sequential appearance of different Nup compositions. Overall, Halo-SiR with Sparse-SIM is a potent tool for extended SR imaging of dynamic structures of NPCs in live cells, and it may also help visualize proteins with limited numbers in general.

Im-SCC-FRET: improved single-cell-based calibration of a FRET system.

We recently developed a SCC-FRET (single-cell-based calibration of a FRET system) method to quantify spectral crosstalk correction parameters (ß and δ) and system calibration parameters (G and k) of a Förster resonance energy transfer (FRET) system by imaging a single cell expressing a standard FRET plasmid with known FRET efficiency (E) and donor-acceptor concentration ratio (RC) (Liu et al., Opt. Express30, 29063 (2022)10.1364/OE.459861). Here we improved the SCC-FRET method (named as Im-SCC-FRET) to simultaneously obtain ß, δ, G, k and the acceptor-to-donor extinction coefficient ratio (ε A ε D), which is a key parameter to calculate the acceptor-centric FRET efficiency (EA), of a FRET system when the range of ß and δ values is set as 0-1. In Im-SCC-FRET, the target function is changed from the sum of absolute values to the sum of squares according to the least squares method, and the initial value of ß and δ estimated by the integral but not the maximum value spectral overlap between fluorophore and filter. Compared with SCC-FRET, the experimental results demonstrate that Im-SCC-FRET can obtain more accurate and stable results for ß, δ, G, and k, and add the ratio ε A ε D, which is necessary for the FRET hybrid assay. Im-SCC-FRET reduces the complexity of experiment preparation and opens up a promising avenue for developing an intelligent FRET correction system.

Mathematical analysis for spatial distribution of vessels, mast cells and adipocytes in superficial fascia.

As a novel origin of adipocytes, the superficial fascia, a typical soft connective tissue, has abundant adipocytes and preadipocytes, accompanied by numerous mast cells. Blood vessels pass through the fascia to form a network structure. The more reasonable statistical analysis methods can provide a new method for in-depth study of soft connective tissue by clarifying the spatial distribution relation between cells (point structure) and blood vessels (linear structure). This study adopted the Guidolin et al. statistical analysis methods used by epidemiology and ecology to quantitatively analyze the distribution pattern and correlations among blood vessels, adipocytes, and mast cells. Image-processing software and self-written computer programs were used to analyze images of whole-mounted fascia, and the relevant data were measured automatically. Voronoi's analysis revealed that the vascular network was non-uniformly distributed. In fascia with average area of 3.75 cm2, quantitative histological analysis revealed 81.16% of mast cells and 74.74% of adipocytes distributed within 60 µm of blood vessels. A Spearman's correlation coefficient (rs) of >0.7 showed the co-distribution of the two types of cells under different areas. Ridge regression analysis further revealed the spatial correlation among blood vessels, adipocytes and mast cells. The combination of classical epidemiological analysis and extended computer program analysis can better analyze the spatial distribution relation between cells and vessels and should provide an effective analysis method for study of the histology and morphology of fascia and related connective tissues.