Pregnancy-induced changes to the gut microbiota drive macrophage pyroptosis and exacerbate septic inflammation.

The physiological and immune changes that occur during pregnancy are associated with worsened disease outcomes during infection and sepsis. How these perturbations exacerbate inflammation has not been explored. Here, using antibiotic treatment and fecal microbial transfers, we showed that sepsis susceptibility is driven by pregnancy-induced changes to gut microbiome in mice and humans. Integrative multiomics and genetically engineered bacteria revealed that reduced Parabacteroides merdae (P. merdae) abundance during pregnancy led to decreased formononetin (FMN) and increased macrophage death. Mechanistically, FMN inhibited macrophage pyroptosis by suppressing nuclear accumulation of hnRNPUL2 and subsequent binding to the Nlrp3 promoter. Treatment with FMN or deletion of murine hnRNPUL2 protected against septic inflammation. Intestinal abundances of P. merdae and FMN inversely correlated with the progression of septic patients. Our data reveal a microbe-immune axis that is disrupted in pregnant septic hosts, highlighting the potential of the FMN-hnRNPUL2-NLRP3 axis in providing promising therapeutic strategies for sepsis.

Architecture-driven quantitative nanoscopy maps cytoskeleton remodeling.

Cytoskeleton remodeling which generates force and orchestrates signaling and trafficking to govern cell migration remains poorly understood, partly due to a lack of an investigation tool with high system flexibility, spatiotemporal resolution, and computational sensitivity. Herein, we developed a multimodal superresolution imaging system-based architecture-driven quantitative (ADQ) framework in spatiotemporal-angular hyperspace to enable both identification of the optimal imaging mode with well-balanced fidelity and phototoxicity and accurate postcharacterization of microtubule remodeling. In the ADQ framework, a pixel/voxel-wise metric reflecting heterogeneous intertubule alignment was proposed with improved sensitivity over previous efforts and further incorporated with temporal features to map dynamic microtubule rearrangements. The ADQ framework was verified by assessing microtubule remodeling in drug-induced (de)polymerization, lysosome transport, and migration. Different remodeling patterns from two migration modes were successfully revealed by the ADQ framework, with a front-rear polarization for individual directed migration and a contact site-centered polarization for cell-cell interaction-induced migration in an immune response model. Meanwhile, these migration modes were found to have consistent orientation changes, which exhibited the potential of predicting migration trajectory.

Untrained neural network enabling fast and universal structured-illumination microscopy.

Structured-illumination microscopy (SIM) offers a twofold resolution enhancement beyond the optical diffraction limit. At present, SIM requires several raw structured-illumination (SI) frames to reconstruct a super-resolution (SR) image, especially the time-consuming reconstruction of speckle SIM, which requires hundreds of SI frames. Considering this, we herein propose an untrained structured-illumination reconstruction neural network (USRNN) with known illumination patterns to reduce the amount of raw data that is required for speckle SIM reconstruction by 20 times and thus improve its temporal resolution. Benefiting from the unsupervised optimizing strategy and CNNs' structure priors, the high-frequency information is obtained from the network without the requirement of datasets; as a result, a high-fidelity SR image with approximately twofold resolution enhancement can be reconstructed using five frames or less. Experiments on reconstructing non-biological and biological samples demonstrate the high-speed and high-universality capabilities of our method.

A bio-inspired co-simulation crawling robot enabled by a carbon dot-doped dielectric elastomer.

Flexible actuation materials play a crucial role in biomimetic robots. Seeking methods to enhance actuation and functionality is one of the directions in which actuators strive to meet the high-performance and diverse requirements of environmental conditions. Herein, by utilizing the method of adsorbing N-doped carbon dots (NCDs) onto SiO2 to form clusters of functional particles, a NCDs@SiO2/PDMS elastomer was prepared and its combined optical and electrical co-stimulation properties were effectively harnessed to develop a biomimetic crawling robot resembling Rhagophthalmus (firefly). The introduction of NCDs@SiO2 cluster particles not only effectively improves the mechanical and dielectric properties of the elastomer but also exhibits fluorescence response and actuation response under the co-stimulation of UV and electricity, respectively. Additionally, a hybrid dielectric elastomer actuator (DEA) with a transparent SWCNT mesh electrode exhibits two notable advancements: an 826% increase in out-of-plane displacement under low electric field stimulation compared to the pure matrix and the ability of NCDs to maintain a stable excited state within the polymer for an extended duration under UV-excitation. Simultaneously, the transparent biomimetic crawling robot can stealthily move in specific environments and fluoresce under UV light.

Exploration of novel 20S proteasome activators featuring anthraquinone structures and their application in hypoxic cardiomyocyte protection.

Under hypoxic conditions, the accumulation of misfolded proteins primarily relies on the autonomous activity of 20S proteasome for degradation. The buildup of toxic proteins in cardiomyocyte contribute to various cardiovascular diseases. Therefore, enhancing the 20S proteasome degradation capacity and restoring protein homeostasis in myocardial cells with small molecule activators represent a promising therapeutic strategy for the treatment of ischemic cardiomyopathy. In this study, the lead compound 8016-8398 was identified through virtual screening, and subsequent structure optimization resulted in a series of highly potent 20S proteasome activators. Intracellular protein degradation assessment revealed that these compounds possessed abilities to alleviate endoplasmic reticulum stress, as demonstrated by the luciferase reporter system. Additionally, selected compound B-03 significantly enhanced the survival rate of hypoxic-damaged cardiomyocytes. Mechanistic investigations verified B-03 rescued hypoxic damaged cardiomyocyte through apoptosis inhibition and proliferation promotion.

Role of VPS39, a key tethering protein for endolysosomal trafficking and mitochondria-lysosome crosstalk, in health and disease.

The coordinated interaction between mitochondria and lysosomes, mainly manifested by mitophagy, mitochondria-derived vesicles, and direct physical contact, is essential for maintaining cellular life activities. The VPS39 subunit of the homotypic fusion and protein sorting complex could play a key role in the regulation of organelle dynamics, such as endolysosomal trafficking and mitochondria-vacuole/lysosome crosstalk, thus contributing to a variety of physiological functions. The abnormalities of VPS39 and related subunits have been reported to be involved in the pathological process of some diseases. Here, we analyze the potential mechanisms and the existing problems of VPS39 in regulating organelle dynamics, which, in turn, regulate physiological functions and disease pathogenesis, so as to provide new clues for facilitating the discovery of therapeutic targets for mitochondrial and lysosomal diseases.

Alternative deep learning method for fast spatial-frequency shift imaging microscopy.

Spatial-frequency shift (SFS) imaging microscopy can break the diffraction limit of fluorescently labeled and label-free samples by transferring the high spatial-frequency information into the passband of microscope. However, the resolution improvement is at the cost of decreasing temporal resolution since dozens of raw SFS images are needed to expand the frequency spectrum. Although some deep learning methods have been proposed to solve this problem, no neural network that is compatible to both labeled and label-free SFS imaging has been proposed. Here, we propose the joint spatial-Fourier channel attention network (JSFCAN), which learns the general connection between the spatial domain and Fourier frequency domain from complex samples. We demonstrate that JSFCAN can achieve a resolution similar to the traditional algorithm using nearly 1/4 raw images and increase the reconstruction speed by two orders of magnitude. Subsequently, we prove that JSFCAN can be applied to both fluorescently labeled and label-free samples without architecture changes. We also demonstrate that compared with the typical spatial domain optimization network U-net, JSFCAN is more robust to deal with deep-SFS images and noisy images. The proposed JSFCAN provides an alternative route for fast SFS imaging reconstruction, enabling future applications for real-time living cell research.

High-throughput multiplexed fluorescence lifetime microscopy.

Fluorescence lifetime microscopy has been widely used in quantifying cellular interaction or histopathological identification of different stained tissues. A novel, to the best of our knowledge, approach for high-throughput multiplexed fluorescence lifetime imaging is presented. To establish a high-throughput fluorescence lifetime acquisition system, a uniformed illumination optical focus array was generated by a novel computer-generated hologram algorithm based on matrix triple product. This, in conjunction with an array detector and multichannel time-correlated single-photon counting, enables the full use of the acquisition ability of each detector. By utilizing interval segmentation of photon time detection, a high-throughput multiplexed fluorescence lifetime imaging is achieved. Experimental results demonstrate that this method achieves a fivefold increase in the collection throughput of fluorescence lifetime and is capable of simultaneous dual-target fluorescence lifetime measurement.

High-speed spatially re-modulated structured illumination microscopy.

Structured illumination microscopy (SIM) allows non-invasive visualization of nanoscale subcellular structures. However, image acquisition and reconstruction become the bottleneck to further improve the imaging speed. Here, we propose a method to accelerate SIM imaging by combining the spatial re-modulation principle with Fourier domain filtering and using measured illumination patterns. This approach enables high-speed, high-quality imaging of dense subcellular structures using a conventional nine-frame SIM modality without phase estimation of the patterns. In addition, seven-frame SIM reconstruction and additional hardware acceleration further improve the imaging speed using our method. Furthermore, our method is also applicable to other spatially uncorrelated illumination patterns, such as distorted sinusoidal, multifocal, and speckle patterns.

Enhanced axial resolution of lattice light sheet microscopy by fluorescence differential detection.

Lattice light-sheet microscopy (LLSM) is promising in long-term biological volumetric imaging due to its high spatiotemporal resolution and low phototoxicity. However, three-dimensional (3D) isotropic spatial resolution remains an unmet goal in LLSM because of its poorer axial resolution. Combing LLSM with fluorescence differential detection, namely LLSDM, has been proposed to improve the axial resolution of LLSM in simulation. It demonstrates the possibility of further enhancing the axial resolution in 3D volumetric imaging with LLSM by specifically discarding the off-focus photons captured using a complementary optical lattice (OL) profile generated with additional 0-π phase modulation at the objective pupil plane. The direct generation of the complementary lattice profile using the binary phase modulator conjugated to the sample plane for amplitude modulation, as used in LLSM, is also permittable. Nevertheless, the previously proposed configuration fails to provide a symmetric complementary lattice pattern along the axial axis, thus leading to the imbalanced off-focus photon suppression in the reconstructed images after subtraction [Opt. Lett.45, 2854 (2020)10.1364/OL.393378]. Here, we modified the LLSDM theory which can produce an ideal complementary lattice pattern with central zero intensity and symmetrically distributed sidelobes. We also analyzed the impact of numerical aperture matching between the original and complementary lattice patterns and presented the consistency between the simulated and experimental results. As demonstrated by imaging the distribution of fluorescent beads and microtubules in fixed U2OS cells, as well as the dynamics of filopodia in live U2OS cells, LLSDM provides about 1.5 times improvement in axial resolution, and higher imaging contrast compared with traditional LLSM.

Ratiometric photon reassignment based on fluorescence lifetime to improve resolution in pulse STED microscopy.

Depletion beams with long pulse durations (∼600ps) have recently been applied in stimulated emission depletion (STED) microscopy to reduce photobleaching and phototoxicity. Therefore, improving the resolution of pulse STED at lower depletion power for the super-resolution imaging of live biological specimens has attracted increasing interest. Herein, we present a simple method termed ratiometric photon reassignment based on the fluorescence lifetime, in which highly spatially resolved long-lifetime fluorophore components in the center are extracted, and short-lifetime fluorophore components in the periphery are discarded to improve resolution without increasing the depletion power. The experimental results demonstrate improved resolution and signal-to-noise ratio compared with traditional time-gated STED. Our proposed method requires lower budget and data processing because, in contrast to the separation of photons by lifetime tuning), lifetime measurements are not required.

Quantitative objective-based ring TIRFM system calibration through back focal plane imaging.

Being the established imaging tool for cell membrane-associated studies, total internal reflection fluorescence microscopy (TIRFM) still has some limitations. The most important one is the inhomogeneous evanescent excitation field mainly caused by the large-angle and fixed-azimuth illumination scheme, which can be eliminated by using ring-shaped illumination (ring TIRFM). However, it is challenging in assembling a ring TIRFM system with precise parameter control that works well. Here we emphasize the quantification of the ring TIRFM system and introduce a robust calibration routine to simultaneously rectify the asymmetry of the spinning light beam and determine the crucial experimental parameter, i.e., the incident angle. The calibration routine requires no specific sample preparation and is entirely based on the automatic back focal plane manipulation, avoiding possible errors caused by the sample difference and manual measurement. Its effectiveness is experimentally demonstrated by both the qualitative and quantitative comparisons of the images acquired using different samples, illumination schemes, and calibration approaches. These characteristics should enable our approach to greatly improve the practicability of TIRFM in life sciences.

Three-dimensional super-resolution imaging of live whole cells using galvanometer-based structured illumination microscopy.

Imaging and tracking three-dimensional (3D) nanoscale organizations and functions of live cells is essential for biological research but it remains challenging. Among different 3D super-resolution techniques, 3D structured illumination microscopy (SIM) has the intrinsic advantages for live-cell studies; it is based on wide-field imaging and does not require high light intensities or special fluorescent dyes to double 3D resolution. However, the 3D SIM system has developed relatively slowly, especially in live imaging. Here, we report a more flexible 3D SIM system based on two galvanometer sets conveniently controlling the structured illumination pattern's period and orientation, which is able to study dynamics of live whole cells with high speed. We demonstrate our microscope's capabilities with strong optical sectioning and lateral, axial, and volume temporal resolution of 104 nm, 320 nm and 4 s, respectively. We do this by imaging nanoparticle and microtubule organizations and mitochondria evolution. These characteristics enable our galvanometer-based 3D SIM system to broaden the accessible imaging content of SIM-family microscopes and further facilitate their applications in life sciences.

Ultra-fast, universal super-resolution radial fluctuations (SRRF) algorithm for live-cell super-resolution microscopy.

For a long term, spatial resolution of fluorescence microscopy was strictly restricted by the diffraction limit. To solve this problem, various super-resolution technologies have been developed. Super-resolution radial fluctuations (SRRF), an emerging type of super-resolution microscopy, directly analyze raw images and generate super-resolution results without fluorophore localization, thereby showing more advantages in handling high-density data. Here, by speeding up the process of the algorithm with graphics processing unit (GPU) and programming with Python language, we expand the universality and improve the computing speed of the SRRF algorithm. We further apply our SRRF algorithm in different live-cell super-resolution microscopy methods with two types of fluorescence fluctuation sources: (i) direct stochastic optical reconstruction microscopy (dSTORM) in which fluorophores themselves blink under specific buffer and laser condition and (ii) structural illumination microscopy (SIM) and modulated Airyscan in which fluorescence fluctuations are artificially introduced with modulated laser illumination. With improved spatiotemporal resolution and image quality, our SRRF algorithm demonstrates its capability in live-cell super-resolution imaging, indicating its wide applications in life sciences.

Transfer and fates of damaged mitochondria: role in health and disease.

Intercellular communication is pivotal in mediating the transfer of mitochondria from donor to recipient cells. This process orchestrates various biological functions, including tissue repair, cell proliferation, differentiation and cancer invasion. Typically, dysfunctional and depolarized mitochondria are eliminated through intracellular or extracellular pathways. Nevertheless, increasing evidence suggests that intercellular transfer of damaged mitochondria is associated with the pathogenesis of diverse diseases. This review investigates the prevalent triggers of mitochondrial damage and the underlying mechanisms of mitochondrial transfer, and elucidates the role of directional mitochondrial transfer in both physiological and pathological contexts. Additionally, we propose potential previously unknown mechanisms mediating mitochondrial transfer and explore their prospective roles in disease prevention and therapy.

Identification of key immune-related genes and potential therapeutic drugs in diabetic nephropathy based on machine learning algorithms.

BACKGROUND: Diabetic nephropathy (DN) is a major contributor to chronic kidney disease. This study aims to identify immune biomarkers and potential therapeutic drugs in DN. METHODS: We analyzed two DN microarray datasets (GSE96804 and GSE30528) for differentially expressed genes (DEGs) using the Limma package, overlapping them with immune-related genes from ImmPort and InnateDB. LASSO regression, SVM-RFE, and random forest analysis identified four hub genes (EGF, PLTP, RGS2, PTGDS) as proficient predictors of DN. The model achieved an AUC of 0.995 and was validated on GSE142025. Single-cell RNA data (GSE183276) revealed increased hub gene expression in epithelial cells. CIBERSORT analysis showed differences in immune cell proportions between DN patients and controls, with the hub genes correlating positively with neutrophil infiltration. Molecular docking identified potential drugs: cysteamine, eltrombopag, and DMSO. And qPCR and western blot assays were used to confirm the expressions of the four hub genes. RESULTS: Analysis found 95 and 88 distinctively expressed immune genes in the two DN datasets, with 14 consistently differentially expressed immune-related genes. After machine learning algorithms, EGF, PLTP, RGS2, PTGDS were identified as the immune-related hub genes associated with DN. In addition, the mRNA and protein levels of them were obviously elevated in HK-2 cells treated with glucose for 24 h, as well as their mRNA expressions in kidney tissues of mice with DN. CONCLUSION: This study identified 4 hub immune-related genes (EGF, PLTP, RGS2, PTGDS), as well as their expression profiles and the correlation with immune cell infiltration in DN.

Killing capacity analysis of tumor-infiltrating cytotoxic lymphocytes and impact on lymph node metastasis in differentiated papillary carcinoma of thyroid with the BRAF V600E mutation.

BACKGROUND: Cytotoxic lymphocytes (CLs) express potent toxins, including perforin (P) and granzyme-B (G), which brings about target cell death. The purpose of this study was to evaluate the killing capacity of tumor-infiltrating CLs by means of P and G analysis, and explore the association with lymph node metastasis in papillary carcinoma of thyroid (PTC) without Hashimoto's thyroiditis (HT). METHODS: Infiltration of lymphocytes in PTC was observed in frozen sections. Both fresh tumor tissues and paracancerous tissues with lymphocyte infiltration were collected and prepared into a single cell suspension. Flow cytometry was used to detect the percentages of CD3+P+, CD3+G+, CD8+P+, and CD8+G+ T lymphocytes (TLs) and CD16-CD56+P+ and CD16-CD56+G+ natural killer (NK) cells. Finally, we investigated differential expression of P and G in NK cells and cytotoxic T lymphocytes (CTLs) in paired tumor tissues (group T, n = 44) and paracancerous tissues (group N, n = 44) from patients with PTC with the BRAF V600E mutation. Furthermore, patients were divided into two groups according to whether cervical central lymph node metastasis (CCLNM) existed: group A (with lymph node metastases, n = 27) and group B (with nonlymph node metastases, n = 17). Patients were also divided into three groups according to the total number of positive CCLNM: group B, group C (with low-level lymph node metastases, less than 5, n = 17) and group D (with high-level lymph node metastases, no less than 5, n = 10). RESULTS: The percentage of CD3+P+ CTLs was significantly higher in group N than in group T (P < 0.05). The percentage of CD8+G+ CTLs was significantly higher in group T than in group N (P < 0.05). The percentages of CD3+G+, CD16-CD56+P+and CD16-CD56+G+ NK cells showed no significant difference in either group T or group N (P > 0.05). The percentages of CD3+P+ CTLs in group A and group C were significantly higher in the paracancerous tissue than in the tumor tissue (P < 0.05). The percentages of CD8+G+ CTLs in group A and group C were significantly higher in the tumor tissues than in the paracancerous tissues (P < 0.05). The percentage of CD16-CD56+G+ NK cells in group D was significantly higher in the tumor tissues than in the paracancerous tissues (P < 0.05). CONCLUSIONS: The killing capacity of infiltrating CLs in PTC differed between tumor tissues and paracancerous tissues. In cases with CCLNM, higher expression of CD16-CD56+G+ NK cells in tumor tissues may be associated with a high risk of lymph node metastasis.

Far-field super-resolution chemical microscopy.

Far-field chemical microscopy providing molecular electronic or vibrational fingerprint information opens a new window for the study of three-dimensional biological, material, and chemical systems. Chemical microscopy provides a nondestructive way of chemical identification without exterior labels. However, the diffraction limit of optics hindered it from discovering more details under the resolution limit. Recent development of super-resolution techniques gives enlightenment to open this door behind far-field chemical microscopy. Here, we review recent advances that have pushed the boundary of far-field chemical microscopy in terms of spatial resolution. We further highlight applications in biomedical research, material characterization, environmental study, cultural heritage conservation, and integrated chip inspection.

Three-dimensional multi-color optical nanoscopy at sub-10-nm resolution based on small-molecule organic probes.

Achieving nanometer-scale resolution remains challenging in expansion microscopy due to photon loss. To address this concern, here we develop a multi-color expansion stimulated emission depletion technique based on small-molecule probes to realize high labeling density and intensity. Our method substantially lowers the barrier to visualizing diverse intracellular proteins and their interactions in three dimensions. It enables us to achieve sub-10-nm resolution in structures such as microfilaments, lysosomes, and mitochondria, providing new insights into cell biology.

Dual inhibition of CHK1/FLT3 enhances cytotoxicity and overcomes adaptive and acquired resistance in FLT3-ITD acute myeloid leukemia.

FLT3 inhibitors (FLT3i) are widely used for the treatment of acute myeloid leukemia (AML), but adaptive and acquired resistance remains a primary challenge. Inhibitors simultaneously blocking adaptive and acquired resistance are highly demanded. Here, we observed the potential of CHK1 inhibitors to synergistically improve the therapeutic effect of FLT3i in FLT3-mutated AML cells. Notably, the combination overcame adaptive resistance. The simultaneous targeting of FLT3 and CHK1 kinases may overcome acquired and adaptive resistance. A dual FLT3/CHK1 inhibitor 30 with a good oral PK profile was identified. Mechanistic studies indicated that 30 inhibited FLT3 and CHK1, downregulated the c-Myc pathway and further activated the p53 pathway. Functional studies showed that 30 was more selective against cells with various FLT3 mutants, overcame adaptive resistance in vitro, and effectively inhibited resistant FLT3-ITD AML in vivo. Moreover, 30 showed favorable druggability without significant blood toxicity or myelosuppression and exhibited a good oral PK profile with a T1/2 over 12 h in beagles. These findings support the targeting of FLT3 and CHK1 as a novel strategy for overcoming adaptive and acquired resistance to FLT3i therapy in AML and suggest 30 as a potential clinical candidate.