Multicolor microscopy tools necessary to localize and visualize the complexity of subcellular systems are limited by current fluorophore technology. While commercial fluorophores cover spectral space from the ultraviolet to the near infrared region and are optimized for conventional bandpass based fluorescence microscopy, they are not ideal for highly multiplexed fluorescence microscopy as they tend to have short Stokes shifts, restricting the number of fluorophores that can be detected in a single sample to four to five. Herein, we synthesized a library of 95 novel boron-dipyrromethene (BODIPY)-based fluorophores and screened their photophysical, optical and spectral properties for their utility in multicolor microscopy. A subset of our BODIPY-based fluorophores yielded varied length Stokes shifts probes, which were used to create a five-color image using a single excitation with confocal laser scanning microscopy for the first time. Combining these novel fluorophores with conventional fluorophores could facilitate imaging in up to nine to ten colors using linear unmixing based microscopy approaches.

Within cells, the spatial compartmentalization of thousands of distinct proteins serves a multitude of diverse biochemical needs. Correlative super-resolution (SR) fluorescence and electron microscopy (EM) can elucidate protein spatial relationships to global ultrastructure, but has suffered from tradeoffs of structure preservation, fluorescence retention, resolution, and field of view. We developed a platform for three-dimensional cryogenic SR and focused ion beam-milled block-face EM across entire vitreously frozen cells. The approach preserves ultrastructure while enabling independent SR and EM workflow optimization. We discovered unexpected protein-ultrastructure relationships in mammalian cells including intranuclear vesicles containing endoplasmic reticulum-associated proteins, web-like adhesions between cultured neurons, and chromatin domains subclassified on the basis of transcriptional activity. Our findings illustrate the value of a comprehensive multimodal view of ultrastructural variability across whole cells.

The low scattering efficiency of Raman scattering makes it challenging to simultaneously achieve good signal-to-noise ratio (SNR), high imaging speed, and adequate spatial and spectral resolutions. Here, we report a noise learning (NL) approach that estimates the intrinsic noise distribution of each instrument by statistically learning the noise in the pixel-spatial frequency domain. The estimated noise is then removed from the noisy spectra. This enhances the SNR by ca. 10 folds, and suppresses the mean-square error by almost 150 folds. NL allows us to improve the positioning accuracy and spatial resolution and largely eliminates the impact of thermal drift on tip-enhanced Raman spectroscopic nanoimaging. NL is also applicable to enhance SNR in fluorescence and photoluminescence imaging. Our method manages the ground truth spectra and the instrumental noise simultaneously within the training dataset, which bypasses the tedious labelling of huge dataset required in conventional deep learning, potentially shifting deep learning from sample-dependent to instrument-dependent.

Background: Cough is the main symptom of mycoplasma pneumoniae (MP) infection. Cough potential protein transient receptor potential A1 (TRPA1) plays an important role in cough reflex. The purpose of this study was to clarify the mechanism of wogonin, the effective component of Qinbai Qingfei concentrated pellet (Qinbai), in the treatment of cough after MP infection. Methods: The Biacore™ system was used to detect whether there was specific binding between Qinbai and cough potential protein TRPA1. Biacore™ fishing technology and UPLC-Q-TOF-MS technology were used during fishing combined active components and identification and analysis of recovered samples. The expression levels of TRPA1, substance P (SP), calcitonin gene-related peptide (CGRP), cough-related proteins, and mRNA in the lung tissues from each group were detected by immunohistochemistry, Western blot, and real-time PCR. Results: Biacore™ results showed that Qinbai had strong specific binding to TRPA1 protein with a binding value of 99.0 resonance unit (RU). The samples obtained from angling were identified and analyzed by UPLC-Q-TOF-MS as wogonin. The results of immunohistochemistry, Western blot, and real-time PCR showed that compared with the model group, the wogonin group had lower expressions of mRNA, TRPA1, SP, and CGRP in the lung tissue of cough mice with MP infection (p < 0.01 or p < 0.05), and the effects were superior to those of azithromycin and pentoxyverine control groups. Conclusion: Wogonin can treat cough after MP infection by affecting the expressions of cough-related proteins, such as TRPA1, SP, and CGRP. This study provided a theoretical foundation for the clinical research of Qinbai.

In order to explore the microstructure evolution of an Mg-RE alloy refined by Al during solution treatment, an Mg-3Y-4Nd-2Al alloy was treated at 545 °C for different time periods. Phase evolution of the alloy was investigated. After solution treatment, the Mg-RE eutectic phase in the Mg-3Y-4Nd-2Al alloy dissolves, the granular Al2RE phase does not change, the acicular Al11RE3 phase breaks into the short rod-like Al2RE phase, and the lamellar Al2RE phase precipitates in the grains. With the extension of solution time, the precipitated phase of the lamellar Al2RE increased at first and then decreased, and its orientation relationship with the matrix is <112>Al2RE//<21¯1¯0>Mg and {111}Al2RE//{0002}Mg. The undissolved granular Al2RE phase can improve the thermal stability of the alloy grain by pinning the grain boundary, and the grain size did not change after solution treatment. Solution treatment significantly improved the plasticity of the alloy. After 48 h of solution treatment, the elongation increased to 17.5% from 8.5% in the as-cast state.

Among the transition metal oxides, the Mn3O4 nanostructure possesses high theoretical specific capacity and lower operating voltage. However, the low electrical conductivity of Mn3O4 decreases its specific capacity and restricts its application in the energy conversion and energy storage. In this work, well-shaped, octahedron-like Mn3O4 nanocrystals were prepared by one-step hydrothermal reduction method. Field emission scanning electron microscope, energy dispersive spectrometer, X-ray diffractometer, X-ray photoelectron spectrometer, high resolution transmission electron microscopy, and Fourier transformation infrared spectrometer were applied to characterize the morphology, the structure, and the composition of formed product. The growth mechanism of Mn3O4 nano-octahedron was studied. Cyclic voltammograms, galvanostatic charge-discharge, electrochemical impedance spectroscopy, and rate performance were used to study the electrochemical properties of obtained samples. The experimental results indicate that the component of initial reactants can influence the morphology and composition of the formed manganese oxide. At the current density of 1.0 A g-1, the discharge specific capacity of as-prepared Mn3O4 nano-octahedrons maintains at about 450 mAh g-1 after 300 cycles. This work proves that the formed Mn3O4 nano-octahedrons possess an excellent reversibility and display promising electrochemical properties for the preparation of lithium-ion batteries.

Formylpeptide receptors (FPRs) as G protein-coupled receptors (GPCRs) can recognize formylpeptides derived from pathogens or host cells to function in host defense and cell clearance. In addition, FPRs, especially FPR2, can also recognize other ligands with a large chemical diversity generated at different stages of inflammation to either promote or resolve inflammation in order to maintain a balanced inflammatory response. The mechanism underlying promiscuous ligand recognition and activation of FPRs is not clear. Here we report a cryo-EM structure of FPR2-Gi signaling complex with a peptide agonist. The structure reveals a widely open extracellular region with an amphiphilic environment for ligand binding. Together with computational docking and simulation, the structure suggests a molecular basis for the recognition of formylpeptides and a potential mechanism of receptor activation, and reveals conserved and divergent features in Gi coupling. Our results provide a basis for understanding the molecular mechanism of the functional promiscuity of FPRs.

Our earlier work generated a powerful platform technology of polymeric scaffolding of stem cells to investigate and treat the injured or diseased central nervous system. However, the reciprocal sequelae between biophysical properties of the polymer and responses of the stem cell have not been examined in situ in lesioned spinal cords. We postulated that implantable synthetic scaffolds, acting through physical features, might affect donor cell behavior and host tissue remodeling. To test this hypothesis, poly(d,l-lactic-co-glycolic acid) (PLGA) in either low/soft or high/hard rigidity was fabricated for carrying adult human bone marrow mesenchymal stromal stem cells (hMSCs). The construct was transplanted into the epicenter of a rat model of acute T9-10 segmental hemisection to evaluate the effect of PLGA rigidity on the therapeutic potential and fate of hMSCs for neural repair. Compared to controls, only treatment with soft PLGA-scaffolded hMSCs significantly improved sensorimotor function via activation of recovery neurobiology mechanisms. The main benefits included inhibiting neuroinflammation and enhancing tissue protection. Also detected in the treated lesion region were expressions of neurotrophic and anti-inflammatory factors together with proliferation of endogenous neural stem cells, impacts likely derived from hMSCs' functional multipotency maintained by soft PLGA-scaffolding. Conversely, hard rigidity PLGA activated mechanotransduction and mesoderm lineage differentiation of hMSCs that ectopically produced bone, cartilage and muscle markers in neural parenchyma. The findings collectively suggested that the physical texture of polymeric scaffolds should be tailored for sustaining the stemness of hMSCs to constructively interact with the spinal cord for functional restoration.

Currently, the overall therapeutic efficiency of mesenchymal stem cells (MSCs) transplantation for the treatment of cardiovascular disease is not satisfactory. The low viability and angiogenic capacity of the implanted cells in the local infarct tissues restrict their further application. Evidence shows that long noncoding RNA H19 (lncRNA-H19) mediates cell survival and angiogenesis. Additionally, it is also involved in MSCs biological activities. This study aimed to explore the functional role of lncRNA-H19 in MSCs survival and angiogenic capacity as well as the underlying mechanism.

Urinary tract infections are predominantly caused by uropathogenic Escherichia coli (UPEC). UPEC infects bladder epithelial cells (BECs) via fusiform vesicles, escapes into the cytosol to evade exocytosis, and establishes intracellular bacterial communities (IBCs) for the next round of infection. The UPEC vesicle escape mechanism remains unclear. Here we show that UPEC senses host immune responses and initiates escape by upregulating a key phospholipase. The UPEC phospholipase PldA disrupts the vesicle membrane, and pldA expression is activated by phosphate reduction in vesicles. The host phosphate transporter PIT1 is located on the fusiform vesicle membrane, transporting phosphate into the cytosol. UPEC infection upregulates PIT1 via nuclear factor κB (NF-κB), resulting in phosphate reduction. Silencing PIT1 blocks UPEC vesicle escape in BECs, inhibits IBC formation in mouse bladders, and protects mice from UPEC infection. Our results shed light on pathogenic bacteria responding to intracellular phosphate shortage and tackling host defense and provide insights for development of new therapeutic agents to treat UPEC infection.

Stress granules (SGs) are cytoplasmic biomolecular condensates containing proteins and RNAs in response to stress. Ras-GTPase-activating protein binding protein 1 (G3BP1) is a core SG protein. Caprin-1 and ubiquitin specific peptidase 10 (USP10) interact with G3BP1, facilitating and suppressing SG formation, respectively. The crystal structures of the nuclear transport factor 2-like (NTF2L) domain of G3BP1 in complex with the G3BP1-interacting motif (GIM) of Caprin-1 and USP10 show that both GIMs bind to the same hydrophobic pocket of G3BP1. Moreover, both GIMs suppressed the liquid-liquid phase separation (LLPS) of G3BP1, suggesting that Caprin-1 likely facilitates SG formation via other mechanisms. Thus, we dissected various domains of Caprin-1 and investigated their role in LLPS in vitro and SG formation in cells. The C-terminal domain of Caprin-1 underwent spontaneous LLPS, whereas the N-terminal domain and GIM of Caprin-1 suppressed LLPS of G3BP1. The opposing effect of the N- and C-terminal domains of Caprin-1 on SG formation were demonstrated in cells with or without the endogenous Caprin-1. We propose that the N- and C-terminal domains of Caprin-1 regulate SG formation in a "yin and yang" fashion, mediating the dynamic and reversible assembly of SGs.

Idiopathic thrombocytopenic purpura (ITP) is a primary autoimmune disease with a decreased platelet count caused by platelet destruction mediated mainly by platelet antibodies. T follicular helper (TFH) cells have demonstrated important roles in autoimmune diseases. The aim of this study is to explore the might role of TFH cells in the patients of ITP.

The signaling adaptor protein Crk has been shown to play an important role in various human cancers. However, its regulatory machinery is not clear. Here, we demonstrated that Crk induced EMT in A549 human lung adenocarcinoma cells through differential regulation of Rac1/Snail and RhoA/Slug, leading to decreased expression of E-cadherin and increased N-cadherin, fibronectin, and MMP2 expression. Cancer cells with mesenchymal features produced TGF-β and also increased the levels of TGF-β receptor. TGF-β increased the endogenous levels of Crk and also augmented Crk-dependent expression of Snail and Slug, and conversely TGF-β receptor inhibitor suppressed the levels of Snail and Slug. Overexpression of Crk was observed at the invasive front of human lung cancer tissues and was significantly associated with poor prognosis. Thus, TGF-β and Crk collaborate to form a positive feedback loop to facilitate EMT, which may lead to the malignancy of human cancers possibly being affected by their microenvironment.

Henoch-Schönlein purpura (HSP) is a common systemic small vessel vasculitis in children with disorder autoimmune responses. T follicular helper (TFH) cells play crucial roles in regulating immune responses. The aim of our study was to investigate the probable role of TFH cells in the pathogenesis of children with HSP. In this study, the frequency of circulating CXCR5(+)CD4(+)TFH cells with inducible costimulator (ICOS) expression in the children with acute HSP was significantly higher than that in healthy controls (HCs) but not CXCR5(+)CD4(+)TFH cells with programmed death-1 (PD-1) expression. Moreover, serum levels of IL-21 and IL-6 cytokines, IgA, and C3 in HSP children were also significantly higher than those in HCs. A positive correlation was observed between the frequencies of circulating ICOS(+)CXCR5(+)CD4(+)TFH cells and the serum IL-21 or IgA levels of acute HSP children, respectively. Additionally, the mRNA expression levels of interleukin- (IL-) 21, IL-6, and transcriptional factors (B-cell lymphoma-6, Bcl-6) were also significantly increased in peripheral blood from acute HSP children compared to HCs. Taken together, these findings suggest that TFH cells and associated molecules might play critical roles in the pathogenesis of HSP, which are possible therapeutic targets in HSP children.

In the neurogenic niches, adult neural stem and/or progenitor cells (NSCs) generate functional neurons throughout life, which has been implicated in learning and memory and affective behaviors. During adult neurogenesis, newborn neurons release feedback signals into the niches to regulate NSC proliferation and differentiation. However, whether and how NSCs contribute to the niche governing newborn neuron development is still unknown. Using a combination of cell ablation, retrovirus-mediated single-cell labeling, and signaling pathway modulation, we show that adult hippocampal NSCs continuously supply pleiotrophin factor to the newborn neurons. Without this feedforward signal, the newborn neurons display defective dendritic development and arborization. Thus, our findings reveal that NSCs behave as a functional niche for newly generated newborn neurons to regulate their maturation.

Human embryonic stem cells (hESCs) play an important role in regenerative medicine due to their potential to differentiate into various functional cells. However, the conventional adherent culture system poses challenges to mass production of high-quality hESCs. Though scientists have made many attempts to establish a robust and economical hESC suspension culture system, there are existing limitations, including suboptimal passage methods and shear force caused by dynamic stirring. Here, we report on an efficient large-scale culture system, which enables long-term, GMP grade, single-cell inoculation, and serial expansion of hESCs with a yield of about 1.5 × 109 cells per 1.5-L culture, while maintaining good pluripotency. The suspension culture system was enlarged gradually from a 100-mm dish to a 1.8-L culture bag with methylcellulose involvement to avoid sphere fusion. Under the optimal experimental protocol, this 3D system resolves current problems that limit mass production and clinical application of hESCs, and thus can be used in commercial-level hESC production for cell therapy and pharmaceutics screening in the future.

Houshiheisan (HSHS), a classic prescription in traditional Chinese medicine (TCM), has remarkable efficacy in the treatment of ischemic stroke.

The ideal orthopedic implant coating is expected to both inhibit microbial infection and promote osseointegration. In this study, Zn ions were immobilized on a Ti substrate via a polydopamine (PDA) chemical surface modification to prepare Ti-PDA-Zn coatings. Scanning electron microscopy (SEM), atomic force microscopy (AFM), energy-dispersive X-ray spectroscope (EDS), X-ray photoelectron spectroscopy (XPS), contact analysis system, and inductively coupled plasma atomic emission spectrometry (ICP-AES) were used to analyze the morphology, composition, wettability, and zinc ions release of the coatings. The Ti-PDA-Zn coatings demonstrated excellent antibacterial activities in vitro against both Staphylococcus aureus and Escherichia coli. The coatings additionally displayed good biocompatibility, as confirmed by cytoskeletal observations and cell viability assays. Furthermore, the in vivo results confirmed the excellent antibacterial properties and improved osseointegration capability of the Ti-PDA-Zn coating in the presence of S. aureus. The present findings indicate that the Ti-PDA-Zn coatings prepared herein have potential application in orthopedic implantation.

Single-walled carbon nanotubes (SWCNTs) incorporated with π-conjugated polymers, have proven to be an effective approach in the production of advanced thermoelectric composites. However, the studied polymers are mainly limited to scanty conventional conductive polymers, and their performances still remain to be improved. Herein, a new planar moiety of platinum acetylide in the π-conjugated system is introduced to enhance the intermolecular interaction with the SWCNTs via π⁻π and d⁻π interactions, which is crucial in regulating the thermoelectric performances of SWCNT-based composites. As expected, SWCNT composites based on the platinum acetylides embedded polymers displayed a higher power factor (130.7 ± 3.8 μW·m-1·K-2) at ambient temperature than those without platinum acetylides (59.5 ± 0.7 μW·m-1·K-2) under the same conditions. Moreover, the strong interactions between the platinum acetylide-based polymers and the SWCNTs are confirmed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) measurements.

Dicotyledonous plants form an apical hook to protect the fragile apical meristem during upward protrusion from the soil. Etiolated pifq (pif1 pif3 pif4 pif5) seedlings display constitutive apical hook opening. Here, we show that PIF proteins control apical hook opening by regulating the expression of Budding Uninhibited by Benzimidazole 3.1 (BUB3.1) and affecting cytokinesis. Consistent with the major function of BUB3.1 in the organization of phragmoplasts during cytokinesis, the phragmoplasts are well formed in dark-grown pifq but not in wild type. DNA staining and flow cytometry analysis further demonstrate that cellular endoreduplication levels are dramatically reduced in pifq. Chemical treatment with caffeine, an inhibitor of phragmoplast-based cytokinesis, shows that cytokinesis is involved in the apical hook opening. Genetically, BUB3.1 is epistatic to PIFq in the regulation of cytokinesis. Our findings reveal an organ-specific role of PIF proteins in regulating cytokinesis by BUB3.1 during apical hook development.