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Single-molecule localization microscopy enables high-resolution imaging of molecular interactions, but discriminating molecular binding types has traditionally relied on complex strategies, such as multiple dyes, time-division techniques, or kinetic analysis, that are asynchronous, invasive, or time-consuming. Here, we uncover previously overlooked spatiotemporal information embedded within diffraction-limited fluorescence, enabling synchronous classification of individual binding event videos using only a single fluorescent dye. Building on this insight, we propose a Temporal-to-Context Convolutional Neural Network (T2C CNN), which integrates long-term spatial convolutions, shallow cross-connected blocks, and a pooling-free structure to enhance contextual representation while preserving fine-grained temporal features. Applied to DNA-PAINT experiments, T2C CNN achieves up to 94.76% classification accuracy and outperforms state-of-the-art video classification models by 15-25 percentage points. Our approach enables rapid and precise binding-type recognition from fluorescence video data, reducing observation time from minutes to seconds and facilitating high-throughput single-molecule imaging without requiring multiple dye channels or extended kinetic measurements. Yin and colleagues propose that diffraction-limited fluorescence videos contain hidden binding-type information. The authors present a deep learning model, T2C CNN, which exploits that hidden information to classify molecular interactions with high accuracy using a single dye in seconds.

Cytokines, crucial in immune modulation, impact disease progression when their secretion is dysregulated. Existing methods for profiling cytokine secretion suffer from time‐consuming and labor‐intensive processes and often fail to capture the dynamic nature of immune responses. Here, iSECRETE, an integrated platform that enables synchronous cell activation, wash‐free, and target‐responsive protein detection for single‐cell IFN‐γ cytokine secretion analysis within 30 min at room temperature is presented. By incorporating a DNA proximity assay (DPA) into a multifunctional microfluidic system, one‐pot homogenous cytokine signal amplification, with a limit of detection of ≈50 secreted molecules per cell is achieved. iSECRETE can robustly handle various sample types that are shown. Two distinct immune activation assay modalities are demonstrated on iSECRETE. Finally, the detection of single‐cell IFN‐γ secretion as an activation hallmark of chimeric antigen receptor T cells within 6 h of exposure to cancer targets is shown. iSECRETE represents the fastest single‐cell sample‐to‐result cytokine secretion assay to date, providing a powerful tool for advancing the understanding of biological phenotypes, functions, and pathways under in vivo‐like conditions. iSECRETE combines the advantages of integrated microfluidics and isothermal DNA proximity assay for a one‐step homogeneous single‐cell cytokine release assay. The assay can analyze whole blood or undiluted cell culture samples and detect activation‐induced cytokine release in live immune cells. iSECRETE is performed on chimeric antigen receptor (CAR) T cells cytotoxicity assay showing CAR T functional cytokine secretion.

Objective This study aimed to investigate the impact of physical activity (PA) on emotional management ability (EMA) in college students and to analyze the chain mediating roles of psychological resilience (PR) and health literacy (HL) in the relationship between PA and EMA. Methods Demographic information was collected using a stratified, cluster, and multi-stage sampling method. Data on PA, PR, HL, and EMA among college students were collected using an online questionnaire. A total of 13,590 valid questionnaires were obtained. Results A significant positive correlation was found between PA and EMA in college students ( r  = 0.177, P  < 0.01). PR and HL played significant mediating roles in the relationship between PA and EMA. The mediation effect consisted of independent mediation by PR and HL and a chain mediation effect between the two. The values of the independent mediation effects and the chain mediation effect were 0.011, 0.010, and 0.004, respectively. The chain mediation effect was lower than each of the individual mediation effects and showed a statistically significant difference compared to the mediation effect of HL ( P  < 0.05). Conclusion PA significantly enhances college students’ EMA through direct effects and a chain mediation pathway involving PR and HL. Coordinated interventions, including regular exercise, PR training, and health education, are recommended to optimize college students’ psychological health.

Background and aimsPancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer-related death worldwide. Neurotransmitter-initiated signalling pathway is profoundly implicated in tumour initiation and progression. Here, we investigated whether dysregulated neurotransmitter receptors play a role during pancreatic tumourigenesis.MethodsThe Cancer Genome Atlas and Gene Expression Omnibus datasets were used to identify differentially expressed neurotransmitter receptors. The expression pattern of gamma-aminobutyric acid type A receptor pi subunit (GABRP) in human and mouse PDAC tissues and cells was studied by immunohistochemistry and western blot analysis. The in vivo implications of GABRP in PDAC were tested by subcutaneous xenograft model and lung metastasis model. Bioinformatics analysis, transwell experiment and orthotopic xenograft model were used to identify the in vitro and in vivo effects of GABRP on macrophages in PDAC. ELISA, co-immunoprecipitation, proximity ligation assay, electrophysiology, promoter luciferase activity and quantitative real-time PCR analyses were used to identify molecular mechanism.ResultsGABRP expression was remarkably increased in PDAC tissues and associated with poor prognosis, contributed to tumour growth and metastasis. GABRP was correlated with macrophage infiltration in PDAC and pharmacological deletion of macrophages largely abrogated the oncogenic functions of GABRP in PDAC. Mechanistically, GABRP interacted with KCNN4 to induce Ca2+ entry, which leads to activation of nuclear factor κB signalling and ultimately facilitates macrophage infiltration by inducing CXCL5 and CCL20 expression.ConclusionsOverexpressed GABRP exhibits an immunomodulatory role in PDAC in a neurotransmitter-independent manner. Targeting GABRP or its interaction partner KCNN4 may be an effective therapeutic strategy for PDAC.

Recently, environmental temperature has been shown to regulate bone homeostasis. However, the mechanisms by which cold exposure affects bone mass remain unclear. In our present study, we observed that exposure to cold temperature (CT) decreased bone mass and quality in mice. Furthermore, a transplant of exosomes derived from the plasma of mice exposed to cold temperature (CT-EXO) can also impair the osteogenic differentiation of BMSCs and decrease bone mass by inhibiting autophagic activity. Rapamycin, a potent inducer of autophagy, can reverse cold exposure or CT-EXO-induced bone loss. Microarray sequencing revealed that cold exposure increases the miR-25-3p level in CT-EXO. Mechanistic studies showed that miR-25-3p can inhibit the osteogenic differentiation and autophagic activity of BMSCs. It is shown that inhibition of exosomes release or downregulation of miR-25-3p level can suppress CT-induced bone loss. This study identifies that CT-EXO mediates CT-induced osteoporotic effects through miR-25-3p by inhibiting autophagy via targeting SATB2, presenting a novel mechanism underlying the effect of cold temperature on bone mass.

Vascular calcification and vascular ageing are “silent” diseases but are highly prevalent in patients with end stage renal failure and type 2 diabetes, as well as in the ageing population. Melatonin (MT) has been shown to induce cardiovascular protection effects. However, the role of MT on vascular calcification and ageing has not been well-identified. In this study, the aortic transcriptional landscape revealed clues for MT related cell-to-cell communication between endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) in vascular calcification and vascular ageing. Furthermore, we elucidated that it was exosomes that participate in the information transportation from ECs to VSMCs. The exosomes secreted from melatonin-treated ECs (MT-ECs-Exos) inhibited calcification and senescence of VSMCs. Mechanistically, miR-302d-5p was highly enriched in MT-ECs-Exos, while depletion of miR-302d-5p blocked the ability of MT-ECs-Exos to suppress VSMC calcification and senescence. Notably, Wnt3 was a bona fide target of miR-302d-5p and modulated VSMC calcification and senescence. Furthermore, we found that maturation of endothelial derived exosomal miR-302d-5p was promoted by WTAP in an N6-methyladenosine (m6A)-dependent manner. Interestingly, MT alleviated vascular calcification and ageing in 5/6-nephrectomy (5/6 NTP) mice, a chronic kidney disease (CKD) induced vascular calcification and vascular ageing mouse model. MT-ECs-Exos was absorbed by VSMCs in vivo and effectively prevented vascular calcification and ageing in 5/6 NTP mice. ECs-derived miR-302d-5p mediated MT induced anti-calcification and anti-ageing effects in 5/6 NTP mice. Our study suggests that MT-ECs-Exos alleviate vascular calcification and ageing through the miR-302d-5p/Wnt3 signaling pathway, dependent on m6A methylation. [Display omitted] •The transcriptional landscape revealed EC-VSMC communications maintained vascular homestasis and protected against the progression of CVD.•Melatonin suppressed calcification and ageing via ECs-derived exosomes in the vascular wall microenvironment.•ECs derived exosomal miR-302d-5p mediated the anti-calcification and anti-ageing effects of melatonin.•Maturation of endothelium derived exosomal cargo was promoted by WTAP/HNRNPA2B1/DGCR8 pathway in a m6A-dependent manner.•Verifying the cell-to-cell communication between ECs and VSMCs by exosomes in regulating vascular calcification and ageing.

Nerve injury induces profound and complex changes at molecular and cellular levels, leading to axonal self-destruction as well as immune and inflammatory responses that may further promote neurodegeneration. To better understand how neural injury changes the proteome within the injured nerve, we set up a mouse model of sciatic nerve injury (SNI) and conducted an unbiased, quantitative proteomic study followed by biochemical assays to confirm some of the changed proteins. Among them, the protein levels of ADP-dependent glucokinase (ADPGK) were significantly increased in the injured sciatic nerve. Further examination indicated that ADPGK was specifically expressed and upregulated in macrophages but not neurons or Schwann cells upon injury. Furthermore, culturing immortalized bone marrow-derived macrophages (iBMDMs) in vitro with the conditioned media from transected axons of mouse dorsal root ganglion (DRG) neurons induced ADPGK upregulation in iBMDMs, suggesting that injured axons could promote ADPGK expression in macrophages non-cell autonomously. Finally, we showed that overexpression of ADPGK per se did not activate macrophages but promoted the phagocytotic activity of lipopolysaccharides (LPS)-treated macrophages. Together, this proteomic analysis reveals interesting changes of many proteins within the injured nerve and our data identify ADPGK as an important in vivo booster of injury-induced macrophage phagocytosis.

For a type-II superconductor in an applied magnetic field greater than the lower critical field H c1 , the magnetic flux will penetrate into the superconductor and form quantized vortices with Andreev bound states in the vortex cores. The characteristics of the bound states are related to the pairing symmetry and band structure of the superconductor. Recently, a new family of high temperature (high- T c ) superconductors, the iron pnictides, has been discovered. Surprisingly, in electron-doped Ba(Fe 1− x Co x ) 2 As 2 , no bound state was found in the vortices. Here, we use a low-temperature scanning tunnelling microscope to study the electronic structure of hole-doped Ba 0.6 K 0.4 Fe 2 As 2 . Two superconducting gaps (with gap values 2 Δ / k B T c ≈2.2 and 5.1, where k B is Boltzmann’s constant) were observed in the superconducting state. By applying magnetic fields, we observed ordered vortices with Andreev bound states in the vortex cores. The bound states and their spatial evolution can be qualitatively explained by our numerical calculations for multiband s -wave superconductivity. The mystery of the missing bound states within a superconducting vortex in a pnictide superconductor has been solved. Not only are bound states present, they also provide information on the gap structure of Ba 0.6 K 0.4 Fe 2 As 2 .