Comprehensive investigation in oncogenic functions and immunological roles of NCBP2 and its validation in prostate cancer.

BACKGROUND: Nuclear cap-binding protein 2 (NCBP2), as the component of the cap-binding complex, participates in a number of biological processes, including pre-mRNA splicing, transcript export, translation regulation and other gene expression steps. However, the role of NCBP2 on the tumor cells and immune microenvironment remains unclear. To systematically analyze and validate functions of NCBP2, we performed a pan-cancer analysis using multiple approaches. METHODS: The data in this study were derived from sequencing, mutation, and methylation data in the TCGA cohort, normal sample sequencing data in the GTEx project, and cell line expression profile data in the CCLE database. RESULTS: Survival analyses including the Cox proportional-hazards model and log-rank test revealed the poor prognostic role of NCBP2 in multiple tumors. We further validated the oncogenic ability of NCBP2 in prostate cancer cell lines, organoids and tumor-bearing mice. A negative correlation was observed between NCBP2 expression and immune score by the ESTIMATE algorithm. Simultaneously, the NCBP2-induced immunosuppressive microenvironment might be related to the decline in CD8+T cells and the increase in regulatory T cells and neutrophils, examined by flow cytometry experiments for NCBP2 overexpressed tumor-bearing mice. CONCLUSION: This research offered strong proof supporting NCBP2 as the prognostic marker and the therapeutic target in the future.

New recognition of the heart-brain axis and its implication in the pathogenesis and treatment of PTSD.

Post-traumatic stress disorder (PTSD) is a complex psychological disorder provoked by distressing experiences, and it remains without highly effective intervention strategies. The exploration of PTSD's underlying mechanisms is crucial for advancing diagnostic and therapeutic approaches. Current studies primarily explore PTSD through the lens of the central nervous system, investigating concrete molecular alterations in the cerebral area and neural circuit irregularities. However, the body's response to external stressors, particularly the changes in cardiovascular function, is often pronounced, evidenced by notable cardiac dysfunction. Consequently, examining PTSD with a focus on cardiac function is vital for the early prevention and targeted management of the disorder. This review undertakes a comprehensive literature analysis to detail the alterations in brain and heart structures and functions associated with PTSD. It also synthesizes potential mechanisms of heart-brain axis interactions relevant to the development of PTSD. Ultimately, by considering cardiac function, this review proposes novel perspectives for PTSD's prophylaxis and therapy.

Influence of substrate temperature on the properties of ZnTe:Cu films prepared by a magnetron co-sputtering method.

Copper-doped Zinc Tellurium (ZnTe:Cu) films were deposited on borosilicate glass using magnetron co-sputtering technique. The influence of the substrate temperature on the structural, morphological, optical and electrical properties of ZnTe:Cu films was investigated by X-ray diffraction (XRD), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), UV-Vis spectrophotometer and Hall effect measurement system. The results indicate that substrate temperature significantly affects the properties of the ZnTe:Cu films. When the substrate temperature increases from room temperature to 600 °C, the (111)-preferred orientation of ZnTe:Cu films is gradually replaced by the (220)-preferred orientation. At high substrate temperatures (≥500 °C), the CuxTe phase appears in the ZnTe:Cu films, resulting in higher carrier concentration (>1019 cm-3) and lower resistivity (<10-2 Ω cm) of the prepared films.

Bioconjugation and Reaction-Induced Tumor Therapy via Alkynamide-Based Thiol-Yne Click Reaction.

Ferroptosis is associated with the occurrence and development of many diseases, which is the result of an imbalance in cellular metabolism and oxidation-reduction balance. Therefore, it is an effective therapeutic strategy that simultaneously regulating the intracellular oxidation-reduction system. Herein, a click reaction of alkynylamide with thiol groups in the presence of amine or in PBS (pH = 7.4) is developed, which can react efficiently with thiol substances, such as cysteine (Cys), glutathione (GSH), and bovine serum albumin (BSA). Notably, MBTB-PA, an aggregation-induced emission (AIE) photosensitizer with an alkynylamide unit, is synthesized and its intracellular behavior is visualized in situ by fluorescence imaging, demonstrating its excellent ability to target the endoplasmic reticulum. Furthermore, MBTB-PA reacted with proteins in tumor cells, consumed reducing substances, and triggered intracellular oxidative stress, resulting in cell death. Based on this reaction therapy strategy, click reaction is combined with photodynamic therapy to achieve effective killing of tumor cells by simultaneously raising the intracellular oxidative state and reducing the reductive state. This work not only develops an application of click reaction of alkynamide with thiol in bioconjugation and anti-tumor therapy, but also provides feasible ideas for organic reactions in the exploration of organisms.

Digital finance, government intervention, and carbon emission efficiency in China.

In accordance with the "dual carbon" objective, China is required to effectively pursue economic expansion and environmental preservation while concurrently enhancing carbon emission efficiency (CEE). This study examines the influence of digital finance on CEE and evaluates the moderating effect of government intervention. The analysis uses panel data collected from 282 cities in China at the prefecture level and above, spanning the period from 2011 to 2021. The findings indicate the following: (1) CEE in China is relatively low, and there are notable regional disparities. Specifically, there is a discernible downward trend in CEE throughout the eastern, central, and western areas. (2) In general, the implementation of digital finance has the potential to enhance the efficiency of carbon emissions. The observed effect is significant in the eastern and central regions but not in the western region. (3) Government subsidies have the potential to amplify digital finance's impact on CEE in the eastern region. Conversely, in the central and western regions, its influence can be increased by environmental regulations. Based on these findings, this study presents recommendations for advancing digital finance, enhancing the targeting and assessment of government subsidies, refining environmental regulations, and encouraging the adoption of green technologies.

Fluorescence dynamics of lysosomal-related organelle flashing in the intestinal cells of Caenorhabditis elegans.

The biological roles of the autofluorescent lysosome-related organelles ("gut granules") in the intestinal cells of many nematodes, including Caenorhabditis elegans, have been shown to play an important role in metabolic and signaling processes, but they have not been fully characterized. We report here a previously undescribed phenomenon in which the autofluorescence of these granules increased and then decreased in a rapid and dynamic manner that may be associated with nutrient availability. We observed that two distinct types of fluorophores are likely present in the gut granules. One displays a "flashing" phenomenon, in which fluorescence decrease is preceded by a sharp increase in fluorescence intensity that expands into the surrounding area, while the other simply decreases in intensity. Gut granule flashing was observed in the different life stages of C. elegans and was also observed in Steinernema hermaphroditum, an evolutionarily distant nematode. We hypothesize that the "flashing" fluorophore is pH-sensitive, and the fluorescence intensity change results from the fluorophore being released from the lysosome-related organelles into the relatively higher pH environment of the cytosol. The visually spectacular dynamic fluorescence phenomenon we describe might provide a handle on the biochemistry and genetics of these lysosome-related organelles.

Click-Reaction-Mediated Chemotherapy and Photothermal Therapy Synergistically Inhibit Breast Cancer in Mice.

The development of functional materials for tumor immunogenicity enhancement is desirable for overcoming the low therapeutic efficiency and easy metastasis during tumor treatments. Herein, the thermoresponsive nanoparticles composed of photothermal agent (PTA) and click reactive reagent are developed for enhanced immunotherapy application. A Ni-bis(dithiolene)-containing PTA with intense near-infrared absorption and efficient photothermal conversion is developed for thermoresponsive nanoparticles construction. The generated heat by encapsulated PTA further induces the phase transition of thermoresponsive nanoparticles with the release of chemotherapy reagent to react with the amino groups on functional proteins, realizing PTT and chemotherapy simultaneously. Moreover, the immunogenic cell death (ICD) of cancer cells evoked by PTT could be further enhanced by the released reactive reagent. As a result, the synergistic effect of photothermal treatment and reaction-mediated chemotherapy can suppress the growth of a primary tumor, and the evoked ICD could further activate the immune response with the suppression of a distant tumor. This synergistic treatment strategy provides a reliable and promising approach for cancer immunotherapy in clinic.

NIR luminogen for low-temperature photothermal therapy by triggering HSP90α down-regulation.

A near-infrared (NIR) luminogen TST was designed and used to efficiently trigger HSP90α protein knockdown through photo-thermal conversion based on a gene interference strategy, by which in vitro and in vivo tumor ablation were significantly acquired at low-temperature.

Spatial Heterogeneity and Scale Effects of Transportation Carbon Emission-Influencing Factors-An Empirical Analysis Based on 286 Cities in China.

In order to scientifically evaluate the characteristics and impact outcomes of transportation carbon emissions, this paper uses the panel statistics of 286 cities to measure transportation carbon emissions and analyze their spatial correlation characteristics. Afterwards, primarily based on the current research, a system of indicators for the impact factors of transportation carbon emissions was established. After that, ordinary least squares regression, geographically weighted regression, and multiscale geographically weighted regression models were used to evaluate and analyze the data, and the outcomes of the multiscale geographically weighted regression model were selected to analyze the spatial heterogeneity of the elements influencing transportation carbon emissions. The effects exhibit that: (1) The spatial characteristics of China's transportation carbon emissions demonstrate that emissions are high in the east, low in the west, high in the north, and low in the south, with high-value areas concentrated in the central cities of Beijing-Tianjin-Hebei, the Yangtze River Delta, the Guangdong-Hong Kong-Macao region, and the Chengdu-Chongqing regions, and the low values concentrated in the Western Sichuan region, Yunnan, Guizhou, Qinghai, and Gansu. (2) The spatial heterogeneity of transportation carbon emissions is on the rise, but the patten of local agglomeration is obvious, showing a clear high-high clustering, and the spatial distribution of high-high agglomeration and low-low agglomeration is positively correlated, with high-high agglomeration concentrated in the eastern region and low-low agglomeration concentrated in the western region. (3) The effects of three variables-namely, GDP per capita, vehicle ownership, and road mileage-have a predominantly positive effect on transportation carbon emissions within the study area, while another three variables-namely, constant term, population density, and number of people employed in transportation industry-have different mechanisms of influence in different regions. Constant term, vehicle ownership, and road mileage have greater impacts on transportation carbon emissions.

Aggregation Effect on Multiperformance Improvement in Aryl-Armed Phenazine-Based Emitters.

The concept of aggregate science was proposed to explain changes in materials performance that accompany the generation of aggregates, but aggregation-triggered multifunction improvements in a class of materials have rarely been reported. Herein, we present the first report of a new class of multifunctional aggregation-induced emission (AIE) luminogens (AIEgens) based on 5,10-diarylphenazine (DPZ) derivates with full-wavelength emission. Intriguingly, multiple properties, such as fluorescence intensity and free radical and type I reactive oxygen species (ROS) efficiencies, could be simultaneously activated from the unimolecular level to the aggregate state. The mechanisms of this multiple performance improvement are discussed in detail based on sufficient performance characterization, and some of the newly prepared AIEgens exhibited toxicity to cancer cells during photodynamic therapy. This work systematically demonstrates the positive effect of aggregation on improving multiple functions of materials, which is expected to promote the development of aggregate science theory for the design of multifunctional materials.

Research Progress of Wide Tunable Bragg Grating External Cavity Semiconductor Lasers.

In this paper, we review the progress of wide tunable Bragg grating external cavity semiconductor lasers (BG-ECSLs). We concentrate on BG-ECSLs based on the wide tunable range for multicomponent detection. Wide tunable BG-ECSLs have many important applications, such as wavelength-division multiplexing (WDM) systems, coherent optical communications, gas detection and atom cooling. Wide tunability, narrow linewidth and a high side-mode suppression ratio BG-ECSLs have attracted much attention for their merits. In this paper, three main structures for achieving widely tunable, narrow linewidth, high side-mode suppression ratio BG-ECSLs are reviewed and compared in detail, such as the volume Bragg grating (VBG) structure, fiber Bragg grating (FBG) structure and waveguide Bragg grating (WBG) structure of ECSLs. The advantages and disadvantages of different structures of BG-ECSLs are analyzed. The results show that WBG-ECSLs are a potential way to realize the integration, small size, wide tuning range, stable spectral output and high side-mode suppression ratio laser output. Therefore, the use of WBG as optical feedback elements is still the mainstream direction of BG-ECSLs, and BG-ECSLs offer a further new option for multicomponent detection and multi-atoms cooling.

Referential modification strategy based on phenolic hydroxyl-containing KSA luminogens for ER-targeting probe construction.

The endoplasmic reticulum (ER) plays essential roles in various physiological processes and is intimately connected to kinds of diseases. The development of ER-targeting theranostic agents is highly demanded for precise treatments, however, the effective and referential strategies for the construction of ER-targeting probes are limited. Herein, we developed series of ER-targeting luminogens based on keto-salicylaldehyde azine (KSA) framework by introducing phenolic hydroxyl group, which present good theranostic performance with selective enrichment in ER. Under systematical structure modulation, the key role of phenolic hydroxyl group at K-terminal in ER-targeting was experimentally confirmed. Besides, the cyanobenzyl moiety at S-terminal can enhance the luminous efficiency and improve cellular uptake ability. Moreover, the generated reactive oxygen species (ROS) of these KSA derivatives can efficiently trigger ER stress to induce the apoptosis of cancer cells, resulting in the effective inhibition of tumor cells both in vitro and in vivo. Therefore, this feasible modification strategy of inserting phenolic hydroxyl group to common multi-aryl-based luminogens provides a reliable and referential approach for ER-targeting probe establishment.

The Relationship of Low-Density-Lipoprotein to Lymphocyte Ratio with Chronic Obstructive Pulmonary Disease.

Background: Chronic Obstructive Pulmonary Disease (COPD) has been a concern all over the world because of its high prevalence and mortality. The ratio of low-density-lipoprotein to lymphocyte (LLR) has been widely used to predict the prognosis of cerebral infarction, but its association with COPD is less known. We aim to explore the relationship between LLR and COPD and to investigate its indicative role in the severity and prognosis of COPD. Methods: In this study, 279 participants (n = 138 with COPD and n = 138 age- and sex-matched health control) were recruited. COPD patients were divided into two groups according to the optimal cut-off value of LLR determined by the receiver operating characteristic curve (ROC). We collected the clinical characteristics, pulmonary function, LLR, neutrophil-lymphocyte ratio (NLR), platelet-lymphocyte ratio (PLR) and other data of all subjects. t-test, Pearson correlation test, logistic regression analysis and other statistical analysis were carried out. Results: Compared with the healthy control group, COPD patients had a significantly higher LLR level (p < 0.001). The disease was more serious in the high LLR group, which was reflected by Global Initiative for Chronic Obstructive Lung Disease (GOLD) and BMI, airway obstruction, dyspnoea, severe exacerbations (BODE) index and St. George's Respiratory Questionnaire (SGRQ) index (p = 0.001, p = 0.013, p = 0.011, respectively). The forced expiration volume in 1 second (FEV1) (p = 0.033) and forced expiratory volume in 1 second in percent of the predicted value (FEV1%) (p = 0.009) in high LLR group were lower. Univariate and multivariate logistic regression analysis showed that LLR was an independent factor affecting the severity of COPD patients (odds ratio [OR] = 2.599, 95% CI: 1.266-5.337, p = 0.009). Conclusion: We found that LLR is a novel biomarker in predicting the severity of patients with COPD. Further studies with larger database were recommended to verify our findings.

Coevolutionary insights between promoters and transcription factors in the plant and animal kingdoms.

The divergence and continuous evolution of plants and animals contribute to ecological diversity. Promoters and transcription factors (TFs) are key determinants of gene regulation and transcription throughout life. However, the evolutionary trajectories and relationships of promoters and TFs are still poorly understood. Here, we conducted extensive analysis of large-scale multi-omics sequences in 420 animal species and 223 plant species spanning nearly a billion years of evolutionary history. Results showed that promoter GC-content and TF isoelectric points, as features/signatures that accompany long biological evolution, exhibited increasing growth in animal cells but a decreasing trend in plant cells. Furthermore, the evolutionary trajectories of promoter and TF signatures in the animal kingdom provided further evidence that Mammalia as well as Aves evolved directly from the ancestor Reptilia. The strong correlation between promoter and TF signatures indicates that promoters and TFs formed antagonistic coevolution in the animal kingdom, but mutualistic coevolution in the plant kingdom. The distinct coevolutionary patterns potentially drive the plant-animal divergence，divergent evolution and ecological diversity.

Single-Cell RNA Sequencing of Peripheral Blood Mononuclear Cells From Acute Myocardial Infarction.

Background: Acute myocardial infarction (AMI) can occur in patients with atherosclerotic disease, with or without plaque rupture. Previous studies have indicated a set of immune responses to plaque rupture. However, the specific circulating immune cell subsets that mediate inflammatory plaque rupture remain elusive. Methods: Ten AMI patients were enrolled in our study (five with and five without plaque rupture; plaque characteristics were identified by optical coherence tomography). By single-cell RNA sequencing, we analyzed the transcriptomic profile of peripheral blood mononuclear cells. Results: We identified 27 cell clusters among 82,550 cells, including monocytes, T cells, NK cells, B cells, megakaryocytes, and CD34+ cells. Classical and non-classical monocytes constitute the major inflammatory cell types, and pro-inflammatory genes such as CCL5, TLR7, and CX3CR1 were significantly upregulated in patients with plaque rupture, while the neutrophil activation and degranulation genes FPR2, MMP9, and CLEC4D were significantly expressed in the intermediate monocytes derived from patients without plaque rupture. We also found that CD4+ effector T cells may contribute to plaque rupture by producing a range of cytokines and inflammatory-related chemokines, while CD8+ effector T cells express more effector molecules in patients without plaque rupture, such as GZMB, GNLY, and PRF1, which may contribute to the progress of plaque erosion. Additionally, NK and B cells played a significant role in activating inflammatory cells and promoting chemokine production in the plaque rupture. Cell-cell communication elaborated characteristics in signaling pathways dominated by inflammatory activation of classical monocytes in patients with plaque rupture. Conclusions: Our studies demonstrate that the circulating immune cells of patients with plaque rupture exhibit highly pro-inflammatory characteristics, while plaque erosion is mainly associated with intermediate monocyte amplification, neutrophil activation, and degranulation. These findings may provide novel targets for the precise treatment of patients with AMI.

The Relationship of Partial Pressure of Carbon Dioxide (PaCO2) with Disease Severity Indicators Such as BODE and GOLD in Hospitalized COPD Patients.

Purpose: This study aimed to investigate the relationship of partial pressure of carbon dioxide (PaCO2) with BODE and GOLD in stable COPD subjects and to explore the predictive value of PaCO2 for severe COPD (BODE index score ≥5 or GOLD index score ≥3). Patients and Methods. In total, 80 participants with COPD and free from other conditions affecting PaCO2 were recruited. Arterial blood gases, BODE, GOLD, SGRQ, lung function, and other data were collected. The BODE index was calculated, and patients were divided into two groups according to the BODE index and PaCO2 median, respectively. We used Pearson's correlation test and the receiver operating characteristic curves to evaluate the utility of PaCO2. Besides, the univariate and multivariate logistic regression analyses were conducted to verify whether PaCO2 was an independent factor associated with BODE grades. Results: COPD subjects with BODE ≥5 and GOLD ≥3 had significantly higher levels of PaCO2 (p = 0.004, p = 0.001, respectively). In the high PaCO2 group, patients underwent poorer outcomes than the low PaCO2 group. PaCO2 was negatively correlated with forced expiratory volume in 1 second in percent of the predicted value (FEV1%) (r = -0.612, p < 0.001). The performance of PaCO2 levels in predicting BODE ≥5 and GOLD ≥3 was 0.748 and 0.755, respectively. The logistic regression analyses proved that PaCO2 was associated with BODE ≥5 in COPD patients (odds ratio = 1.160, 95% CI: 1.025-1.313, p = 0.019). Conclusions: A higher level of PaCO2 was associated with a higher index for BODE or GOLD in COPD and had the predictive value for severe COPD.

Associations between bone mineral density and chronic obstructive pulmonary disease.

OBJECTIVE: To assess the relationship between chronic obstructive pulmonary disease (COPD) severity and bone mineral density (BMD) in the whole body and different body areas. METHODS: This retrospective, cross-sectional study included patients with COPD. Demographic and lung function data, COPD severity scales, BMD, and T scores were collected. Patients were grouped by high (≥-1) and low (<-1) T scores, and stratified by body mass index, airway obstruction, dyspnoea, and exercise capacity (BODE) index. The relationship between whole-body BMD and BODE was evaluated by Kendall's tau-b correlation coefficient. Risk factors associated with COPD severity were identified by univariate analyses. BMD as an independent predictor of severe COPD (BODE ≥5) was verified by multivariate logistic regression. BMD values in different body areas for predicting severe COPD were assessed by receiver operating characteristic curves. RESULTS: Of 88 patients with COPD, lung-function indicators and COPD severity were significantly different between those with high and low T scores. Whole-body BMD was inversely related to COPD severity scales, including BODE. Multivariate logistic regression revealed that BMD was independently associated with COPD severity. The area under the curve for pelvic BMD in predicting severe COPD was 0.728. CONCLUSION: BMD may be a novel marker in predicting COPD severity, and pelvic BMD may have the strongest relative predictive power.

Photo-Enhanced Chemotherapy Performance in Bladder Cancer Treatment via Albumin Coated AIE Aggregates.

The implementation of cisplatin-based neoadjuvant chemotherapy (NAC) plays a key role in conjunction with surgical resection in preventing bladder cancer progression and recurrence. However, the significant dose-dependent toxic side effects of NAC are still a major challenge. To solve this problem, we developed a photoenhanced cancer chemotherapy (PECC) strategy based on AIEgen ((E)-3-(2-(2-(5-(4-(diphenylamino)phenyl)thiophen-2-yl)vinyl)-1,1-dimethyl-1H-3λ4-benzo[e]indol-3-yl)propane-1-sulfonate), which is abbreviated as BITT. Multifunctional BITT@BSA-DSP nanoparticles (NPs) were employed with an albumin-based nanocarrier decorated with the cisplatin(IV) prodrug and loaded to produce strong near-infrared fluorescence imaging (NIR FLI), and they exhibited good photoenhancement performance via photodynamic therapy (PDT) and photothermal therapy (PTT). In vitro results demonstrated that BITT@BSA-DSP NPs could be efficiently taken up by bladder cancer cells and reduced to release Pt (II) under reductase, ensuring the chemotherapy effect. Furthermore, both in vitro and in vivo evaluation verified that the integration of NIR FL imaging-guided PECC efficiently promoted the sensitivity of bladder cancer to cisplatin chemotherapy with negligible side effects. This work provides a promising strategy to enhance the sensitivity of multiple cancers to chemotherapy drugs and even achieve effective treatments for drug-resistant cancers.

miR-129-5p restores cardiac function in rats with chronic heart failure by targeting the E3 ubiquitin ligase Smurf1 and promoting PTEN expression.

Chronic heart failure (CHF) is a prevalent health concern with complex pathogenesis. This current study set out to estimate the function of the miR-129-5p/Smurf1/PTEN axis on cardiac function injury in CHF. The model of CHF in rats was established. The cardiac function indexes, myocardial tissue damage, and oxidative stress-related factors in CHF rats were evaluated after the interference of Smurf1/miR-129-5p/PTEN. The targeting relationships between miR-129-5p and Smurf1 and between PTEN and Smurf1 were verified. It was found that that after modeling, cardiac functions were impaired, heart/left ventricular/lung weight and the myocardial structure was destroyed, and the degree of fibrosis of myocardial tissue was increased. After Smurf1 knockdown, the cardiac function, myocardial structure, and oxidative stress were improved, and the fibrosis in myocardial tissue was decreased. Smurf1 was a target of miR-129-5p. miR-129-5p could annul the protective effect of Smurf1 silencing on CHF rats. Smurf1 inhibited PTEN expression by promoting PTEN ubiquitination, while miR-129-5p enhanced PTEN expression by inhibiting Smurf1. Meanwhile, overexpression of PTEN annulled the cardiac dysfunction in CHF rats induced by Smurf1. In conclusion, miR-129-5p targeted Smurf1 and repressed the ubiquitination of PTEN, and promoted PTEN expression, thus improving the cardiac function of CHF rats.

Imaging Neuropeptide Release at Drosophila Neuromuscular Junction with a Genetically Engineered Neuropeptide Release Reporter.

Despite the important roles of neuropeptides in a variety of physiological processes, there still lacks a method to probe neuropeptide release events in vivo with satisfying temporal and spatial resolution. Neuropeptide Release Reporter (NPRR) was recently introduced as a novel genetically encoded indicator of neuropeptide release with a high temporal resolution and peptide specificity based on GCaMP molecule. Here we describe a method for using NPRR to image selective neuropeptide release at Drosophila neuromuscular junction in semi-dissected larvae. This method provides a quantitative analysis of activity-dependent neuropeptide release as real-time changes in fluorescence intensity of GCaMP reporter with sub-second temporal resolution and single bouton specificity.