Independent activation of CREB3L2 by glucose fills a regulatory gap in mouse ß-cells by co-ordinating insulin biosynthesis with secretory granule formation.

OBJECTIVE: Although individual steps have been characterized, there is little understanding of the overall process whereby glucose co-ordinates the biosynthesis of insulin with its export out of the endoplasmic reticulum (ER) and incorporation into insulin secretory granules (ISGs). Here we investigate a role for the transcription factor CREB3L2 in this context. METHODS: MIN6 cells and mouse islets were analysed by immunoblotting after treatment with glucose, fatty acids, thapsigargin and various inhibitors. Knockdown of CREB3L2 was achieved using si or sh constructs by transfection, or viral delivery. In vivo metabolic phenotyping was conducted after deletion of CREB3L2 in ß-cells of adult mice using Ins1-CreER+. Islets were isolated for RNAseq and assays of glucose-stimulated insulin secretion (GSIS). Trafficking was monitored in islet monolayers using a GFP-tagged proinsulin construct that allows for synchronised release from the ER. RESULTS: With a Km ≈3.5 mM, glucose rapidly (T1/2 0.9 h) increased full length (FL) CREB3L2 followed by a slower rise (T1/2 2.5 h) in its transcriptionally-active cleavage product, P60 CREB3L2. Glucose stimulation repressed the ER stress marker, CHOP, and this was partially reverted by knockdown of CREB3L2. Activation of CREB3L2 by glucose was not due to ER stress, however, but a combination of O-GlcNAcylation, which impaired proteasomal degradation of FL-CREB3L2, and mTORC1 stimulation, which enhanced its conversion to P60. cAMP generation also activated CREB3L2, but independently of glucose. Deletion of CREB3L2 inhibited GSIS ex vivo and, following a high-fat diet (HFD), impaired glucose tolerance and insulin secretion in vivo. RNAseq revealed that CREB3L2 regulated genes controlling trafficking to-and-from the Golgi, as well as a broader cohort associated with ß-cell compensation during a HFD. Although post-Golgi trafficking appeared intact, knockdown of CREB3L2 impaired the generation of both nascent ISGs and proinsulin condensates in the Golgi, implying a defect in ER export of proinsulin and/or its processing in the Golgi. CONCLUSION: The stimulation of CREB3L2 by glucose defines a novel, rapid and direct mechanism for co-ordinating the synthesis, packaging and storage of insulin, thereby minimizing ER overload and optimizing ß-cell function under conditions of high secretory demand. Upregulation of CREB3L2 also potentially contributes to the benefits of GLP1 agonism and might in itself constitute a novel means of treating ß-cell failure.

Activatable BODIPY-chromene NIR-II probes with small spectral crosstalk enable high-contrast in vivo bioimaging.

Herein, we design a novel "crossbreeding" dye (BC-OH) within the second near-infrared (NIR-II) window based on BODIPY and chromene chromophores. BC-OH can serve as a platform to construct activatable NIR-II probes with small spectral crosstalk, thereby making a breakthrough in imaging in vivo H2O2 fluctuation in an APAP-induced liver injury model with high signal-to-background ratio.

ß-cell function is regulated by metabolic and epigenetic programming of islet-associated macrophages, involving Axl, Mertk, and TGFß receptor signaling.

We have exploited islet-associated macrophages (IAMs) as a model of resident macrophage function, focusing on more physiological conditions than the commonly used extremes of M1 (inflammation) versus M2 (tissue remodeling) polarization. Under steady state, murine IAMs are metabolically poised between aerobic glycolysis and oxidative phosphorylation, and thereby exert a brake on glucose-stimulated insulin secretion (GSIS). This is underpinned by epigenetic remodeling via the metabolically regulated histone demethylase Kdm5a. Conversely, GSIS is enhanced by engaging Axl receptors on IAMs, or by augmenting their oxidation of glucose. Following high-fat feeding, efferocytosis is stimulated in IAMs in conjunction with Mertk and TGFß receptor signaling. This impairs GSIS and potentially contributes to ß-cell failure in pre-diabetes. Thus, IAMs serve as relays in many more settings than currently appreciated, fine-tuning insulin secretion in response to dynamic changes in the external environment. Intervening in this nexus might represent a means of preserving ß-cell function during metabolic disease.

Bent-to-planar Si-rhodamines: a distinct rehybridization lights up NIR-II fluorescence for tracking nitric oxide in the Alzheimer's disease brain.

An ongoing revolution in fluorescence-based technologies has transformed the way we visualize and manipulate biological events. An enduring goal in this field is to explore high-performance fluorogenic scaffolds that show tunability and capability for in vivo analysis, especially for small-molecular near-infrared (NIR) fluorophores. We present a unique bent-to-planar rehybridization design strategy for NIR fluorogenic scaffolds, thus yielding a palette of switchable bent/planar Si-rhodamines that span from visible to NIR-II wavelengths. We demonstrate that the rehybridization of meso-nitrogen in this innovative NIR scaffold Cl-SiRhd results in flipping between the disruption and recovery of the polymethine π-electron system, thereby significantly altering the spectral wavelength with crosstalk-free responses. Using elaborately lighting-up NIR-II probes with ultra-large Stokes shifts (ca. 250 nm), we successfully achieve real-time in situ monitoring of biological events in live cells, zebrafish, and mice. Notably, for the first time, the light-up NIR-II probe makes a breakthrough in directly in situ tracking nitric oxide (NO) fluctuations in the brains of mice with Alzheimer's disease. This de novo bent-to-planar rehybridization strategy of NIR-II probes opens up exciting opportunities for expanding the in vivo imaging toolbox in both life science research and clinical applications.

[The application progress on diagnostic scales of laryngopharyngeal reflux disease].

At present, objective methods for diagnosing laryngopharyngeal reflux disease(LPRD) are not minimally invasive, effective, and economical. Diagnostic scales are widely used worldwide due to the advantages of inexpensive, noninvasive, and easy to operate. The reflux symptom index(RSI) and the reflux finding score(RFS) are preferred to use in clinical diagnosis. However, many controversies have appeared in the application of RSI and RFS in recent years, causing many troubles to clinical diagnosis. Therefore, this review briefly discusses the problems of RSI and RFS in clinical applications to provide reference for diagnosing LPRD accurately.

Fluorescent Probes Based on AIEgen-Mediated Polyelectrolyte Assemblies for Manipulating Intramolecular Motion and Magnetic Relaxivity.

Uniting photothermal therapy (PTT) with magnetic resonance imaging (MRI) holds great potential in nanotheranostics. However, the extensively utilized hydrophobicity-driven assembling strategy not only restricts the intramolecular motion-induced PTT, but also blocks the interactions between MR agents and water. Herein, we report an aggregation-induced emission luminogen (AIEgen)-mediated polyelectrolyte nanoassemblies (APN) strategy, which bestows a unique "soft" inner microenvironment with good water permeability. Femtosecond transient spectra verify that APN well activates intramolecular motion from the twisted intramolecular charge transfer process. This de novo APN strategy uniting synergistically three factors (rotational motion, local motion, and hydration number) brings out high MR relaxivity. For the first time, APN strategy has successfully modulated both intramolecular motion and magnetic relaxivity, achieving fluorescence lifetime imaging of tumor spheroids and spatio-temporal MRI-guided high-efficient PTT.

Preparation of near-infrared AIEgen-active fluorescent probes for mapping amyloid-ß plaques in brain tissues and living mice.

Fibrillar aggregates of the amyloid-ß protein (Aß) are the main component of the senile plaques found in brains of patients with Alzheimer's disease (AD). Development of probes allowing the noninvasive and high-fidelity mapping of Aß plaques in vivo is critical for AD early detection, drug screening and biomedical research. QM-FN-SO3 (quinoline-malononitrile-thiophene-(dimethylamino)phenylsulfonate) is a near-infrared aggregation-induced-emission-active fluorescent probe capable of crossing the blood-brain barrier (BBB) and ultrasensitively lighting up Aß plaques in living mice. Herein, we describe detailed procedures for the two-stage synthesis of QM-FN-SO3 and its applications for mapping Aß plaques in brain tissues and living mice. Compared with commercial thioflavin (Th) derivatives ThT and ThS (the gold standard for detection of Aß aggregates) and other reported Aß plaque fluorescent probes, QM-FN-SO3 confers several advantages, such as long emission wavelength, large Stokes shift, ultrahigh sensitivity, good BBB penetrability and miscibility in aqueous biological media. The preparation of QM-FN-SO3 takes ~2 d, and the confocal imaging experiments for Aß plaque visualization, including the preparation for mouse brain sections, take ~7 d. Notably, acquisition and analyses for in vivo visualization of Aß plaques in mice can be completed within 1 h and require only a basic knowledge of spectroscopy and chemistry.

Designing of 3D MnO2-graphene catalyst on sponge for abatement temperature removal of formaldehyde.

The Mn-based catalysts, with low cost and high activity, are believed to be the effective composites for eliminating in-door formaldehyde (HCHO), while the powdered form nanosized catalysts are hardly to apply for practical application. Herein, hetero-structure of nanosheets manganese oxide (MnO2) encapsulating N-doping graphene sphere (GS) were deposited in network-like sponge for constructing 3D catalyst. The prepared MnO2-GS-Sponge composite catalyst exhibited excellent performance for removing HCHO at room temperature compared with GS and commercial MnO2. The MnO2-GS with larger specific surface area (209.1 m2·g-1) was dispersed evenly in 3D network of sponge, which facilitated exposing more activate sites and achieving fast transport kinetics accelerating catalytic reaction for converting 97.1 % of 100 ppm of HCHO continuously to CO2 for 120 h. Moreover, rely on the chemisorption of amino groups on N-doping GS surface, HCHO could be enriched even at low concentrations and efficient elimination (from 1000 ppb to12 ppb, at 35 â„ƒ in 48 h). The average oxidation state and infrared spectra analysis suggested that abundant oxygen vacancies on MnO2-GS-Sponge could be identified as surface-active sites of converting HCHO into the intermediates of dioxymethylene and formate. This work might inspire the designing 3D composite material for potential application in other fields of environmental engineering or energy industrial.

A pH-activated fluorescent probe via transformation of azo and hydrazone forms for lysosomal pH imaging.

We developed a fluorescent probe Sth-NH by introducing a 6-hydroxypyridone skeleton. The presence of an active proton enables the probe to transform from a deprotonated azo form to a hydrazone form in a strongly acidic environment to realize fluorescence light-up behavior, thus monitoring the lower lysosomal pH of cancer cells and distinguishing them from normal cells.

An AIE-active probe for monitoring calcium-rich biological environment with high signal-to-noise and long-term retention in situ.

Fluorescent probe is a first-line method for qualitative and quantitative detection of calcium ions (Ca2+) in organisms. However, the high affinity and aggregate-caused quenching (ACQ) characteristics of commercially available probes have restricted the detection limit to low concentrations from nM to µM, unavailable to detect higher Ca2+ concentrations from µM to mM in situ. Here, we develop a Ca2+ probe of TCM-4COOH with aggregation-induced emission (AIE) activity and desirable affinity, exhibiting a linear response to concentrated Ca2+ at mM level. The rapid binding between the TCM-4COOH and Ca2+ results in dramatic enhancement in fluorescence with high S/N ratio, and the nature that the chelates are not easy to diffuse from the cells endows the probe with long-term imaging ability in organisms. In the molecular design, the multiple iminodiacetic carboxyl groups ensure the good water solubility and pH biocompatibility of TCM-4COOH, resulting in negligible background fluorescence and high signal-to-noise (S/N) ratio. Moreover, the relatively dispersed carboxyl groups and the electron-withdrawing effect of TCM building block jointly adjust the probe affinity to Ca2+, thereby broadening the upper detection limit. In addition, to obtain better cell membrane penetrability, TCM-4COOH was modified with acetoxymethyl ester, which unit can be cleaved by endogenous esterase to release TCM-4COOH, so as to detect intracellular calcium ions. Benefit from the reasonable design of fluorophore and chelating groups, the AIE-active sensor TCM-4COOH can achieve long-term in-situ retention in visualizing calcium-overloaded cells and bone microcracks, especially providing a unique platform to broaden the upper limit of Ca2+ detection in biological environments.

Arcuate NPY is involved in salt-induced hypertension via modulation of paraventricular vasopressin and brain-derived neurotrophic factor.

Chronic high salt intake is one of the leading causes of hypertension. Salt activates the release of the key neurotransmitters in the hypothalamus such as vasopressin to increase blood pressure, and neuropepetide Y (NPY) has been implicated in the modulation of vasopressin levels. NPY in the hypothalamic arcuate nucleus (Arc) is best known for its control in appetite and energy homeostasis, but it is unclear whether it is also involved in the development of salt-induced hypertension. Here, we demonstrate that wild-type mice given 2% NaCl salt water for 8 weeks developed hypertension which was associated with marked downregulation of NPY expression in the hypothalamic Arc as demonstrated in NPY-GFP reporter mice as well as by in situ hybridization analysis. Furthermore, salt intake activates neurons in the hypothalamic paraventricular nucleus (PVN) where mRNA expression of brain-derived neurotrophic factor (BDNF) and vasopressin was found to be upregulated, leading to elevated serum vasopressin levels. This finding suggests an inverse correlation between the Arc NPY level and expression of vasopressin and BDNF in the PVN. Specific restoration of NPY by injecting AAV-Cre recombinase into the Arc only of the NPY-targeted mutant mice carrying a loxP-flanked STOP cassette reversed effects of salt intake on vasopressin and BDNF expression, leading to a normalization of salt-dependent blood pressure. In summary, our study uncovers an important Arc NPY-originated neuronal circuitry that could sense and respond to peripheral electrolyte signals and thereby regulate hypertension via vasopressin and BDNF in the PVN.

Sequence-Activated Fluorescent Nanotheranostics for Real-Time Profiling Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC), as one of the most malignant tumors with dense desmoplastic stroma, forms a specific matrix barrier to hinder effective diagnosis and therapy. To date, a paramount challenge is in the search for intelligent nanotheranostics for such hypopermeable tumors, especially in breaking the PDAC-specific physical barrier. The unpredictable in vivo behaviors of nanotheranostics, that is, real-time tracking where, when, and how they cross the physical barriers and are taken up by tumor cells, are the major bottleneck. Herein, we elaborately design sequence-activated nanotheranostic TCM-U11&Cy@P with dual-channel near-infrared fluorescence outputs for monitoring in vivo behaviors in a sequential fashion. This nanotheranostic with a programmable targeting capability effectively breaks through the PDAC barriers. Ultimately, the released aggregation-induced emission (AIE) particle TCM-U11 directly interacts with PDAC cells and penetrates into the deep tissue. Impressively, this fluorescent nanotheranostic intraoperatively can map human clinical PDAC specimens with high resolution. We believe that this unique sequence-activated fluorescent strategy expands the repertoire of nanotheranostics in the treatment of hypopermeable tumors.

Harnessing α-l-fucosidase for in vivo cellular senescence imaging.

Precise detection of cellular senescence may allow its role in biological systems to be evaluated more effectively, while supporting studies of therapeutic candidates designed to evade its detrimental effect on physical function. We report here studies of α-l-fucosidase (α-fuc) as a biomarker for cellular senescence and the development of an α-fuc-responsive aggregation induced emission (AIE) probe, termed QM-NHαfuc designed to complement more conventional probes based on ß-galactosidase (ß-gal). Using QM-NHαfuc, the onset of replicative-, reactive oxygen species (ROS)-, ultraviolet A (UVA)-, and drug-induced senescence could be probed effectively. QM-NHαfuc also proved capable of identifying senescent cells lacking ß-gal expression. The non-invasive real-time senescence tracking provided by QM-NHαfuc was validated in an in vivo senescence model. The results presented in this study lead us to suggest that the QM-NHαfuc could emerge as a useful tool for investigating senescence processes in biological systems.

NAD+ supplement potentiates tumor-killing function by rescuing defective TUB-mediated NAMPT transcription in tumor-infiltrated T cells.

Although tumor-infiltrating lymphocytes (TILs) maintain their ability to proliferate, persist, and eradicate tumors, they are frequently dysfunctional in situ. By performing both whole-genome CRISPR and metabolic inhibitor screens, we identify that nicotinamide phosphoribosyltransferase (NAMPT) is required for T cell activation. NAMPT is low in TILs, and its expression is controlled by the transcriptional factor Tubby (TUB), whose activity depends on the T cell receptor-phospholipase C gamma (TCR-PLCγ) signaling axis. The intracellular level of NAD+, whose synthesis is dependent on the NAMPT-mediated salvage pathway, is also decreased in TILs. Liquid chromatography-mass spectrometry (LC-MS) and isotopic labeling studies confirm that NAD+ depletion led to suppressed glycolysis, disrupted mitochondrial function, and dampened ATP synthesis. Excitingly, both adoptive CAR-T and anti-PD1 immune checkpoint blockade mouse models demonstrate that NAD+ supplementation enhanced the tumor-killing efficacy of T cells. Collectively, this study reveals that an impaired TCR-TUB-NAMPT-NAD+ axis leads to T cell dysfunction in the tumor microenvironment, and an over-the-counter nutrient supplement of NAD+ could boost T-cell-based immunotherapy.

Enzyme-activatable fluorescent probes for ß-galactosidase: from design to biological applications.

ß-Galactosidase (ß-gal), a typical hydrolytic enzyme, is a vital biomarker for cell senescence and primary ovarian cancers. Developing precise and rapid methods to monitor ß-gal activity is crucial for early cancer diagnoses and biological research. Over the past decade, activatable optical probes have become a powerful tool for real-time tracking and in vivo visualization with high sensitivity and specificity. In this review, we summarize the latest advances in the design of ß-gal-activatable probes via spectral characteristics and responsiveness regulation for biological applications, and particularly focus on the molecular design strategy from turn-on mode to ratiometric mode, from aggregation-caused quenching (ACQ) probes to aggregation-induced emission (AIE)-active probes, from near-infrared-I (NIR-I) imaging to NIR-II imaging, and from one-mode to dual-mode of chemo-fluoro-luminescence sensing ß-gal activity.

Circularly Polarized Fluorescence Resonance Energy Transfer (C-FRET) for Efficient Chirality Transmission within an Intermolecular System.

The occurrence and transmission of chirality is a fascinating characteristic of nature. However, the intermolecular transmission efficiency of circularly polarized luminescence (CPL) remains challenging due to poor through-space energy transfer. We report a unique CPL transmission from inducing the achiral acceptor to emit CPL within a specific liquid crystal (LC)-based intermolecular system through a circularly polarized fluorescence resonance energy transfer (C-FRET), wherein the luminescent cholesteric LC is employed as the chirality donor, and rationally designed achiral long-wavelength aggregation-induced emission (AIE) fluorophore acts as the well-assembled acceptor. In contrast to photon-release-and-absorption, the chirality transmission channel of C-FRET is highly dependent upon the energy resonance in the highly intrinsic chiral assembly of cholesteric LC, as verified by deliberately separating the achiral acceptor from the chiral donor to keep it far beyond the resonance distance. This C-FRET mode provides a de novo strategy concept for high-level information processing for applications such as high-density data storage, combinatorial logic calculation, and multilevel data encryption and decryption.

Fluorescence umpolung enables light-up sensing of N-acetyltransferases and nerve agents.

Intramolecular charge transfer (ICT) is a fundamental mechanism that enables the development of numerous fluorophores and probes for bioimaging and sensing. However, the electron-withdrawing targets (EWTs)-induced fluorescence quenching is a long-standing and unsolved issue in ICT fluorophores, and significantly limits the widespread applicability. Here we report a simple and generalizable structural-modification for completely overturning the intramolecular rotation driving energy, and thus fully reversing the ICT fluorophores' quenching mode into light-up mode. Specifically, the insertion of an indazole unit into ICT scaffold can fully amplify the intramolecular rotation in donor-indazole-π-acceptor fluorophores (fluorescence OFF), whereas efficiently suppressing the rotation in their EWT-substituted system (fluorescence ON). This molecular strategy is generalizable, yielding a palette of chromophores with fluorescence umpolung that spans visible and near-infrared range. This strategy expands the bio-analytical toolboxes and allows exploiting ICT fluorophores for light-up sensing of EWTs including N-acetyltransferases and nerve agents.

Peripheral-specific Y1 receptor antagonism increases thermogenesis and protects against diet-induced obesity.

Obesity is caused by an imbalance between food intake and energy expenditure (EE). Here we identify a conserved pathway that links signalling through peripheral Y1 receptors (Y1R) to the control of EE. Selective antagonism of peripheral Y1R, via the non-brain penetrable antagonist BIBO3304, leads to a significant reduction in body weight gain due to enhanced EE thereby reducing fat mass. Specifically thermogenesis in brown adipose tissue (BAT) due to elevated UCP1 is enhanced accompanied by extensive browning of white adipose tissue both in mice and humans. Importantly, selective ablation of Y1R from adipocytes protects against diet-induced obesity. Furthermore, peripheral specific Y1R antagonism also improves glucose homeostasis mainly driven by dynamic changes in Akt activity in BAT. Together, these data suggest that selective peripheral only Y1R antagonism via BIBO3304, or a functional analogue, could be developed as a safer and more effective treatment option to mitigate diet-induced obesity.

Harnessing Hypoxia-Dependent Cyanine Photocages for In†Vivo Precision Drug Release.

Photocaging holds promise for the precise manipulation of biological events in space and time. However, current near-infrared (NIR) photocages are oxygen-dependent for their photolysis and lack of timely feedback regulation, which has proven to be the major bottleneck for targeted therapy. Herein, we present a hypoxia-dependent photo-activation mechanism of dialkylamine-substituted cyanine (Cy-NH) accompanied by emissive fragments generation, which was validated with retrosynthesis and spectral analysis. For the first time, we have realized the orthogonal manipulation of this hypoxia-dependent photocaging and dual-modal optical signals in living cells and tumor-bearing mice, making a breakthrough in the direct spatiotemporal control and in vivo feedback regulation. This unique photoactivation mechanism overcomes the limitation of hypoxia, which allows site-specific remote control for targeted therapy, and expands the photo-trigger toolbox for on-demand drug release, especially in a physiological context with dual-mode optical imaging under hypoxia.

Rational Design of Near-Infrared Cyanine-Based Fluorescent Probes for Rapid In Vivo Sensing Cysteine.

Cysteine (Cys) is well-known to be an important biothiol and related to many diseases. However, the in situ trapping of endogenous Cys is still handicapped by a lack of straightforward methods combined with long-wavelength emission and high-performance response. In this work, we described the rational design strategy of cyanine-based near-infrared (NIR) probes for the rapid detection of mitochondrial Cys in living cells and mice. We focus on how to improve the response rate via regulating the electron density of the recognition units in probes. The obtained three probes all displayed remarkable fluorescence enhancement at 780 nm. From screening the obtained probes, it was found that the probe Cy-S-diOMe with electron-donating recognition unit displayed the fastest response rate, the lowest detection limit, and the highest signal-to-noise ratio. More importantly, Cy-S-diOMe was successfully applied to monitor Cys in tumor-bearing mice (within merely 5 min). This paradigm by modulation of the response rate in the cyanine dyes provides a promising methodology for the design of high-performance cyanine-based NIR probes.