Comprehensive insights into fluorescent probes for the determination nitric oxide for diseases diagnosis.

Nitric oxide (NO) plays an important role in multiple physiological processes of the body involved in regulation, such as cardiovascular relaxation, neural homeostasis, and immune regulation, etc. The real-time monitoring of NO is of great significance in the investigation of related disease mechanisms and the evaluation of pharmacodynamics. Fluorescent probes are considered as a highly promising approach for pharmaceutical analysis and bioimaging due to their non-invasive character, real-time detection, and high sensitivity. However, there are still some challenges in the determination of biological nitric oxide with fluorescent probes, such as low anti-interference ability, poor function modifiability, and low organ specificity. Therefore, it would be beneficial to develop a new generation of fluorescent probes for real-time bioimaging of NO in vivo after this systematic summary.

Practical NIR Assay Derived from Cyanine to Evaluate Intracellular H2S in Living Cell Imaging.

To monitor the biological function of H2S in real time, this investigation demonstrated the design and synthesis of a novel fluorescent probe integrated with cyanine and 2,4-dinitrophenol for the qualitative and quantitative detection of H2S. An NIR sensitive sensor (FS-HS-1) was provided with a straightforward process. Spectroscopy experiments elucidated that FS-HS-1 could selectively detect H2S in a PBS solution (containing 40% acetonitrile) with a 111-fold fluorescence enhancement at 715 nm (ex. 605 nm). The response towards NaHS occurred in less than 2 min, and the detection limit was confirmed to be as low as 4.47 ± 0.11 nmol/L. Furthermore, the probe is capable of monitoring changes in exogenous H2S concentrations within living cells with confocal and 2P imaging.

Cryo-EM structures of the human PA200 and PA200-20S complex reveal regulation of proteasome gate opening and two PA200 apertures.

Proteasomes are highly abundant and conserved protease complexes that eliminate unwanted proteins in the cells. As a single-chain ATP-independent nuclear proteasome activator, proteasome activator 200 (PA200) associates with 20S core particle to form proteasome complex that catalyzes polyubiquitin-independent degradation of acetylated histones, thus playing a pivotal role in DNA repair and spermatogenesis. Here, we present cryo-electron microscopy (cryo-EM) structures of the human PA200-20S complex and PA200 at 2.72 Å and 3.75 Å, respectively. PA200 exhibits a dome-like architecture that caps 20S and uses its C-terminal YYA (Tyr-Tyr-Ala) to induce the α-ring rearrangements and partial opening of the 20S gate. Our structural data also indicate that PA200 has two openings formed by numerous positively charged residues that respectively bind (5,6)-bisdiphosphoinositol tetrakisphosphate (5,6[PP]2-InsP4) and inositol hexakisphosphate (InsP6) and are likely to be the gates that lead unfolded proteins through PA200 and into the 20S. Besides, our structural analysis of PA200 found that the bromodomain (BRD)-like (BRDL) domain of PA200 shows considerable sequence variation in comparison to other human BRDs, as it contains only 82 residues because of a short ZA loop, and cannot be classified into any of the eight typical human BRD families. Taken together, the results obtained from this study provide important insights into human PA200-induced 20S gate opening for substrate degradation and the opportunities to explore the mechanism for its recognition of H4 histone in acetylation-mediated proteasomal degradation.

Recent Progress on Fluorescent Probes in Heavy Metal Determinations for Food Safety: A Review.

One of the main challenges faced in food safety is the accumulation of toxic heavy metals from environmental sources, which can sequentially endanger human health when they are consumed. It is invaluable to establish a practical assay for the determination of heavy metals for food safety. Among the current detection methods, technology based on fluorescent probes, with the advantages of sensitivity, convenience, accuracy, cost, and reliability, has recently shown pluralistic applications in the food industry, which is significant to ensure food safety. Hence, this review systematically presents the recent progress on novel fluorescent probes in determining heavy metals for food safety over the past five years, according to fluorophores and newly emerging sensing cores, which could contribute to broadening the prospects of fluorescent materials and establishing more practical assays for heavy metal determinations.

Zebularine elevates STING expression and enhances cGAMP cancer immunotherapy in mice.

DNA methylation abnormality is closely related to tumor occurrence and development. Chemical inhibitors targeting DNA methyltransferase (DNMTis) have been used in treating cancer. However, the impact of DNMTis on antitumor immunity has not been well elucidated. In this study, we show that zebularine (a demethylating agent) treatment of cancer cells led to increased levels of interferon response in a cyclic guanosine monophosphate-AMP (cGAMP) synthase (cGAS)- and stimulator of interferon genes (STING)-dependent manner. This treatment also specifically sensitized the cGAS-STING pathway in response to DNA stimulation. Incorporation of zebularine into genomic DNA caused demethylation and elevated expression of a group of genes, including STING. Without causing DNA damage, zebularine led to accumulation of DNA species in the cytoplasm of treated cells. In syngeneic tumor models, administration of zebularine alone reduced tumor burden and extended mice survival. This effect synergized with cGAMP and immune checkpoint blockade therapy. The efficacy of zebularine was abolished in nude mice and in cGAS-/- or STING-/- mice, indicating its dependency on host immunity. Analysis of tumor cells indicates upregulation of interferon-stimulated genes (ISGs) following zebularine administration. Zebularine promoted infiltration of CD8 T cells and natural killer (NK) cells into tumor and therefore suppressed tumor growth. This study unveils the role of zebularine in sensitizing the cGAS-STING pathway to promote anti-tumor immunity and provides the foundation for further therapeutic development.

Propagation of Gaussian Schell-model beams through a jet engine exhaust.

Theoretical predictions of light beam interactions with jet engine exhaust are of importance for optimization of various optical systems, including LIDARs, imagers and communication links operating in the vicinity of aircrafts and marine vessels. Here we extend the analysis previously carried out for coherent laser beams propagating in jet engine exhaust, to the broad class of Gaussian Schell-Model (GSM) beams, being capable of treating any degree of coherence in addition to size and radius of curvature. The analytical formulas for the spectral density (SD) and the spectral degree of coherence (DOC) of the GSM beam are obtained and analyzed on passage through a typical jet engine exhaust region. It is shown that for sources with high coherence, the transverse profiles of the SD and the DOC of the GSM beams gradually transition from initially circular to elliptical shape upon propagation at very short ranges. However, such transition is suppressed for sources with lower coherence and disappears in the incoherent source limit, implying that the GSM source with low source coherence is an excellent tool for mitigation of the jet engine exhaust-induced anisotropy of turbulence. The physical interpretation and the illustration are included.

Melanosome evolution indicates a key physiological shift within feathered dinosaurs.

Inference of colour patterning in extinct dinosaurs has been based on the relationship between the morphology of melanin-containing organelles (melanosomes) and colour in extant bird feathers. When this relationship evolved relative to the origin of feathers and other novel integumentary structures, such as hair and filamentous body covering in extinct archosaurs, has not been evaluated. Here we sample melanosomes from the integument of 181 extant amniote taxa and 13 lizard, turtle, dinosaur and pterosaur fossils from the Upper-Jurassic and Lower-Cretaceous of China. We find that in the lineage leading to birds, the observed increase in the diversity of melanosome morphologies appears abruptly, near the origin of pinnate feathers in maniraptoran dinosaurs. Similarly, mammals show an increased diversity of melanosome form compared to all ectothermic amniotes. In these two clades, mammals and maniraptoran dinosaurs including birds, melanosome form and colour are linked and colour reconstruction may be possible. By contrast, melanosomes in lizard, turtle and crocodilian skin, as well as the archosaurian filamentous body coverings (dinosaur 'protofeathers' and pterosaur 'pycnofibres'), show a limited diversity of form that is uncorrelated with colour in extant taxa. These patterns may be explained by convergent changes in the key melanocortin system of mammals and birds, which is known to affect pleiotropically both melanin-based colouration and energetic processes such as metabolic rate in vertebrates, and may therefore support a significant physiological shift in maniraptoran dinosaurs.

TargetLink, a new method for identifying the endogenous target set of a specific microRNA in intact living cells.

MicroRNAs are small non-coding RNAs acting as posttranscriptional repressors of gene expression. Identifying mRNA targets of a given miRNA remains an outstanding challenge in the field. We have developed a new experimental approach, TargetLink, that applied locked nucleic acid (LNA) as the affinity probe to enrich target genes of a specific microRNA in intact cells. TargetLink also consists a rigorous and systematic data analysis pipeline to identify target genes by comparing LNA-enriched sequences between experimental and control samples. Using miR-21 as a test microRNA, we identified 12 target genes of miR-21 in a human colorectal cancer cell by this approach. The majority of the identified targets interacted with miR-21 via imperfect seed pairing. Target validation confirmed that miR-21 repressed the expression of the identified targets. The cellular abundance of the identified miR-21 target transcripts varied over a wide range, with some targets expressed at a rather low level, confirming that both abundant and rare transcripts are susceptible to regulation by microRNAs, and that TargetLink is an efficient approach for identifying the target set of a specific microRNA in intact cells. C20orf111, one of the novel targets identified by TargetLink, was found to reside in the nuclear speckle and to be reliably repressed by miR-21 through the interaction at its coding sequence.

GLP-1 Receptor Mediated Targeting of a Fluorescent Zn(2+) Sensor to Beta Cell Surface for Imaging Insulin/Zn(2+) Release.

The pancreatic islet beta cell plays an essential role in maintaining the normal blood glucose level by releasing insulin. Loss of functional beta cell mass leads to diabetes­a disease affecting ∼9% of the population worldwide. There has been great interest and intense effort in developing imaging probes for monitoring islet beta cells, and glucagon-like peptide-1 receptor (GLP-1R) has emerged as a valuable biomarker for targeting beta cells. However, efforts thus far in GLP-1R mediated beta cell labeling and imaging has largely, if not exclusively, focused on developing imaging probes for monitoring beta cell mass, and few studies have investigated imaging beta cell function (insulin release) through GLP-1R. We now report the design and synthesis of a bioconjugate, ZIMIR-Ex4(9-39), that consists of a fluorescent Zn(2+) sensor and a truncated exendin 4 peptide for imaging insulin/Zn(2+) release in islet beta cells. In vitro, the conjugate bound to Zn(2+) with high affinity and displayed a robust fluorescence enhancement upon Zn(2+) chelation. When added to beta cells at submicromolar concentration, ZIMIR-Ex4(9-39) rapidly labeled cell surface in minutes to report the dynamics of insulin/Zn(2+) release with high spatiotemporal resolution. Future explorations of this approach may lead to probes for tracking beta cell function using different imaging modalities.

Imaging dynamic insulin release using a fluorescent zinc indicator for monitoring induced exocytotic release (ZIMIR).

Current methods of monitoring insulin secretion lack the required spatial and temporal resolution to adequately map the dynamics of exocytosis of native insulin granules in intact cell populations in three dimensions. Exploiting the fact that insulin granules contain a high level of Zn(2+), and that Zn(2+) is coreleased with insulin during secretion, we have developed a fluorescent, cell surface-targeted zinc indicator for monitoring induced exocytotic release (ZIMIR). ZIMIR displayed a robust fluorescence enhancement on Zn(2+) chelation and bound Zn(2+) with high selectivity against Ca(2+) and Mg(2+). When added to cultured ß cells or intact pancreatic islets at low micromolar concentrations, ZIMIR labeled cells rapidly, noninvasively, and stably, and it reliably reported changes in Zn(2+) concentration near the sites of granule fusion with high sensitivity that correlated well with membrane capacitance measurement. Fluorescence imaging of ZIMIR-labeled ß cells followed the dynamics of exocytotic activity at subcellular resolution, even when using simple epifluorescence microscopy, and located the chief sites of insulin release to intercellular junctions. Moreover, ZIMIR imaging of intact rat islets revealed that Zn(2+)/insulin release occurred largely in small groups of adjacent ß cells, with each forming a "secretory unit." Concurrent imaging of ZIMIR and Fura-2 showed that the amplitude of cytosolic Ca(2+) elevation did not necessarily correlate with insulin secretion activity, suggesting that events downstream of Ca(2+) signaling underlie the cell-cell heterogeneity in insulin release. In addition to studying stimulation-secretion coupling in cells with Zn(2+)-containing granules, ZIMIR may find applications in ß-cell engineering and screening for molecules regulating insulin secretion on high-throughput platforms.

Frequency-dependent mitochondrial Ca(2+) accumulation regulates ATP synthesis in pancreatic ß cells.

Pancreatic ß cells respond to increases in glucose concentration with enhanced metabolism, the closure of ATP-sensitive K(+) channels and electrical spiking. The latter results in oscillatory Ca(2+) influx through voltage-gated Ca(2+) channels and the activation of insulin release. The relationship between changes in cytosolic and mitochondrial free calcium concentration ([Ca(2+)]cyt and [Ca(2+)]mit, respectively) during these cycles is poorly understood. Importantly, the activation of Ca(2+)-sensitive intramitochondrial dehydrogenases, occurring alongside the stimulation of ATP consumption required for Ca(2+) pumping and other processes, may exert complex effects on cytosolic ATP/ADP ratios and hence insulin secretion. To explore the relationship between these parameters in single primary ß cells, we have deployed cytosolic (Fura red, Indo1) or green fluorescent protein-based recombinant-targeted (Pericam, 2mt8RP for mitochondria; D4ER for the ER) probes for Ca(2+) and cytosolic ATP/ADP (Perceval) alongside patch-clamp electrophysiology. We demonstrate that: (1) blockade of mitochondrial Ca(2+) uptake by shRNA-mediated silencing of the uniporter MCU attenuates glucose- and essentially blocks tolbutamide-stimulated, insulin secretion; (2) during electrical stimulation, mitochondria decode cytosolic Ca(2+) oscillation frequency as stable increases in [Ca(2+)]mit and cytosolic ATP/ADP; (3) mitochondrial Ca(2+) uptake rates remained constant between individual spikes, arguing against activity-dependent regulation ("plasticity") and (4) the relationship between [Ca(2+)]cyt and [Ca(2+)]mit is essentially unaffected by changes in endoplasmic reticulum Ca(2+) ([Ca(2+)]ER). Our findings thus highlight new aspects of Ca(2+) signalling in ß cells of relevance to the actions of both glucose and sulphonylureas.

Synthesis of a new fluorophore: wavelength-tunable bisbenzo[f]isoindolylidenes.

The creation of new functional molecules is a central task in chemical synthesis. Herein, we report the synthesis of a new type of fluorophore, bisbenzo[f]isoindolylidenes, from easily accessible dipropargyl benzenesulfonamides. Wavelength-tunable fluorophores emitting strong fluorescence of green to red light were obtained in this reaction. Late-stage modifications and incorporation of bioactive molecules into these fluorophores give rise to potential applications in biological studies. Detailed computational and experimental studies were conducted to elucidate the mechanism, and suggest a reaction sequence involving Garratt-Braverman type cyclization, isomerization, fragmentation, dimerization and oxidation.

DWL-4-140: A allene small molecule targeting STING that alleviates lupus-like phenotype in Trex1-/- mice.

The innate immune system plays a critical role in the host response against pathogenic microbial infection. However, aberrant activation of the innate immune pathways is a characteristic feature of various diseases. Thus, targeted drugs must be developed based on the understanding of the innate immune signaling pathways. This study demonstrated that an allene small molecule (DWL-4-140) can efficiently and selectively exert regulatory effects on the stimulator of interferon genes (STING), resulting in the downregulation of DNA-induced interferon responses. Mechanistically, DWL-4-140 targeted the cyclized nucleotide-binding domain (CBD) of STING, inhibiting the assembly of the STING multimeric complex and the recruitment of downstream signaling mediators. In addition to downregulating the 10-carboxymethyl-9-acridanone-induced production of inflammatory factors, DWL-4-140 alleviated the pathological features of Trex1 deletion-induced lupus in mice. Thus, this study demonstrated that DWL-4-140 pharmacologically inhibits STING with potential therapeutic applications in auto-inflammatory diseases.

Synthesis of unsymmetrically tetrasubstituted pyrroles and studies of AIEE in pyrrolo[1,2-a]pyrimidine derivatives.

Pyrroles are among the most important heterocycles in pharmaceuticals and agrochemicals. Construction of pyrrole scaffolds with different substituents and a free NH group, however, is challenging. Herein, a metal-free method for the synthesis of unsymmetrically tetrasubstituted NH-pyrroles using a consecutive chemoselective double cyanation is reported. The desired pyrroles were obtained with yields up to 99% and good functional group tolerance. Mechanistic studies identified a reaction mechanism that features a subtle sequence of first cyano-addition and migration, followed by cyano-addition and aromatization to afford the pyrrole skeleton. Pyrrolo[1,2-a]pyrimidines are synthesized as the synthetic applications of NH-pyrroles, and these pyrrolo[1,2-a]pyrimidines exhibit unpredicted time-dependent aggregation-induced emission enhancement (AIEE) properties.

Iron-Catalyzed Asymmetric Decarboxylative Azidation.

The first iron-catalyzed asymmetric azidation of benzylic peresters has been reported with trimethylsilyl azide (TMSN3) as the azido source. Hydrocarbon radicals that lack of strong interactions were capable to be enantioselectively azidated. The reaction features good functional group tolerance, high yields, and mild conditions. The chiral benzylic azides can further be used in click reaction, phosphoramidation, and reductive amination, which demonstrate the synthetic values of this reaction.

Gentamicin Combined With Hypoionic Shock Rapidly Eradicates Aquaculture Bacteria in vitro and in vivo.

Bacterial pathogens are a major cause of infectious diseases in aquatic animals. The abuse of antibiotics in the aquatic industry has led to the proliferation of antibiotic resistance. It is therefore essential to develop more effective and safer strategies to increase the efficacy and extend the life span of the antibiotics used in aquaculture. In this study, we show that six aquaculture bacterial pathogens (i.e., Aeromonas hydrophila, Vibrio alginolyticus, Edwardsiella tarda, Streptococcus iniae, Vibrio harveyi, and Vibrio fluvialis) in the stationary phase can be rapidly killed after immersion in gentamicin- or neomycin-containing, ion-free solutions for a few minutes. Such hypoionic shock treatment enhances the bacterial uptake of gentamicin in an ATP-dependent manner. Importantly, we demonstrate, as a proof of concept, that gentamicin under hypoionic shock conditions can effectively kill A. hydrophila in vivo in a skin infection model of zebrafish (Danio rerio), completely curing the infected fish. Given that pathogenic bacteria generally adhere to the skin surface and gills of aquatic animals, our strategy is of potential significance for bacterial infection control, especially for small-scale economic fish farming and ornamental fish farming. Further, the combined treatment can be completed within 5 min with a relatively small volume of solution, thus minimizing the amount of residual antibiotics in both animals and the environment.

Asymmetric radical carboesterification of dienes.

The straightforward strategy of building a chiral C-O bond directly on a general carbon radical center is challenging and stereocontrol of the reactions of open-chain hydrocarbon radicals remains a largely unsolved problem. Advance in this elementary step will spur the development of asymmetric radical C-O bond construction. Herein, we report a copper-catalyzed regioselective and enantioselective carboesterification of substituted dienes using alkyl diacyl peroxides as the source of both the carbon and oxygen substituents. The participation of external acids in this reaction substantially extends its applicability and leads to structurally diverse allylic ester products. This work represents the advance in the key elementary reaction of intermolecular enantioselective construction of C-O bond on open-chain hydrocarbon radicals and may lead to the discovery of other asymmetric radical reactions.

ZIGIR, a Granule-Specific Zn2+ Indicator, Reveals Human Islet α Cell Heterogeneity.

Numerous mammalian cells contain abundant Zn2+ in their secretory granules, yet available Zn2+ sensors lack the desired specificity and sensitivity for imaging granular Zn2+. We developed a fluorescent zinc granule indicator, ZIGIR, that possesses numerous desired properties for live cell imaging, including >100-fold fluorescence enhancement, membrane permeability, and selective enrichment to acidic granules. The combined advantages endow ZIGIR with superior sensitivity and specificity for imaging granular Zn2+. ZIGIR enables separation of heterogenous ß cells based on their insulin content and sorting of mouse islets into pure α cells and ß cells. In human islets, ZIGIR facilitates sorting of endocrine cells into highly enriched α cells and ß cells, reveals unexpectedly high Zn2+ activity in the somatostatin granule of some δ cells, and uncovers variation in the glucagon content among human α cells. We expect broad applications of ZIGIR for studying Zn2+ biology and Zn2+-rich secretory granules and for engineering ß cells with high insulin content for treating diabetes.

Celastrol ameliorates autoimmune disorders in Trex1-deficient mice.

Celastrol is one of most potent bioactive molecule isolated from the medicinal plant Tripterygium wilfordii (Thunder God Vine) and is well known for its potential therapeutic value against various chronic diseases including the autoimmune diseases, such as systemic lupus erythematosus and Aicardi-Goutieres syndrome, or other interferonopathies. However, the underlying mechanism of celastrol function remains unclear. Here we showed that celastrol caused inhibition of interferon regulatory factor 3 (IRF3) activation leading to the down-regulation of the interferon response triggered by cytosolic nucleic acids in vitro and in vivo. Moreover, celastrol treatment markedly ameliorates the autoimmune phenotypes including myocarditis, aberrant interferon response and autoantibody production, as well as the excessive T-cell activation in Trex1-/- autoimmune disease mouse model. Collectively, our results indicate that celastrol inhibits interferon response by targeting IRF3 activation and may be used as an effective treatment for interferon response-dependent autoimmune diseases.

The ratiometric fluorescent probes for monitoring the reactive inorganic sulfur species (RISS) signal in the living cell.

RSSs (reactive sulfur species) and their metabolites, such as H2S, Sn2-, SO32-/HSO3-, S2O42- and S2O52- (Reactive Inorganic Sulfur Species, RISSs), play a crucial role in the cushion against oxidative stress and the other physiological events. The molecular mechanisms how they affect cellular signaling and other physiological events remain largely unknown. To address their physiological functions, the techniques that can track their levels should be invaluable. Herein, six coumarin hemicyanine scaffolds (CH-RISSs) were synthesized and their fast and strong responses upon H2S, Sn2-, SO32-, HSO3-, S2O42- and S2O52- (Reactive Inorganic Sulfur Species, RISSs) were clarified in the absorption (colorimetric) and fluorescence (ratiometric) spectra, which showed good stability in the physiological pH (7.4). Upon the analytes, the maxima absorption of CH-RISSs switched from ~580 nm to ~400 nm in the absorption spectra. The fluorescence of CH-RISSs depleted at 650-660 nm and increased at 480-505 nm upon the RISSs. Both of coumarin hemicyanine structures with C12 alkyl chain (CH-RISS-3 and CH-RISS-6) showed quick and robust ratiometric fluorescence switch in the living cell imaging. Access to the fluorescent probes for RISSs sets the stage for applying the developing technologies to probe reactive sulfur biology in living systems.