Functionalized α-Cyanostilbene Derivatives for Detection of Hypoxia or Proteostasis Imbalance in Live Cells.

α-Cyanostilbene represents one of the easily functionalized aggregation-induced emission (AIE) scaffolds. It has been widely adopted for the construction of fluorescent materials for broad applications. Here, we further expanded the utilization of α-cyanostilbene derivatives for the detection of hypoxia or proteostasis imbalance in live cells. Four different amine containing donors were introduced to construct α-cyanostilbene derivatives (R-ASC) with donor-acceptor scaffolds. Equipped with the cysteine (Cys) reactive group, maleimide (MI), R-ASC-MI shows fluorescence turn-on property upon binding with unfolded proteins in vitro and in live cells under proteostatic stress. By virtue of R-ASC-MI, the level of unfolded protein loads in cells can be quantified by flow cytometry, or visualized under microscope. Furthermore, we also characterized the performance of R-ASC-NO2, synthetic precursors of R-ASC-MI, in cellular hypoxia. R-ASC-NO2 revealed upregulated activities of nitroreductase, as well as increased hydrophobicity in live cells, under either chemical (NaN3) induced or atmospheric (1% O2) hypoxia. Together, the advantages of easy modification and high signal-to-noise ratio of new α-cyanostilbene derivatives reported in this work highlight the great potential of α-cyanostilbene in constructing functional biosensors and many other domains.

The fluorescence toolbox for visualizing autophagy.

Autophagy is an adaptive catabolic process functioning to promote cell survival in the event of inappropriate living conditions such as nutrient shortage and to cope with diverse cytotoxic insults. It is regarded as one of the key survival mechanisms of living organisms. Cells undergo autophagy to accomplish the lysosomal digestion of intracellular materials including damaged proteins, organelles, and foreign bodies, in a bulk, non-selective or a cargo-specific manner. Studies in the past decades have shed light on the association of autophagy pathways with various diseases and also highlighted the therapeutic value of autophagy modulation. Hence, it is crucial to develop effective approaches for monitoring intracellular autophagy dynamics, as a comprehensive account of methodology establishment is far from complete. In this review, we aim to provide an overview of the major current fluorescence-based techniques utilized for visualizing, sensing or measuring autophagic activities in cells or tissues, which are categorized firstly by targets detected and further by the types of fluorescence tools. We will mainly focus on the working mechanisms of these techniques, put emphasis on the insight into their roles in biomedical science and provide perspectives on the challenges and future opportunities in this field.

Achieving Efficient Multichannel Conductance in Through-Space Conjugated Single-Molecule Parallel Circuits.

Constructing single-molecule parallel circuits with multiple conduction channels is an effective strategy to improve the conductance of a single molecular junction, but rarely reported. We present a novel through-space conjugated single-molecule parallel circuit (f-4Ph-4SMe) comprised of a pair of closely parallelly aligned p-quaterphenyl chains tethered by a vinyl bridge and end-capped with four SMe anchoring groups. Scanning-tunneling-microscopy-based break junction (STM-BJ) and transmission calculations demonstrate that f-4Ph-4SMe holds multiple conductance states owing to different contact configurations. When four SMe groups are in contact with two electrodes at the same time, the through-bond and through-space conduction channels work synergistically, resulting in a conductance much larger than those of analogous molecules with two SMe groups or the sum of two p-quaterphenyl chains. The system is an ideal model for understanding electron transport through parallel π-stacked molecular systems and may serve as a key component for integrated molecular circuits with controllable conductance.

Remarkable Multichannel Conductance of Novel Single-Molecule Wires Built on Through-Space Conjugated Hexaphenylbenzene.

Through-bond conjugated molecules are the major frameworks for traditional molecular wires, while through-space conjugated units are rarely utilized and studied although they have shown unique conducting potential. Herein, we present novel single-molecule wires built on through-space conjugated hexaphenylbenzene. Their conductance, measured by the scanning tunneling microscopy based break-junction technique, increases with the improvement of through-space conjugation and finally reaches a remarkable value (12.28 nS) which greatly exceeds that of conventional through-bond conjugated counterpart (2.45 nS). The multichannel conducting model by integrating through-space and through-bond conjugations could be a promising strategy for the further design of robust single-molecule wires with advanced conductance and stability.

Barbituric Acid Based Fluorogens: Synthesis, Aggregation-Induced Emission, and Protein Fibril Detection.

Fluorescent dyes, especially those emitting in the long wavelength region, are excellent candidates in the area of bioassay and bioimaging. In this work, we report a series of simple organic fluorescent dyes consisting of electron-donating aniline groups and electron-withdrawing barbituric acid groups. These dyes are very easy to construct while emitting strongly in the red region in their solid state. The photophysical properties of these dyes, such as solvatochromism and aggregation-induced emission, are systematically characterized. Afterward, the structure-property relationships of these barbituric acid based fluorogens are discussed. Finally, we demonstrate their potential applications for protein amyloid fibril detection.

Role of human amnion-derived mesenchymal stem cells in promoting osteogenic differentiation by influencing p38 MAPK signaling in lipopolysaccharide -induced human bone marrow mesenchymal stem cells.

Periodontitis is a chronic inflammatory disease induced by bacterial pathogens, which not only affect connective tissue attachments but also cause alveolar bone loss. In this study, we investigated the anti-inflammatory effects of Human amnion-derived mesenchymal stem cells (HAMSCs) on human bone marrow mesenchymal stem cells (HBMSCs) under lipopolysaccharide (LPS)-induced inflammatory conditions. Proliferation levels were measured by flow cytometry and immunofluorescence staining of 5-ethynyl-2'-deoxyuridine (EdU). Osteoblastic differentiation and mineralization were investigated using chromogenic alkaline phosphatase activity (ALP) activity substrate assays, Alizarin red S staining, and RT-PCR analysis of HBMSCs osteogenic marker expression. Oxidative stress induced by LPS was investigated by assaying reactive oxygen species (ROS) level and superoxide dismutase (SOD) activity. Here, we demonstrated that HAMSCs increased the proliferation, osteoblastic differentiation, and SOD activity of LPS-induced HBMSCs, and down-regulated the ROS level. Moreover, our results suggested that the activation of p38 MAPK signal transduction pathway is essential for reversing the LPS-induced bone-destructive processes. SB203580, a selective inhibitor of p38 MAPK signaling, significantly suppressed the anti-inflammatory effects in HAMSCs. In conclusion, HAMSCs show a strong potential in treating inflammation-induced bone loss by influencing p38 MAPK signaling.

MiR-224-5p inhibits osteoblast differentiation and impairs bone formation by targeting Runx2 and Sp7.

Osteoporosis is a complicated multifactorial disorder characterized by low bone mass and deteriorated bone microarchitecture with an elevated fracture risk. MicroRNAs play important roles in osteoblastic differentiation. In the present study, we found that miR-224-5p was markedly downregulated during the osteogenic differentiation of C2C12 cells. Overexpression of miR-224-5p in C2C12 cells inhibited osteoblast activity, as indicated by reduced ALP activity, matrix mineralization and the expression of osteogenic marker genes. Moreover, we demonstrated that Runx2 and Sp7 were direct targets of miR-224-5p. Furthermore, the specific inhibition of miR-224-5p by femoral bone marrow cavity injection with miR-224-5p antagomir prevented ovariectomy-induced bone loss. Finally, we found that the levels of miR-224-5p were markedly elevated in the sera of patients with osteoporosis. Collectively, this study revealed that miR-224-5p negatively regulates osteogenic differentiation by targeting Runx2 and Sp7. It also highlights the potential use of miR-224-5p as a therapeutic target and diagnostic biomarker for osteoporosis. Supplementary information: The online version contains supplementary material available at 10.1007/s10616-023-00593-z.

A novel red-emitting aggregation-induced emission probe for determination of ß-glucosidase activity.

ß-Glucosidase (ß-Glu) is a ubiquitous enzyme which has multiple roles in medical diagnosis, food production, agriculture, etc. Existing ß-Glu assays have limitations such as complex operation, long running time, and high background noise. Here we report a red-emissive probe TBPG for measuring the activity of ß-Glu. The probe was synthesized through conjugating a ß-Glu targeting glucoside to an aggregation-induced emission (AIE) fluorophore. In the presence of ß-Glu, TBPG was hydrolyzed and exhibited a fluorescence turn-on process. The detection conditions including time, temperature, pH value, buffer, and probe concentration were optimized systematically. Afterwards, fluorescence titration was conducted showing an excellent linearity (R2 = 0.998), a wide linear dynamic range (0-5.0 U/mL), and a limit of detection as low as 0.6 U/L. The detection specificity and ion interference were evaluated by adding various biological species and ions to probe without or with ß-Glu. Next, we demonstrate the applicability of probe TBPG in determining the ß-Glu activity in living cells using confocal microscopy and flow cytometry. Finally, this newly established assay was applied to real soil samples. Comparable results were obtained as the commercial assay, manifesting its great potential in soil enzyme analysis.

METTL3-mediated m6A modification of IGFBP7-OT promotes osteoarthritis progression by regulating the DNMT1/DNMT3a-IGFBP7 axis.

Osteoarthritis (OA) is the most common degenerative disorder, affecting approximately half of the elderly population. In this study, we find that the expressions of long noncoding RNA (lncRNA) IGFBP7-OT and its maternal gene, IGFBP7, are upregulated and positively correlated in osteoarthritic cartilage. Overexpression of IGFBP7-OT significantly inhibits chondrocyte viability, promotes chondrocyte apoptosis, and reduces extracellular matrix components, whereas IGFBP7-OT knockdown has the opposite effects. IGFBP7-OT overexpression promotes cartilage degeneration and markedly aggravates the monosodium iodoacetate-induced OA phenotype in vivo. Further mechanistic research reveals that IGFBP7-OT promotes OA progression by upregulating IGFBP7 expression. Specifically, IGFBP7-OT suppresses the occupancy of DNMT1 and DNMT3a on the IGFBP7 promoter, thereby inhibiting methylation of the IGFBP7 promoter. The upregulation of IGFBP7-OT in OA is partially controlled by METTL3-mediated N6-methyladenosine (m6A) modification. Collectively, our findings reveal that m6A modification of IGFBP7-OT promotes OA progression by regulating the DNMT1/DNMT3a-IGFBP7 axis and provide a potential therapeutical target for OA treatment.

ALKBH5-mediated m6A demethylation of HS3ST3B1-IT1 prevents osteoarthritis progression.

HS3ST3B1-IT1 was identified as a downregulated long noncoding RNA in osteoarthritic cartilage. However, its roles and mechanisms in the pathogenesis of osteoarthritis (OA) are unclear. In this study, we demonstrated that the expressions of HS3ST3B1-IT1 and its maternal gene HS3ST3B1 were downregulated and positively correlated in osteoarthritic cartilage. Overexpression of HS3ST3B1-IT1 significantly increased chondrocyte viability, inhibited chondrocyte apoptosis, and upregulated extracellular matrix (ECM) proteins, whereas HS3ST3B1-IT1 knockdown had the opposite effects. In addition, HS3ST3B1-IT1 significantly ameliorated monosodium-iodoacetate-induced OA in vivo. Mechanistically, HS3ST3B1-IT1 upregulated HS3ST3B1 expression by blocking its ubiquitination-mediated degradation. Knockdown of HS3ST3B1 reversed the effects of HS3ST3B1-IT1 on chondrocyte viability, apoptosis, and ECM metabolism. AlkB homolog 5 (ALKBH5)-mediated N6-methyladenosine (m6A) demethylation stabilized HS3ST3B1-IT1 RNA. Together, our data revealed that ALKBH5-mediated upregulation of HS3ST3B1-IT1 suppressed OA progression by elevating HS3ST3B1 expression, suggesting that HS3ST3B1-IT1/HS3ST3B1 may serve as potential therapeutic targets for OA treatment.

Aggregation-Induced Emission (AIE), Life and Health.

Light has profoundly impacted modern medicine and healthcare, with numerous luminescent agents and imaging techniques currently being used to assess health and treat diseases. As an emerging concept in luminescence, aggregation-induced emission (AIE) has shown great potential in biological applications due to its advantages in terms of brightness, biocompatibility, photostability, and positive correlation with concentration. This review provides a comprehensive summary of AIE luminogens applied in imaging of biological structure and dynamic physiological processes, disease diagnosis and treatment, and detection and monitoring of specific analytes, followed by representative works. Discussions on critical issues and perspectives on future directions are also included. This review aims to stimulate the interest of researchers from different fields, including chemistry, biology, materials science, medicine, etc., thus promoting the development of AIE in the fields of life and health.

Low temperature delays degreening of apple fruit by inhibiting pheophorbide a oxygenase (PAO) pathway and chlorophyll oxidation during ripening.

The effects of low temperature (LT) on chlorophyll (Chl) degradation in peel of apple fruit during ripening were investigated. Apples collected at commercial maturity were stored at 4 ± 0.5°C. Our data indicated that LT treatment reduced respiration rate and ethylene production and slowed down softening of apple fruit during ripening. The LT treatment delayed increase in L*, a*, and b* values and decrease in Chl content compared with controls. The LT treatment reduced hydrogen peroxide (H2 O2 ) and malondialdehyde (MDA) contents and decelerated superoxide anion (O2 ·- ) production rate in chloroplast of peel compared with controls during ripening. The LT treatment differentially reduced activities of pheophytin pheophorbide hydrolase (PPH), Mg-dechelatase (MDcase), chlorophyll-degrading peroxidase (Chl-POX), and Chl oxidase, while enhanced SOD activity in chloroplast of peel during ripening. Expression levels of MdHCARa, MdNYC1, MdNYC3, MdNYE1, MdRCCR2, MdPPH1, MdPAO6, MdPAO8, and MdNOL2 in peel were differentially reduced by LT treatment during ripening. Our results indicated that LT treatment might delay Chl degradation through inhibiting PAO pathway and Chl oxidation during ripening of apple fruit. PRACTICAL APPLICATIONS: The LT is a common practice used to extend storage life of apple fruit. Degreening caused by Chl degradation is an integral part of fruit ripening, and elucidating its mechanism is an important subject for fruit quality maintenance. Our data indicated that LT delayed degreening of apple fruit by inhibiting PAO pathway and Chl oxidation during ripening. These results will provide useful information for clarifying molecular mechanisms of LT in regulation of degreening and also for quality maintenance of apple fruit.

Red blood cell membrane-camouflaged nanoparticles loaded with AIEgen and Poly(I : C) for enhanced tumoral photodynamic-immunotherapy.

Red blood cell (RBC)-mimicking nanoparticles (NPs) offer a promising platform for drug delivery because of their prolonged circulation time, reduced immunogenicity and specific targeting ability. Herein, we report the design and preparation of RBC membrane-bound NPs (M@AP), for tumoral photodynamic-immunotherapy. The M@AP is formed by self-assembly of the positively charged aggregation-induced emission luminogen (AIEgen) (named P2-PPh3) and the negatively charged polyinosinic : polycytidylic acid (Poly(I : C)), followed by RBC membrane encapsulation. P2-PPh3 is an AIE-active conjugated polyelectrolyte with additional photosensitizing ability for photodynamic therapy (PDT), while Poly(I : C) serves as an immune-stimulant to stimulate both tumor and immune cells to activate immunity, and thus reduces tumor cell viability. When applied in tumor-bearing mice, the M@AP NPs are enriched in both the tumor region as a result of an enhanced permeability and retention (EPR) effect, and the spleen because of the homing effect of the RBC-mimicking shell. Upon light irradiation, P2-PPh3 promotes strong ROS generation in tumor cells, inducing the release of tumor antigens (TA). The anti-tumor immunity is further enhanced by the presence of Poly(I : C) in M@AP. Thus, this strategy combines the PDT properties of the AIE-active polyelectrolyte and immunotherapy properties of Poly(I : C) to achieve synergistic activation of the immune system for anti-tumor activity, providing a novel strategy for tumor treatment.

Effects of 1-MCP treatment on sprouting and preservation of ginger rhizomes during storage at room temperature.

The purpose of this study was to explore the effects of 1-MCP on the sprouting and preservation of ginger rhizomes during storage at room temperature. Ginger rhizomes were treated with 1 µL L-1 1-methylcyclopropene (1-MCP) and stored at 23 ± 0.2 °C. Our data showed that application of 1-MCP reduced the rate of sprouting during storage compared with the control rhizome. Respiration rate and the reducing sugar content were also reduced following 1-MCP treatment, while the starch content increased. 1-MCP treatment increased the total phenol content and inhibited polyphenol oxidase (PPO) activity. 1-MCP treatment was also associated with a higher ascorbic acid content but a reduced crude fiber content. The generation of superoxide anion free radicals (O2-), hydrogen peroxide (H2O2) and malondialdehyde (MDA) was lower following 1-MCP treatment, while the activities of catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) were higher compared with the controls. These results suggested that application of 1-MCP could reduce sprouting rates, decrease the accumulation of ROS, and maintain the quality of ginger rhizomes during storage at room temperature. It would be useful to further explore the role and mechanisms of action of ethylene in regulating the sprouting of ginger rhizomes.

Aggregation-Induced Emission Photosensitizers: From Molecular Design to Photodynamic Therapy.

Photodynamic therapy (PDT) has emerged as a promising noninvasive treatment option for cancers and other diseases. The key factor that determines the effectiveness of PDT is the photosensitizers (PSs). Upon light irradiation, the PSs would be activated, produce reactive oxygen species (ROS), and induce cell death. One of the challenges is that traditional PSs adopt a large flat disc-like structure, which tend to interact with the adjacent molecules through strong π-π stacking that reduces their ROS generation ability. Aggregation-induced emission (AIE) molecules with a twisted configuration to suppress strong intermolecular interactions represent a new class of PSs for image-guided PDT. In this Miniperspective, we summarize the recent progress on the design rationale of AIE-PSs and the strategies to achieve desirable theranostic applications in cancers. Subsequently, approaches of combining AIE-PS with other imaging and treatment modalities, challenges, and future directions are addressed.

Optimising molecular rotors to AIE fluorophores for mitochondria uptake and retention.

Molecular rotors exhibit fluorescence enhancement in a confined environment and thus have been used extensively in biological imaging. However, many molecular rotors suffer from small Stokes shift and self-aggregation caused quenching. In this work, we have synthesised a series of red emissive molecular rotors based on cationic α-cyanostilbene. Profoundly enhanced aggregation-induced emission (AIE) properties and greatly widened Stokes shifts can be achieved by molecular engineering. With specificity to stain mitochondria, we demonstrate a simple approach to achieve cell uptake and retention upon tuning the pyridinium substituent of the dyes.

Effects of calcium chloride treatment on softening in red raspberry fruit during low-temperature storage.

Fruit softening is an inevitable event during ripening of red raspberry fruit even when stored at low temperature. In this research, the effects of CaCl2 treatment on softening of red raspberry during storage at 4°C were studied. The results indicated that CaCl2 treatment effectively delayed the decrease of firmness and reduced the respiration rate of red raspberry fruit during storage. The CaCl2 -treated fruit maintained higher protopectin content and lower soluble pectin content compared with controls. The cellulose and starch contents in the fruit treated with CaCl2 kept higher than in the control during storage. Moreover, CaCl2 treatment decreased activities of polygalacturonase (PG), pectin methylesterase (PME), and cellulase (Cx) mainly at the early stage of softening. Application of CaCl2 lead to the decreased activities of amylase (AM) and ß-galactosidase (ß-gal) compared with controls during the entire storage periods. These results indicated that CaCl2 treatment might delay postharvest softening of red raspberry fruit stored at low-temperature by retarding cell wall degradation and starch hydrolysis. PRACTICAL APPLICATIONS: Red raspberry fruit is very popular with consumers because of its high-nutritional value and anticancer effects. However, it has a very short postharvest life and softens easily even when stored at low temperature, which limits its distribution to distant market. Our data indicated that CaCl2 treatment delayed postharvest softening of red raspberry fruit stored at low temperature. The results could provide preliminary yet essential information to research community to further study the molecular mechanisms of softening in red raspberry fruit, and also provide reference data for maintaining quality of postharvest red raspberry fruit.

A Maleimide-functionalized Tetraphenylethene for Measuring and Imaging Unfolded Proteins in Cells.

Collapse of the protein homeostasis (proteostasis) can lead to accumulation and aggregation of unfolded proteins, which has been found to associate with a number of disease conditions including neurodegenerative diseases, diabetes and inflammation. Here we report a maleimide-functionalized tetraphenylethene (TPE)-derivatized fluorescent dye, TPE-NMI, which shows fluorescence turn-on property upon reacting with unfolded proteins in vitro and in live cells under proteostatic stress conditions. The level of unfolded proteins can be measured by flow cytometry and visualized with confocal microscopy.

Synthesis, aggregation-enhanced emission, polymorphism and piezochromism of TPE-cored foldamers with through-space conjugation.

A series of new folded tetraphenylethene derivatives with different substituents are stereoselectively synthesized, which exhibit interesting through-space conjugation, aggregation-enhanced emission, polymorphism and piezochromism properties.

In vitro and in vivo studies of surface-structured implants for bone formation.

BACKGROUND AND METHODS: Micronanoscale topologies play an important role in implant osteointegration and determine the success of an implant. We investigated the effect of three different implant surface topologies on osteoblast response and bone regeneration. In this study, implants with nanotubes and micropores were used, and implants with flat surfaces were used as the control group. RESULTS: Our in vitro studies showed that the nanostructured topologies improved the proliferation, differentiation, and development of the osteoblastic phenotype. Histological analysis further revealed that the nanotopology increased cell aggregation at the implant-tissue interfaces and enhanced bone-forming ability. Pushout testing indicated that the nanostructured topology greatly increased the bone-implant interfacial strength within 4 weeks of implantation. CONCLUSION: Nanotopography may improve regeneration of bone tissue and shows promise for dental implant applications.