Fluorescence Image Analysis Research Articles

Disrupting CENP-N mediated SEPT9 methylation as a strategy to inhibit aerobic glycolysis and liver metastasis in colorectal cancer.

Colorectal cancer (CRC) is a prevalent malignancy with a high mortality rate, primarily due to liver metastasis. This study explores the role of centromere protein N (CENP-N) in mediating the methylation of septin 9 (SEPT9) and its subsequent effects on aerobic glycolysis and liver metastasis in CRC. We employed in vitro and in vivo experiments, including single-cell RNA sequencing, methylation-specific PCR (MSP), ChIP assays, and various functional assays to assess the impact of CENP-N and SEPT9 on CRC cell proliferation, migration, invasion, and metabolic reprogramming. Our data reveal that CENP-N directly interacts with SEPT9, enhancing its methylation at specific lysine residues. This modification significantly upregulates key glycolytic enzymes, thereby promoting aerobic glycolysis, CRC cell proliferation, and migration. In vivo studies further demonstrate that the CENP-N/SEPT9 axis facilitates liver metastasis of CRC, as confirmed by fluorescence imaging and histological analysis. This study identifies a novel pathway where CENP-N-mediated methylation of SEPT9 drives metabolic reprogramming and metastasis in CRC. These findings suggest potential therapeutic targets for inhibiting CRC progression and liver metastasis, offering new insights into CRC pathogenesis.

Highly fluorescent nitrogen doped carbon dots as analytical probe for sensitive detection of curcumin through smartphone integrated 3D-printed platform: A new horizon in food safety

COVID-19 pandemic has significantly influenced the dietary habits of humans, emphasizing the incorporation of natural ingredients to enhance immunity towards viral and bacterial infections. Curcumin (Cur), a widely used traditional medicine in various Asian countries and a natural coloring agent, has gained popularity, leading to surge in its usage specially in post COVID-19 era. This surge has led to increased scrutiny of the potential side effects of excessive Cur use, with recent reports suggesting it may result in inactivation of DNA and reduce adenosine triphosphate levels, leading to health risks. In this work, we synthesized highly fluorescent nitrogen-doped carbon dots with a photoluminescence quantum yield of 72.9 % for the sensitive and selective detection of Cur. The developed fluorescent probe exhibits excellent sensory response towards Cur within a concentration range of 0.081–51.45 µM, achieving an ultra-low detection limit of 15.91 nM. The sensor was successfully tested on real food samples like ginger powder, turmeric powder, and curry powder, demonstrating good recovery rates. To assess the practicality of the sensor system, we developed a 3D-printed smartphone-integrated device platform for curcumin detection through fluorescence image analysis. This developed platform exhibited promising results, achieving a limit of detection (LoD) of 132.28 nM across a curcumin concentration range of 0.13–54.00 µM. This device platform holds significant potential for the development of efficient sensors for real-time detection of Cur in food samples.

Rosmarinic acid attenuates glioblastoma cells and spheroids’ growth and EMT/stem-like state by PTEN/PI3K/AKT downregulation and ERK-induced apoptosis

BackgroundGlioblastoma (GB) is a highly malignant type of brain cancer with a poor prognosis. Therapeutic strategies for GB are still limited. Rosmarinic acid (RA), a polyphenolic compound, is a promising experimental anticancer agent, but its specific protein targets for GB remain unclear. PurposeThis study aimed to elucidate the anticancer effects of RA in 2D- and 3D-GB cells and the underlying mechanisms. Methods3D-tumor spheroids (mimics in vivo tumors) were obtained by the hanging-drop/agarose method. RA's anti-glioma activity on U-87MG (p53-wt/PTEN-mt) and LN229 (p53-mt/PTEN-wt) cells was evaluated through cell viability, colony-formation, migration/invasion/angiogenesis assays, fluorescence imaging, and spheroid growth analysis. The underlying mechanism of the anticancer effects of RA was investigated by Western blot and immunofluorescence analysis. The MEK inhibitor U0126 was used to block ERK phosphorylation. ResultsRA treatments exerted anti-proliferative and pro-apoptotic effects on human GB cells. RA dose-dependently reduced angiogenesis and intracellular ROS levels, suppressed glioma growth, and migration/invasion in 2D-culture and cancer stem cell (CSC)-like 3D-spheroid culture (SPC). Repeated therapy in SPC was more effective by leading to disrupted structure than a single treatment. Treatments in SPC also suppressed epithelial-mesenchymal transition (EMT) and CSC-like properties. Strikingly, RA downregulated the SIRT1/FOXO1/NF-κB axis independently of p53 or PTEN function in both gliomas. Immunofluorescence labeling revealed decreased SIRT1 and NF-κB-p65 and increased FOXO1 and GAPDH proteins in nuclear location (associated with apoptosis). Surprisingly, RA increased p-ERK1/2 levels, but priming with U0126 abolished RA-mediated p-ERK upregulation; thus, autophagy and apoptosis induction in GB cells were prevented, and the growth of GB spheroids accelerated. Specifically, RA also inhibited the PTEN/PI3K/AKT pathway in U-87MG cells. Due to genetic differences in cells, U-87MG cells were more sensitive to RA treatments than LN229 cells. Meanwhile, our positive control drug trial results with FDA-approved temozolomide (TMZ) used in GB treatment showed that our test compound rosmarinic acid exhibited higher therapeutic effects than TMZ at lower doses. ConclusionSuppression of EMT, downregulation of SIRT1/FOXO1/NF-κB axis, inhibition of PTEN/PI3K/AKT signaling pathway, and ERK-induced apoptosis and autophagy were determined to be involved in stopping glioma progression. Our findings for the first time, revealed that RA may have potential therapeutic use by having multiple targets in human brain cancer with further clinical studies.

Targeting Myeloid-Derived Suppressor Cells via Dual-Antibody Fluorescent Nanodiamond Conjugate.

Fluorescent nanodiamonds (FNDs) are carbon-based nanomaterials that emit bright, photostable fluorescence and exhibit a modifiable surface chemistry. Myeloid-derived suppressor cells (MDSCs) are an immunosuppressive cell population known to expand in cancer patients and contribute to worse patient outcomes. To target MDSC, glycidol-coated FND were conjugated with antibodies against the murine MDSC markers, CD11b and GR1 (dual-Ab FND). In vitro, dual-Ab FND uptake by murine MDSC was significantly higher than IgG-coated FND (94.7% vs. 69.0%, p < 0.05). In vivo, intra-tumorally injected dual-Ab FND primarily localized to the tumor 2 and 24 h post-injection, as measured by in vivo fluorescence imaging and flow cytometry analysis of the spleen and tumor. Dual-Ab FND were preferentially taken up by intra-tumoral MDSC, representing 87.1% and 83.0% of FND+ cells in the tumor 2 and 24 h post-injection, respectively. Treatment of mice with anti-PD-L1 immunotherapy prior to intra-tumoral injection of dual-Ab FND did not significantly alter the uptake of FND by MDSC. These results demonstrate the ability of our novel dual-antibody conjugated FND to target MDSC and reveal a potential strategy for targeted delivery to other specific immune cell populations in future cancer research.

Sensitive Cancer Hypoxia Detection via a Dual-Locking Fluorescence Response System Using Two Hypoxia Indicators.

Hypoxia is intricately associated with various diseases, including ischemia, vascular disorders, and cancer. Particularly in cancer cells, hypoxia promotes tumor growth, cell proliferation, migration, and invasion and enhances treatment resistance, making its detection crucial for cancer diagnosis and therapy. However, methods for detecting hypoxia are limited, often relying on single-detection systems. In this study, we developed a dual-lock-based fluorescent probe that selectively exhibits strong green fluorescence under hypoxic conditions due to simultaneous activity of nitroreductases (NTRs) and hydrogen sulfide (H2S), with a high signal-to-background ratio. The biocompatibility and photophysical properties of the probes were thoroughly investigated through both extracellular and intracellular experimental analyses. Among the synthesized naphthalimide-based probes, the dual-detection probe DNNC demonstrated excellent selectivity and sensitivity to the simultaneous activity of NTR/H2S compared to other single-detection probes. The performance of DNNC was applied to various organ-derived cancer cells and tumor tissue models such as HeLa cell sparoids, enabling spatiotemporal confocal fluorescence imaging and quantitative analysis of hypoxic levels in cancer. Our development of DNNC is expected to significantly advance cancer diagnosis and treatment by molecularly detecting hypoxia associated with cancer aggressiveness, therapy resistance, and unfavorable prognosis.

Uptake of substances into living mammalian cells by microwave induced perturbation of the plasma membrane

Delivering foreign molecules and genetic material into cells is a crucial process in life sciences and biotechnology, resulting in great interest in effective cell transfection methods. Importantly, physical transfection methods allow delivery of molecules of different chemical composition and are, thus, very flexible. Here, we investigated the influence of microwave radiation on the transfection and survival of mammalian cells. We made use of an optimized microwave-poration device and analyzed its performance (frequency and electric field strength) in comparison with simulations. We, then, tested the effect of microwave irradiation on cells and found that 18 GHz had the least impact on cell survival, viability, cell division and genotoxicity while 10 GHz drastically impacted cell physiology. Using live-cell fluorescence microscopy and image analysis, we tested the uptake of small chemical substances, which was most efficient at 18 GHz and correlated with electric field strength and frequency. Finally, we were able to obtain cellular uptake of molecules of very different chemical composition and sizes up to whole immunoglobulin antibodies. In conclusion, microwave-induced poration enables the uptake of widely different substances directly into mammalian cells growing as adherent cultures and with low physiological impact.

A digital aptasensor for the ultrasensitive detection of T-2 toxin by using single molecule array

T-2 toxin is a low molecular weight, refractory mycotoxin, often contaminating cereal-based foods, and can cause detrimental effects on human health such as nausea, vomiting, and long-term immune suppression. Therefore, highly sensitive detection platform for T-2 toxin test at the molecular level is an urgent need for food safety and public health. In this work, a digital aptasensor is developed for the ultrasensitive detection of T-2 toxin by combining aptamer and single-molecule array technology. Initially, T-2 toxin competed with the biotin-labeled complementary sequence probe to bind the T-2 toxin aptamer. The free biotin-labeled probe was then hybridized with a capture probe modified on the magnetic beads to form a stable double-stranded structure. After combining with streptavidin-enzyme, the structure was used for fluorescence imaging analysis via single-molecule microwell arrays. By correlating the concentration of T-2 toxin with the number of fluorescent microtiter wells, the ultra-sensitive detection of the T-2 toxin was realized. The developed digital aptasensor has demonstrated remarkable sensitivity toward T-2 toxin, with a detection range between 0.16 and 300 pM (approximately 0.075–140 pg/mL) and a detection limit of 44.5 fM (approximately 20.8 fg/mL). Additionally, 87.0 %-103.1 % recoveries were obtained from spiked samples in corn. This work offers a highly effective approach to detecting trace amounts of T-2 toxin, which has the potential to be used for enhancing food safety protocols, providing a powerful tool for industries and regulatory agencies to protect public health.

High-Throughput Screening of Antiviral Compounds Using a Recombinant Hepatitis B Virus and Identification of a Possible Infection Inhibitor, Skimmianine.

We developed a novel hepatitis B virus (HBV) infection-monitoring system using a luminescent, 11-amino acid reporter (HiBiT). We performed high-throughput antiviral screening using this system to identify anti-HBV compounds. After the infection of primary human hepatocytes with the recombinant virus HiBiT-HBV, which contains HiBiT at its preS1, 1262 compounds were tested in a first screening using extracellular HiBiT activity as an indicator of viral infection. Following a second screening, we focused on the compound skimmianine, which showed a potent antiviral effect. When skimmianine was added at the same time as HiBiT-HBV infection, skimmianine inhibited HiBiT activity with EC50 of 0.36 pM, CC50 of 1.67 μM and a selectivity index (CC50:EC50 ratio) of 5,100,000. When skimmianine was added 72 h after HiBiT-HBV infection, the EC50, CC50 and selectivity index were 0.19 μM, 1.87 μM and 8.79, respectively. Time-lapse fluorescence imaging analysis using another recombinant virus, ReAsH-TC155HBV, with the insertion of tetra-cysteine within viral capsid, revealed that skimmianine inhibited the accumulation of the capsid into hepatocytes. Furthermore, skimmianine did not inhibit either attachment or internalization. These results imply that skimmianine inhibits the retrograde trafficking of the virus after internalization. This study demonstrates the usefulness of the recombinant virus, HiBiT-HBV, for high-throughput screening to identify anti-HBV compounds.

Aptamer-Loop DNA Nanoflower Recognition and Multicolor Fluorescent Carbon Quantum Dots Labeling System for Multitarget Living Cell Imaging.

Visualization of multiple targets in living cells is important for understanding complex biological processes, but it still faces difficulties, such as complex operation, difficulty in multiplexing, and expensive equipment. Here, we developed a nanoplatform integrating a nucleic acid aptamer and DNA nanotechnology for living cell imaging. Aptamer-based recognition probes (RPs) were synthesized through rolling circle amplification, which were further self-assembled into DNA nanoflowers encapsulated by an aptamer loop. The signal probes (SPs) were obtained by conjugation of multicolor emission carbon quantum dots with oligonucleotides complementary to RPs. Through base pairing, RPs and SPs were hybridized to generate aptamer sgc8-, AS1411-, and Apt-based imaging systems. They were used for individual/simultaneous imaging of cellular membrane protein PTK7, nucleolin, and adenosine triphosphate (ATP) molecules. Fluorescence imaging and intensity analysis showed that the living cell imaging system can not only specifically recognize and efficiently bind their respective targets but also provide a 5-10-fold signal amplification. Cell-cycle-dependent distribution of nucleolin and concentration-dependent fluorescence intensity of ATP demonstrated the utility of the system for tracking changes in cellular status. Overall, this system shows the potential to be a simple, low-cost, highly selective, and sensitive living cell imaging platform.

Renal Clearable H‐Dots Leveraging Ligand Complexation for Enhanced Active Tumor Targeting

The use of ligand conjugation onto nanoparticle surfaces as an active targeting strategy has gained significant attention in the pursuit of improving tumor‐specific delivery and retention. However, the chemical conjugation of targeting moieties often induces alterations in the physicochemical properties of nanoparticles, including size, conformation, charge‐to‐mass ratio, and hydrophilicity/lipophilicity, resulting in unexpected biodistribution and pharmacokinetic profiles. Here, the enhanced active targeting efficiency achieved by integrating cyclic arginine–glycine–aspartic acid (cRGD) peptides onto ultrasmall nanocarrier H‐dot while preserving its essential physicochemical and pharmacokinetic attributes is investigated. The resulting cRGD/H‐dots demonstrate improved cellular uptake via integrin αvβ3 receptors, accompanied by negligible cytotoxicity. Notably, the active targeting efficacy of cRGD/H‐dots compared to unmodified H‐dots (1.2%ID/g, two‐fold increase) is quantitatively evaluated, validated through fluorescence imaging and histological analysis. The findings highlight that cRGD/H‐dots offer enhanced tumor targetability and prolonged tumoral retention while maintaining active renal clearance of unbound molecules.

Nanoscale single-vesicle analysis: High-throughput approaches through AI-enhanced super-resolution image analysis

The analysis of membrane vesicles at the nanoscale level is crucial for advancing the understanding of intercellular communication and its implications for health and disease. Despite their significance, the nanoscale analysis of vesicles at the single particle level faces challenges owing to their small size and the complexity of biological fluids. This new vesicle analysis tool leverages the single-molecule sensitivity of super-resolution microscopy (SRM) and the high-throughput analysis capability of deep-learning algorithms. By comparing classical clustering methods (k-means, DBSCAN, and SR-Tesseler) with deep-learning-based approaches (YOLO, DETR, Deformable DETR, and Faster R–CNN) for the analysis of super-resolution fluorescence images of exosomes, we identified the deep-learning algorithm, Deformable DETR, as the most effective. It showed superior accuracy and a reduced processing time for detecting individual vesicles from SRM images. Our findings demonstrate that image-based deep-learning-enhanced methods from SRM images significantly outperform traditional coordinate-based clustering techniques in identifying individual vesicles and resolving the challenges related to misidentification and computational demands. Moreover, the application of the combined Deformable DETR and ConvNeXt-S algorithms to differently labeled exosomes revealed its capability to differentiate between them, indicating its potential to dissect the heterogeneity of vesicle populations. This breakthrough in vesicle analysis suggests a paradigm shift towards the integration of AI into super-resolution imaging, which is promising for unlocking new frontiers in vesicle biology, disease diagnostics, and the development of vesicle-based therapeutics.

An investigation of nano- and micron-sized carriers based on calcium carbonate and polylactic acid for oral administration of siRNA

ABSTRACT Background Oral delivery of small interfering RNAs (siRNAs) draws significant attention, but the gastrointestinal tract (GIT) has many biological barriers that limit the drugs’ bioavailability. The aim of this work was to investigate the potential of micro- and nano-sized CaCO3 and PLA carriers for oral delivery of siRNA and reveal a relationship between the physicochemical features of these carriers and their biodistribution. Research design and methods In vitro stability of carriers was investigated in simulated gastric and intestinal fluids. Toxicity and cellular uptake were investigated on Caco-2 cells. The biodistribution profiles of the developed CaCO3 and PLA carriers were examined using different visualization methods, including SPECT, fluorescence imaging, radiometry, and histological analysis. The delivery efficiency of siRNA loaded carriers was investigated both in vitro and in vivo. Results Micro-sized carriers were accumulated in the stomach and later localized in the colon tissues. The nanoscale particles (100–250 nm) were distributed in the colon tissues. nPLA was also detected in small intestine. The developed carriers can prevent siRNA from premature degradation in GIT media. Conclusion Our results reveal how the physicochemical properties of the particles, including their size and material type can affect their biodistribution profile and oral delivery of siRNA.

Selective Cellular Uptake and Druggability Efficacy through Functionalized Chitosan-Conjugated Polyamidoamine (PAMAM) Dendrimers.

Nanotechnology has ushered in significant advancements in drug design, revolutionizing the prevention, diagnosis, and treatment of various diseases. The strategic utilization of nanotechnology to enhance drug loading, delivery, and release has garnered increasing attention, leveraging the enhanced physical and chemical properties offered by these systems. Polyamidoamine (PAMAM) dendrimers have been pivotal in drug delivery, yet there is room for further enhancement. In this study, we conjugated PAMAM dendrimers with chitosan (CS) to augment cellular internalization in tumor cells. Specifically, doxorubicin (DOX) was initially loaded into PAMAM dendrimers to form DOX-loaded PAMAM (DOX@PAMAM) complexes via intermolecular forces. Subsequently, CS was linked onto the DOX-loaded PAMAM dendrimers to yield CS-conjugated PAMAM loaded with DOX (DOX@CS@PAMAM) through glutaraldehyde crosslinking via the Schiff base reaction. The resultant DOX@CS@PAMAM complexes were comprehensively characterized using Fourier-transform infrared (FTIR) spectroscopy, transmission electron microscopy (TEM), and dynamic light scattering (DLS). Notably, while the drug release profile of DOX@CS@PAMAM in acidic environments was inferior to that of DOX@PAMAM, DOX@CS@PAMAM demonstrated effective acid-responsive drug release, with a cumulative release of 70% within 25 h attributed to the imine linkage. Most importantly, DOX@CS@PAMAM exhibited significant selective cellular internalization rates and antitumor efficacy compared to DOX@PAMAM, as validated through cell viability assays, fluorescence imaging, and flow cytometry analysis. In summary, DOX@CS@PAMAM demonstrated superior antitumor effects compared to unconjugated PAMAM dendrimers, thereby broadening the scope of dendrimer-based nanomedicines with enhanced therapeutic efficacy and promising applications in cancer therapy.

Borneol and lactoferrin dual-modified crocetin-loaded nanoliposomes enhance neuroprotection in HT22 cells and brain targeting in mice

Crocetin (CCT), a natural bioactive compound extracted and purified from the traditional Chinese medicinal herb saffron, has been shown to play a role in neurodegenerative diseases, particularly depression. However, due to challenges with solubility, targeting, and bioavailability, formulation development and clinical use of CCT are severely limited. In this study, we used the emulsification-reverse volatilization method to prepare CCT-loaded nanoliposomes (CN). We further developed a borneol (Bor) and lactoferrin (Lf) dual-modified CCT-loaded nanoliposome (BLCN) for brain-targeted delivery of CCT. The results of transmission electron microscope (TEM) and particle size analysis indicated that the size of BLCN (∼140 nm) was suitable for transcellular transport across olfactory axons (∼200 nm), potentially paving a direct path to the brain. Studies on lipid solubility, micropolarity, and hydrophobicity showed that BLCN had a relatively high Lf grafting rate (81.11 ± 1.33 %) and CCT entrapment efficiency (83.60 ± 1.04 %) compared to other liposomes, likely due to Bor improving the lipid solubility of Lf, and the combination promoting the orderly arrangement of liposome membrane molecules. Microplate reader and fluorescence microscopy analysis showed that BLCN efficiently promoted the endocytosis of fluorescent coumarin 6 into HT22 cells with a maximal fluorescence intensity of (13.48 ± 0.80 %), which was significantly higher than that of CCT (5.73 ± 1.17 %) and CN (12.13 ± 1.01 %). BLCN also exhibited sustained function, remaining effective for more than 12 h after reaching a peak at 1 h in cells, while CN showed a significant decrease after 4 h. The uptake mechanisms of BLCN in HT22 cells mainly involve energy-dependent, caveolae-mediated, and microtubule-mediated endocytosis, as well as micropinocytosis. Furthermore, BLCN displayed a significant neuroprotective effect on HT22 cells in glutamate-, corticosterone-, and H2O2-induced models. Tissue fluorescence image analysis of mice showed that BLCN exhibited substantial retention of fluorescent DiR in the brain after nasal administration for 12 h. These findings suggest that CCT has the potential for cellular uptake, neuroprotection, and targeted delivery to the brain following intranasal administration when encapsulated in Bor and Lf dual-modified nanoliposomes.

Utilizing Laser-Induced Fluorescence to Evaluate the Efficiency of Carbon Dioxide and Diode Lasers in Dentin Sealing after Tooth Preparation: An in-vitro Study.

Adhesive dentistry has made it feasible to design restorations with high aesthetic qualities and little invasiveness. However, the freshly cut dentin after the tooth preparation needs to be sealed to prevent its contaminations, bacterial leakage, and hypersensitivity. Consequently, the immediate dentin sealing (IDS) method has been advised. This study examines different dentinal tubule sealing methods via CO2 laser, diode laser (980-nm) and a two-step self-etch adhesive system applied directly to the fresh cut dentin preceding the placement of the provisional phase. The sealing efficiency of each laser and bond system was evaluated based on the laser-induced fluorescence (LIF) properties and image analysis by scanning electron microscopy. Moreover, the obtained LIF spectra were evaluated using partial least square progression. A two-step adhesive containing a high concentration of S-PRG fillers produced a thick layer that was not perfectly uniform at all sites due to uneven filler distribution in the bond with totally and partially closed dentinal tubules. However, the peaks of the LIF spectra dropped after applying laser because of its sealing effectiveness. Accordingly, CO2 and diode lasers have strong evidence in dentinal tubule sealing and a definitive treatment modality for dentinal hypersensitivity. Moreover, IDS with an adhesive system is superior in occluding dentinal tubules in a biomimetic manner based on its filler content and bioactive properties.

Synchronously in vivo real-time monitoring bacterial load and temperature with evaluating immune response to decipher bacterial infection.

Determining the precise course of bacterial infection requires abundant in vivo real-time data. Synchronous monitoring of the bacterial load, temperature, and immune response can satisfy the shortage of real-time in vivo data. Here, we conducted a study in the joint-infected mouse model to synchronously monitor the bacterial load, temperature, and immune response using the second near-infrared (NIR-II) fluorescence imaging, infrared thermography, and immune response analysis for 2 weeks. Staphylococcus aureus (S. aureus) was proved successfully labeled with glucose-conjugated quantum dots in vitro and in subcutaneous-infected model. The bacterial load indicated by NIR-II fluorescence imaging underwent a sharp drop at 1 day postinfection. At the same time, the temperature gap detected through infrared thermography synchronously brought by infection reached lowest value. Meanwhile, the flow cytometry analysis demonstrated that immune response including macrophage, neutrophil, B lymphocyte, and T lymphocyte increased to the peak at 1 day postinfection. Moreover, both M1 macrophage and M2 macrophage in the blood have an obvious change at ~1 day postinfection, and the change was opposite. In summary, this study not only obtained real-time and long-time in vivo data on the bacterial load, temperature gap, and immune response in the mice model of S. aureus infection, but also found that 1 day postinfection was the key time point during immune response against S. aureus infection. Our study will contribute to synchronously and precisely studying the complicated complex dynamic relationship after bacterial infection at the animal level.

Protective Effects of Changbudodam-tang on Cell Death Signals on the Bone Marrow-Derived Human Mesenchymal Stem Cells via Regulation of MKK7/JNK/c-Jun Signaling Pathway.

Polycystic ovary syndrome (PCOS) is one of the most common disorders and it shows up to 20% prevalence in reproductive-aged women populations, but no cures are available to date. We aimed to investigate the protective effects of Changbudodam-tang (CBD) on cell death signaling pathways, inflammation, and oxidative stress observed in Bone-Marrow derived human mesenchymal stem cell (BM-hMSC) by means of PCOS therapeutics in the future. BM-hMSCs were applied with cell deaths and injuries. Apoptosis and pyroptosis signals were quenched with their related signaling pathways using quantitative PCR, Western blot, and fluorescence image analysis. Our data clearly displayed hydrogen peroxide- and nigericin-treated cell death signaling pathways via regulations of mitochondrial integrity and interleukin (IL)-1β at the cellular levels (p < 0.01 or 0.001). We further observed that pre-treatment with CBD showed protective effects against oxidative stress by enhancement of antioxidant components at the cellular level, with respect to both protein and mRNA expression levels (p < 0.05, 0.01 or 0.001). The mechanisms of CBD were examined by Western blot analysis, and it showed anti-cell death, anti-inflammatory, and antioxidant effects via normalizations of the Jun N-terminal kinase/mitogen-activated protein kinase kinase 7/c-Jun signaling pathways. This study confirmed the pharmacological properties of CBD by regulation of cellular oxidation and the inflammation-provoked cell death condition of BM-hMSCs, which is mediated by the MKK7/JNK/c-Jun signaling pathway.

Modulation of Photosystem II Function in Celery via Foliar-Applied Salicylic Acid during Gradual Water Deficit Stress.

Water deficit is the major stress factor magnified by climate change that causes the most reductions in plant productivity. Knowledge of photosystem II (PSII) response mechanisms underlying crop vulnerability to drought is critical to better understanding the consequences of climate change on crop plants. Salicylic acid (SA) application under drought stress may stimulate PSII function, although the exact mechanism remains essentially unclear. To reveal the PSII response mechanism of celery plants sprayed with water (WA) or SA, we employed chlorophyll fluorescence imaging analysis at 48 h, 96 h, and 192 h after watering. The results showed that up to 96 h after watering, the stroma lamellae of SA-sprayed leaves appeared dilated, and the efficiency of PSII declined, compared to WA-sprayed plants, which displayed a better PSII function. However, 192 h after watering, the stroma lamellae of SA-sprayed leaves was restored, while SA boosted chlorophyll synthesis, and by ameliorating the osmotic potential of celery plants, it resulted in higher relative leaf water content compared to WA-sprayed plants. SA, by acting as an antioxidant under drought stress, suppressed phototoxicity, thereby offering PSII photoprotection, together with enhanced effective quantum yield of PSII photochemistry (ΦPSII) and decreased quantity of singlet oxygen (1O2) generation compared to WA-sprayed plants. The PSII photoprotection mechanism induced by SA under drought stress was triggered by non-photochemical quenching (NPQ), which is a strategy to protect the chloroplast from photo-oxidative damage by dissipating the excess light energy as heat. This photoprotective mechanism, triggered by NPQ under drought stress, was adequate in keeping, especially in high-light conditions, an equal fraction of open PSII reaction centers (qp) as of non-stress conditions. Thus, under water deficit stress, SA activates a regulatory network of stress and light energy partitioning signaling that can mitigate, to an extent, the water deficit stress on PSII functioning.

Tailored design of NHS–SS–NHS cross-linked chitosan nano-hydrogels for enhanced anti-tumor efficacy by GSH-responsive drug release

The traditional chemotherapeutic agents’ disadvantages such as high toxicity, untargeting and poor water solubility lead to disappointing chemotherapy effects, which restricts its clinical application. In this work, novel size-appropriate and glutathione (GSH)-responsive nano-hydrogels were successfully prepared via the active ester method between chitosan (containing –NH2) and cross-linker (containing NHS). Especially, the cross-linker was elaborately designed to possess a disulfide linkage (SS) as well as two terminal NHS groups, namely NHS–SS–NHS. These functionalities endowed chitosan-based cross-linked scaffolds with capabilities for drug loading and delivery, as well as a GSH-responsive mechanism for drug release. The prepared nano-hydrogels demonstrated excellent performance applicable morphology, excellent drug loading efficiency (∼22.5%), suitable size (∼100 nm) and long-term stability. The prepared nano-hydrogels released over 80% doxorubicin (DOX) after incubation in 10 mM GSH while a minimal DOX release less than 25% was tested in normal physiological buffer (pH = 7.4). The unloaded nano-hydrogels did not show any apparent cytotoxicity to A 549 cells. In contrast, DOX-loaded nano-hydrogels exhibited marked anti-tumor activity against A 549 cells, especially in high GSH environment. Finally, through fluorescent imaging and flow cytometry analysis, fluorescein isothiocyanate-labeled nano-hydrogels show obvious specific binding to the GSH high-expressing A549 cells and nonspecific binding to the GSH low-expressing A549 cells. Therefore, with this cross-linking approach, our present finding suggests that cross-linked chitosan nano-hydrogel drug carrier improves the anti-tumor effect of the A 549 cells and may serve as a potential injectable delivery carrier.

Fabrication and characteristics of multifunctional hydrogel dressings using dopamine modified hyaluronic acid and phenylboronic acid modified chitosan.

The healing of damaged skin is a complex and dynamic process, and the multi-functional hydrogel dressings could promote skin tissue healing. This study, therefore, explored the development of a composite multifunctional hydrogel (HDCP) by incorporating the dopamine modified hyaluronic acid (HA-DA) and phenylboronic acid modified chitosan (CS-PBA) crosslinked using boric acid ester bonds. The integration of HA-DA and CS-PBA could be confirmed using the Fourier transform infrared spectrometer and 1H nuclear magnetic resonance analyses. The fabricated HDCP hydrogels exhibited porous structure, elastic solid behavior, shear-thinning, and adhesion properties. Furthermore, the HDCP hydrogels exhibited antibacterial efficacy against Gram-negative Escherichia coli (E. coli) and Gram-positive Staphylococcus aureus (S. aureus). Subsequently, the cytocompatibility of the HDCP hydrogels was verified through CCK-8 assay and fluorescent image analysis following co-cultivation with NIH-3T3 cells. This research presents an innovative multifunctional hydrogel that holds promise as a wound dressing for various applications within the realm of wound healing.