Intracellular Detection Research Articles

Exploring the anticancer and antioxidant potential of gold nanoparticles synthesized from Pterocarpus marsupium bark extract against oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is a disease of significant concern with higher mortality rates. Conventional treatment approaches have several drawbacks, leading to the opening of new research avenues in the field of nanoparticle-based cancer therapeutics. The study aimed at the synthesis of gold nanoparticles (Pm-AuNPs) from the aqueous bark extract of Pterocarpus marsupium, followed by its characterization and in vitro anticancer evaluation against OSCC. The synthesized Pm-AuNPs were characterized using UV–visible spectroscopy, particle size analyser, zeta potential, FTIR and SEM techniques. The anticancer potential of the Pm-AuNPs was evaluated against OSCC cell lines (SCC29b, SSC154 and OECM-1) through in vitro assays. The IC50 value was found to be 25 ± 1.2, 45 ± 1.5 and 75 ± 2.1 µg/mL for the three OSCC cell lines, elucidating Pm-AuNPs cytotoxic effects and mechanism of action. Intracellular ROS and SOX detection, mitochondrial transmembrane potential analysis and apoptosis detection were used to confirm the activity of Pm-AuNPs against OSCC. Acute toxicity studies on Wistar rats confirmed the non-toxic nature of the Pm-AuNPs at a higher dose concentration up to 2000 mg/kg body weight. The findings underscore Pm-AuNPs as promising candidates for future anticancer therapeutics, providing insights into their mechanism of action and therapeutic efficacy against OSCC.

Turn-On Fluorescence Probe for Cancer-Related γ-Glutamyltranspeptidase Detection.

The design and development of fluorescent materials for detecting cancer-related enzymes are crucial for cancer diagnosis and treatment. Herein, we present a substituted rhodamine derivative for the chromogenic and fluorogenic detection of the cancer-relevant enzyme γ-glutamyltranspeptidase (GGT). Initially, the probe is non-chromic and non-emissive due to its spirolactam form, which hinders extensive electronic delocalization over broader pathway. However, selective enzymatic cleavage of the side-coupled group triggers spirolactam ring opening, resulting in electronic flow across the rhodamine skeleton, and reduces the band gap for low-energy electronic transitions. This transformation turns the reaction mixture from colorless to intense pink, with prominent UV and fluorescence bands. The sensor's selectivity was tested against various human enzymes, including urease, alkaline phosphatase, acetylcholinesterase, tyrosinase, and cyclooxygenase, and showed no response. Absorption and fluorescence titration analyses of the probe upon incremental addition of GGT into the probe solution revealed a consistent increase in both absorption and emission spectra, along with intensified pink coloration. The cellular toxicity of the receptor was evaluated using the MTT assay, and bioimaging analysis was performed on BHK-21 cells, which produced bright red fluorescence, demonstrating the probe's excellent cell penetration and digestion capabilities for intracellular analytical detection. Molecular docking results supported the fact that probe-4 made stable interactions with the GGT active site residues.

Gold nanoclusters stabilized with dopa-containing ligands: Catalyst-indicator integrated probe for tumor cell screening

Elevated hydrogen peroxide (H2O2) levels not only inflict cellular damage but also serve as a harbinger for various diseases. Tumor cells, in particular, often exhibit an abundance of H2O2. Hence, the detection of this pivotal molecule assumes paramount importance in monitoring physiological states and expediting cancer diagnosis. To this end, we have ingeniously devised an enzyme-free and monomeric system for intracellular H2O2 detection. Our astute selection of dopa-containing peptidomimetics, replete with ortho-bisphenol and amino acid moieties, has engendered the synthesis of distinctive fluorescent gold nanoclusters (AuNCs). These nanoclusters not only function as a peroxidase-like catalyst, catalyzing the decomposition of H2O2 into hydroxyl radicals (·OH), but also serve as an indicator, with their fluorescence quenched in response to varying H2O2 concentrations. Experimental results evince that our GDpE-AuNCs exhibit remarkable sensitivity, boasting a detection limit of 0.49 μM and a linear range of 5–1000 μM. Moreover, the amalgamation of catalyst and indicator within a single structure, facilitating efficient cellular uptake, engenders intracellular H2O2 detection and discernment of tumor cells. This pioneering approach bequeaths a valuable assay probe for monitoring physiological states and ushering in early disease diagnosis.

Aggregation-induced emission luminogens for intracellular bacteria imaging and elimination

Intracellular bacterial infections are a serious threat to human health due to their ability to escape immunity and develop drug resistance. Recent attention has been devoted to identifying and ablating intracellular bacteria with fluorescence probes. Aggregation-induced emission luminogens (AIEgens) photosensitizers as fluorescence probes possess excellent photostability and rapid response, which have emerged as powerful fluorescent tools for intracellular bacterial detection and antibacterial therapy. This review is intended to highlight the current advances in AIEgens on intracellular bacteria imaging and elimination, which covers topics from intracellular AIE mechanism, intracellular bacteria imaging of AIEgens to the elimination of intracellular bacteria with AIEgens. AIEgens utilized different interactions to detect intracellular bacteria, emitting bright light due to restricted intramolecular movement to visualize intracellular bacteria. Photosensitive AIEgens generate reactive oxygen species (ROS) in the aggregate state to elimate intracellular bacteria. Moreover, the prospects and application of AIEgens in intracellular bacteria imaging and elimination are also discussed, which provides insights for the development of AIE-based diagnostic and therapeutic materials and technologies.

Endogenous enzyme-activated AND-gate DNA nanomachines for intracellular miRNA detection and cell-selective imaging

The occurrence and development of tumors are accompanied by the abnormal expression of specific microRNAs (miRNAs). Therefore, miRNAs are considered as an important biomarker. The establishment of efficient, simple and sensitive miRNA imaging methods in living cells will contribute to the early diagnosis, treatment and drug development of diseases. In this study, we developed an endogenous enzyme-initiated AND logic circuit using gold nanocubes (AuNCs) as carriers for simultaneous detection of miRNA-21 and miRNA-210 in cells. Apurinic/apyrimidinic endonuclease 1 (APE1) and telomerase (TE), which are overexpressed in cancer cells, act as control switches in a logic circuit that enables sensitive in situ analysis of intracellular miRNAs without additional external intervention. At the same time, due to the lack of necessary enzymes as activation switches, the DNA circuit in normal cells remains in an inactive state. This strategy effectively reduces the risk of false positive signal generation. Our research results show that the logic circuit can not only distinguish between cancer cells and normal cells, and able to distinguish between different types of cancer cells. This finding provides a promising approach to accurately identify cell types.

Green synthesis of Au nanoparticles by Scutellaria barbata extract for chemo-photothermal anticancer therapy

IntroductionThe combination of photothermal therapy and chemotherapy has emerged as a promising strategy for cancer treatment. The green synthesis of gold nanoparticles (Au NPs) using extracts from traditional Chinese medicine Scutellaria barbata D. Don (Scu) has revealed an economical, sustainable, and eco-friendly approach for the outstanding anticancer activities. MethodsScu extract was mixed with HAuCl4 solution to fabricate Scu-Au NPs. The synthesis process was optimized by varying the reaction temperature, reaction time, and HAuCl4 concentration. The changes in the components of Scu before and after synthesis were analyzed using the 3,5-dinitrosalicylic acid (DNS) reduction test, the rutin colorimetric method, and the Folin–Ciocalteu method. The morphology, size, and structure of Scu-Au NPs were characterized by TEM, DLS, XRD, and XPS. The photothermal characteristics induced by near-infrared (NIR) irradiation were assessed by continuously monitoring the temperature elevation. The anticancer activity and mechanisms of Scu-Au NPs were investigated using MTT assay, cell uptake studies, ROS level detection, cell apoptosis and necrosis assays, flow cytometry analysis, intracellular Caspase-3 protein activity fluorescence detection, and Western blot analysis on A549, 4T1, and RAW264.7 cells. ResultsScu-Au NPs were successfully fabricated using Scu extract as a reducing agent. The Scu-Au NPs were spherical with a size of 50 nm. During the synthesis process, the levels of reducing sugars, flavonoids, and polyphenols in the Scu extract decreased by 80.8%, 54.3%, and 70.1%, respectively. Scu-Au NPs demonstrated excellent photothermal responsiveness, thermostability, and biocompatibility. For chemo-photothermal anticancer therapy, the cytotoxicity of Scu-Au NPs on 4T1 cells reached approximately 85% upon exposure to 808 nm NIR irradiation. The anticancer activity was attributed to the induction of apoptosis and necrosis, achieved by increasing intracellular ROS levels, causing cell cycle arrest at the S phase, and modulating the expression of apoptosis-related proteins. DiscussionOur synthesized Scu-Au NPs exhibit favorable photothermal conversion characteristics, demonstrating excellent therapeutic efficacy and safety for cancer treatment.

Cytolysin A is an intracellularly induced and secreted cytotoxin of typhoidal Salmonella

Typhoidal Salmonella enterica serovars, such as Typhi and Paratyphi A, cause severe systemic infections, thereby posing a significant threat as human-adapted pathogens. This study focuses on cytolysin A (ClyA), a virulence factor essential for bacterial dissemination within the human body. We show that ClyA is exclusively expressed by intracellular S. Paratyphi A within the Salmonella-containing vacuole (SCV), regulated by the PhoP/Q system and SlyA. ClyA localizes in the bacterial periplasm, suggesting potential secretion. Deletion of TtsA, an essential Type 10 Secretion System component, completely abolishes intracellular ClyA detection and its presence in host cell supernatants. Host cells infected with wild-type S. Paratyphi A contain substantial ClyA, with supernatants capable of lysing neighboring cells. Notably, ClyA selectively lyses macrophages and erythrocytes while sparing epithelial cells. These findings identify ClyA as an intracellularly induced cytolysin, dependent on the SCV environment and secreted via a Type 10 Secretion System, with specific cytolytic activity.

Cuproptosis in microsatellite stable colon cancer cells affects the cytotoxicity of CD8+T through the WNT signaling pathway

The microsatellite stable (MSS) colon cancer (CC) has long been considered resistant to immunotherapy. Cuproptosis, as a novel form of cell death, may interact with tumor immunity. This project focused on the impact of cuproptosis on the cytotoxicity of CD8+T in MSS CC, aiming to provide effective clues for improving the treatment strategy of MSS CC. The study developed an MSS CC cuproptosis model using 50 nM elesclomol and 1 μM CuCl2. Cuproptotic SW480 cells were directly co-cultured with CD8+ T cells. Cuproptosis levels were assessed via intracellular copper ion detection, Western blot, and confocal laser scanning microscopy. CCK-8, Hochest/PI staining, CFSE cell proliferation assay, LDH cytotoxicity detection, and ELISA were used to evaluate CD8+ T cell immune activity and cytotoxicity. Transcriptome sequencing and bioinformatics analysis identified regulated signals in cuproptotic SW480 cells. A rescue experiment utilized a WNT pathway activator (BML-284). PD-L1 expression in cells/membranes was analyzed using qRT-PCR, Western blot, and flow cytometry. NSG mice were immunoreconstituted, and the effects of cuproptosis on immune infiltration and cancer progression in MSS CC mice were assessed using ELISA and immunohistochemistry (IHC). Treatment with 50 nM elesclomol and 1 μM CuCl2 significantly increased cuproptosis in SW480 cells. Co-culture with CD8+ T cells enhanced their cytotoxicity. Sequencing revealed cuproptosis-mediated modulation of immune and inflammatory pathways, including WNT signaling. Rescue experiments showed downregulation of WNT signaling in cuproptotic SW480 cells. Indirectly, CD8+ T cell immune function was enhanced by reducing PD-L1 expression. In mice, cuproptosis resulted in increased infiltration of CD8+ T cells in tumor tissue, leading to delayed cancer progression compared to the control group. Cuproptosis in MSS CC cells enhances the cytotoxicity of CD8+ T cells, which may be achieved through downregulation of the WNT signaling pathway and decreased expression of PD-L1. In the future, drugs that can induce cuproptosis may be a promising approach to improve MSS CC immunotherapy.

Yeast Surface-Displayed Quenchbody as a Novel Whole-Cell Biosensor for One-Step Detection of Influenza A (H1N1) Virus.

Timely surveillance of airborne pathogens is essential to preventing the spread of infectious diseases and safeguard human health. Methods for sensitive, efficient, and cost-effective detection of airborne viruses are needed. With advances in synthetic biology, whole-cell biosensors have emerged as promising platforms for environmental monitoring and medical diagnostics. However, the current design paradigm of whole-cell biosensors is mostly based on intracellular detection of analytes that can transport across the cell membrane, which presents a critical challenge for viral pathogens and large biomolecules. To address this challenge, we developed a new type of whole-cell biosensor by expressing and displaying VHH-based quenchbody (Q-body) on the surface of the yeast Saccharomyces cerevisiae for simple one-step detection of influenza A (H1N1) virus. Seventeen VHH antibody fragments targeting the hemagglutinin protein H1N1-HA were displayed on the yeast cells and screened for the H1N1-HA binding affinity. The functionally displayed VHHs were selected to create surface-displayed Q-body biosensors. The surface-displayed Q-body exhibiting the highest quenching and dequenching efficiency was identified. The biosensor quantitatively detected H1N1-HA in a range from 0.5 to 16 μg/mL, with a half-maximal concentration of 2.60 μg/mL. The biosensor exhibited high specificity for H1N1-HA over other hemagglutinin proteins from various influenza A virus subtypes. Moreover, the biosensor succeeded in detecting the H1N1 virus at concentrations from 2.4 × 104 to 1.5 × 107 PFU/mL. The results from this study demonstrated a new whole-cell biosensor design that circumvents the need for transport of analytes into biosensor cells, enabling efficient detection of the target virus particles.

A reverberating triple AND logic gate nanomachine for intracellular multi-enzyme tracking and MicroRNA detection

With the rapid advancement of molecular biology and nanotechnology, DNA logic gates have emerged as a method to simulate complex chemical reaction networks, which is crucial for distinguishing tumor cells from normal cells in early cancer diagnosis and treatment. The current challenges include the compositional differences in biomarkers between tumors and normal tissues and achieving high specific imaging in low-expression miRNAs within cells. We developed a triple AND logic-gate nanomachine that utilizes the high expression of matrix metalloproteinase (MMPs) in tumor metastasis and the specific overexpression of telomerase in cancer cells to initiate a first-stage signal and accurately identify tumor cells. This system then interacts with miRNA-21 to trigger a second-stage signal, enabling precise detection and signal amplification through the activity of purine-free/pyrimidine-free endonuclease 1 (APE1). This system can not only achieve efficient target recognition in complex bio-logical samples, but also provide a strong detection signal through continuous signal amplification, greatly improving the accuracy and reliability of diagnosis. This research provides a new molecular tool for cancer diagnosis, facilitating early detection and improving patient treatment outcomes and quality of life. Additionally, the principles and methods of this technique can be applied to the detection of other disease markers and tumor cell subtypes.

Protocols for circulating neutrophil depletion in neonatal C57Bl/6 mice.

Murine neonatal neutrophil depletion strategies have problems with achieving deep circulating neutrophil clearance and accurate residual neutrophil fraction detection. Boivin et al. demonstrated an isotype switch method of achieving profound neutrophil clearance using a combination of anti-Ly6G and anti-rat κ Ig light chain antibodies in adult C57Bl/6 mice, proven by extra- and intracellular Ly6G detection by flow cytometry. We adapted this technique to neonatal mice testing four neutrophil depletion strategies. Four protocols were tested: P3 Ly6G and P1-3 Ly6G (anti-Ly6G on postnatal days (P) 3 and 1-3 respectively), and P3 Dual and P1-3 Dual (anti-Ly6G and anti-rat κ Ig light chain on P3 and P1-3 respectively). Intracellular and extracellular Ly6G presence was detected using flow cytometry. Isotype control antibodies were used as controls. P1-3 Dual achieved significantly better neutrophil depletion than the P1-3 Ly6G or P3 Ly6G protocols (97% vs 74% and 97% vs 50%, respectively). The P3 Dual protocol alone was enough to achieve significantly better neutrophil clearance (93%) than any of the Ly6G alone protocols. The Ly6G alone protocols led to near-total elimination of extracellular Ly6G, but there was a significant presence of intracellular Ly6G in the CD45+ cell population, which evaded detection by extracellular Ly6G antibody-based detection methods. Anti-Ly6G antibody alone is ineffective in producing effective circulating neutrophil clearance in neonatal mice. Dual antibody-based neutrophil depletion strategies achieve>90% clearance in P4 mice. A single day of dual antibody treatment achieves 93% neutrophil depletion in neonatal mice and is an alternative to daily injections. Dual antibody-based neutrophil depletion effectively induces circulating neutrophil clearance in neonatal mice.

Biological Evaluation of Lysionotin: a Novel Inhibitor of 5-Lipoxygenase for Anti-glioma.

To explore the potential mechanism of lysionotin in treating glioma. First, target prediction based on Bernoulli Naïve Bayes profiling and pathway enrichment was used to predict the biological activity of lysionotin. The binding between 5-lipoxygenase (5-LO) and lysionotin was detected by surface plasmon resonance (SPR) and molecular docking, and the inhibitory effects of lysionotin on 5-LO and proliferation of glioma were determined using enzyme inhibition assay in vitro and cell viability analysis, respectively. Furthermore, the pharmaceutical effect of lysionotin was explored by cell survival rate analysis and liquid chromatography with tandem mass spectrometry (LC-MS/MS). The protein expression, intracellular calcium ion concentration and cytoskeleton detection were revealed by Western blot, flow cytometry and fluorescence labeling, respectively. Target prediction and pathway enrichment revealed that lysionotin inhibited 5-LO, a key enzyme involved in the arachidonic acid metabolism pathway, to inhibit the proliferation of glioma. Molecular docking results demonstrated that 5-LO can be binding to lysionotin through hydrogen bonds, forming bonds with His600, Gln557, Asn554, and His372. SPR analysis further confirmed the interaction between 5-LO and lysionotin. Furthermore, enzyme inhibition assay in vitro and cell survival rate analysis revealed that 50% inhibition concentration of lysionotin and the median effective concentration of lysionotin were 90 and 16.58 µmol/L, respectively, and the results of LC-MS/MS showed that lysionotin inhibited the production of 5S-hydroperoxy-eicosatetraenoic acid (P<0.05), and moreover, the LC-MS/MS results indicated that lysionotin can enter glioma cells well (P<0.01) and inhibit their proliferation. Western blot analysis demonstrated that lysionotin can inhibit the expression of 5-LO (P<0.05) and downstream leukotriene B4 receptor (P<0.01). In addition, the results showed that lysionotin affected intracellular calcium ion concentration by inhibiting 5-LO to affect the cytoskeleton, as determined by flow cytometry and fluorescence labeling. Lysionotin binds to 5-LO could suppress glioma by inhibiting arachiodonic acid metabolism pathway.

Biotin-Modified PEGylated Protein-Stabilized gold Nanoclusters: A strategy for selective and efficient intracellular transport of glucose oxidase for glucose detection

Although bovine serum albumin-stabilized gold nanoclusters (BSA-AuNCs) have various applications in biological environments., limited research has been carried out on their potential for intracellular delivery of enzymes and highly selective intracellular detection of specific analytes. We developed biotin-capped polyethylene glycol (PEG)-functionalized bovine serum albumin-stabilized gold nanoclusters (biotin-AuNCs) for enhanced optical properties and bioimaging applications. Biotin-AuNCs exhibited superior performance to BSA-AuNCs, with higher quantum yield (∼14 %), improved photostability, and enhanced resistance to trypsin digestion. These nanoclusters enabled selective imaging of biotin receptor-positive cells such as HeLa and MCF-7, while forming nanocomposites (GOx-AuNCs) with avidin-glucose oxidase (GOx) for intracellular glucose detection. The GOx-AuNCs showed selective imaging capabilities and facilitated glucose sensing through GOx-catalyzed H2O2 production, leading to fluorescence quenching of the biotin-AuNCs. This method provided a reliable indicator for intracellular glucose levels, establishing a linear calibration curve between GOx-AuNC-labeled cells’ luminescence intensity and intracellular glucose concentration. Additionally, we developed dual-emission probes by modifying red-emitting GOx-AuNCs with green-emitting BDP-FL for ratiometric imaging of intracellular glucose concentration, enhancing glucose sensing precision in biological environments.

Novel mycophenolic acid precursor-based fluorescent probe for intracellular H2O2 detection in living cells and Daphnia magna and Zebrafish model systems.

Innovative for the scientific community and attracting attention in the extensive biomedical field are novel compact organic chemosensing systems built upon unique core molecular frameworks. These systems may demonstrate customized responses and may be adaptable to analytes, showing promise for potential in vivo applications. Our recent investigation focuses on a precursor of Mycophenolic acid, resulting in the development of LBM (LOD = 13 nM) - a specialized probe selective for H2O2. This paper details the synthesis, characterization, and thorough biological assessments of LBM. Notably, we conducted experiments involving living cells, daphnia, and zebrafish models, utilizing microscopy techniques to determine probe nontoxicity and discern distinct patterns of probe localization. Localization involved the distribution of the probe in the Zebrafish model within the gut, esophagus, and muscles of the antennae.

Lyso-H2S: A Mycophenolic Acid-Derived Probe for Ultra-Low Toxicity, Intracellular H2S Detection, and Zebrafish Model Validation.

In several biological processes, H2S is known to function as an endogenous gaseous agent. It is very necessary to monitor H2S and relevant physiological processes in vivo. Herein, a new type of fluorophore with a reliable leaving group allows for excited-state intramolecular transfer characteristics (ESIPT), inspired by mycophenolic acid. A morpholine ring was connected at the maleimide position of the probe to target the lysosome. Subsequently, the dinitrophenyl group known for a photoinduced electron transfer (PET) effect, was connected to allow for an effective "turn-on" probe Lyso-H2S. Lyso-H2S demonstrated strong selectivity towards H2S, a large Stokes shift (111 nm), and an incredibly low detection limit (41.8 nM). The imaging of endogenous and exogenous H2S in living cells (A549 cell line) was successfully achieved because of the specificity and ultra-low toxicity (100 % cell viability at 50 μM concentration of Lyso-H2S.) Additionally, Lyso-H2S was also employed to visualize the activity of H2S in the gallbladder and intestine in a living zebrafish model. This is the first report of a fluorescent probe to track H2S sensing in specific organ systems to our knowledge.

Dual-Function DNA Nanowires with Self-Feedback Amplification and Efficient Signal Transduction for Intracellular Imaging of MicroRNA-155.

Intracellular detection and imaging of microRNAs (miRNAs) with low expression usually face the problem of unsatisfactory sensitivity. Herein, a novel dual-function DNA nanowire (DDN) with self-feedback amplification and efficient signal transduction was developed for the sensitive detection and intracellular imaging of microRNA-155 (miRNA-155). Target miRNA-155 triggered catalytic hairpin assembly (CHA) to generate plenty of double-stranded DNA (dsDNA), and a trigger primer exposed in dsDNA initiated a hybridization chain reaction (HCR) between four well-designed hairpins to produce DDN, which was encoded with massive target sequences and DNAzyme. On the one hand, target sequences in DDN acted as self-feedback amplifiers to reactivate cascaded CHA and HCR, achieving exponential signal amplification. On the other hand, DNAzyme encoded in DDN acted as signal transducers, successively cleaving Cy5 and BHQ-2 labeled substrate S to obtain a significantly enhanced fluorescence signal. This efficient signal transduction coupling self-feedback amplification greatly improved the detection sensitivity with a limit of detection of 160 aM for miRNA-155, enabling ultrasensitive imaging of low-abundance miRNA-155 in living cells. The constructed DDN creates a promising fluorescence detection and intracellular imaging platform for low-expressed biomarkers, exhibiting tremendous potential in biomedical studies and clinical diagnosis of diseases.

P-593 Inflammaging in the ovary: focus on foamy cells generation

Abstract Study question Could the dysregulation of the inflammatory response in the aged ovary be associated with foamy cells generation and be ameliorated using an alternative therapeutic treatment? Summary answer Aged ovarian macrophages show an inflammatory profile associated with incorporation of lipid droplets that can be reduced after culture with platelet-rich plasma from fertile women. What is known already The concept of ‘inflammaging’ represents the inflammation associated to aging, which is systemic, chronic and linked to the increase of pro-inflammatory mediators. This microenvironment is associated with the presence of ovarian foamy macrophages, a unique subset with intracytoplasmic lipids accumulation. Here, we investigate the mechanisms underlying the exacerbated inflammatory response during ovarian aging, focusig on the generation of foamy macrophages and their impact in oocyte quality. Since, oocytes develop in the context of ovarian follicles, we obtained follicular fluid (FF) from women with different ages, to investigate ovarian immune microenvironment and their modulation after in vitro platelet-rich plasma (PRP) treatment. Study design, size, duration Mononuclear cells were recovered from FF from women of different ages and IL-1β production and macrophages and lymphocytes profiles tested by immune-staining and FACS analysis. To test foamy cells generation, we measured surface and intracellular CD68 expression (scavenger receptor involved in lipid accumulation). Immune cells profiles were correlated with clinic parameters as number of oocytes, M2 and atretic follicles. Finally, isolated ovarian macrophages were treated with PRP and modulation of macrophage profile was tested. Participants/materials, setting, methods We studied FF (n = 70) from women from ovodonation (21-29y), with cryopreservation desire (30-35y) and ART indication (36-42y). The San Isidro Ethics Committee, Buenos Aires, Argentina, approved this study. Mononuclear cells were isolated using Ficoll-Hypaque and macrophages profile (IL-1β+, CD68+) and the novel lymphocyte regulatory subpopulation (CD3+CD4-CD8-) tested by flow cytometry. Lipid droplets were measured using BODIPY 493/503 probe. Ovarian macrophages were isolated by adherence and treated with PRP (obtained from fertile women) for 5 days. Main results and the role of chance FF macrophages from aged women (35-42y) showed a significant increase in IL-1β production in comparison with the youngest group (21-29y), that also correlates negatively with the number of M2 oocytes recovered. Focusing on macrophage characterization, FF from aged women displayed an increased frequency of CD68+ macrophages in comparison with young women. Since CD68 shuttles between the cytoplasm and the plasmatic membrane to incorporate lipids, we evaluated lipid accumulation in FF macrophages to generate foamy cells. Interestingly, we found an increase of lipid droplets in FF macrophages from aged women in comparison with the younger group. Therefore, we discriminated surface and intracellular CD68 detection and found higher intracellular CD68 expression in aged macrophages, correlating with lipid droplets accumulation. Then, we isolated ovarian macrophages from aged women and treated with fertile PRP to evaluate its modulatory effects and observed a reduction in the frequency of CD68+IL-1β cells in comparison with those cultures without treatment. Finally, we focused on a novel T lymphocyte regulatory subpopulation, CD3+CD4-CD8- (DNT), that is emerging as effector cells capable of mediating immune tolerance in the female reproductive system. We found that FF from aged women displayed a reduced frequency of DNT, in comparison with FF from younger women. Limitations, reasons for caution The present results were obtained using FF samples from women from different ages. Even though the samples represent the ovarian microenvironment, it is difficult to study the human ovary per se and further studies are necessary to elucidate whether these mechanisms operate similarly in vivo. Wider implications of the findings We demonstrated that dysregulation of the inflammatory response during aging leads to the generation of foamy cells and reduction of DNT cells associated to the regulation of the immune ovarian microenvironment impacting in oocyte quality. In vitro PRP treatment displayed immunomodulatory effects, suggesting its relevance as a potential therapeutic treatment. Trial registration number not applicable

Discovery of Positive Allosteric Modulators of α 7 nAChR by an Ensemble-based Virtual Screening Method, Molecular Dynamics Simulation, and in vitro Biological Activity Testing

The [Formula: see text]7 nicotinic acetylcholine receptor (nAChR) is a neurotransmitter receptor and a promising therapeutic target for neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease. In this study, we used deep learning-based classification modeling, molecular docking, and intracellular calcium signal detection experiments to identify positive allosteric modulators (PAMs) of the [Formula: see text]7 nAChR from natural tobacco products. First, we used a classification prediction model based on the direct message-passing neural network (D-MPNN) and molecular docking to predict potential [Formula: see text]7 PAMs. Subsequently, absorption, distribution, metabolism, excretion, and toxicity (ADMET) predictions were performed to determine its pharmacological properties and minimal toxic side effects, leading to the selection of three natural compounds for in vitro activity validation. Calcium signaling assay experiments revealed that diosmetin can enhance nicotine-induced calcium ion signaling. 100 ns molecular dynamics (MD) simulations indicated that diosmetin can form a dense and stable complex with [Formula: see text]7 nAChR. This study provides valuable insights for the design of more effective and selective ligands for the [Formula: see text]7 nAChR.

Sensitive fluorescence detection of miRNA-124 in cardiomyocytes under oxidative stress using a nucleic acid probe

MicroRNAs (miRNAs) are small noncoding RNAs of 18–25 bases. miRNAs are also important new biomarkers that can be used for disease diagnosis in the future. Studies have shown that miR-124 levels are significantly elevated during acute myocardial infarction (AMI) and play a key role in the cardiovascular system. A variety of methods have been established to detect myocardial infarction-related miRNAs. However, most require complex miRNA extraction and isolation, and these methods are virtually undetectable when RNA levels are low in the sample. It may lead to biased results. Thus, it is necessary to develop a technique that can detect miRNA without extracting it, which means that intracellular detection is of great significance. Here, we improved the traditional silicon spheres and obtained a biosensor that could effectively capture and detect specific noncoding nucleic acids through the layer-by-layer assembly method. The sensor is protected by hyaluronic acid so it can successfully escape the lysosome into the cell and achieve detection. With the help of a full-featured microplate reader, we determined that the detection limit of the biosensor could reach 1 fM, meeting the needs of intracellular detection. At the same time, we prepared an oxidative stress cardiomyocyte infarction model and successfully captured the overexpressed miR-124 in the infarcted cells to achieve in situ detection. This study could provide a new potential tool to develop miRNAs for sensitive diagnosis in AMI, and the proposed strategy implies its potential for biomedical research.

Nasopharyngeal carcinoma cell screening based on nuclear targeting Surface-Enhanced Raman Scattering (SERS) detection

BackgroundNasopharyngeal carcinoma (NPC) is a malignant epithelial carcinoma arising from the nasopharyngeal mucosal lining. Diagnosis of NPC at early stage can improve the outcome of patients and facilitate reduction in cancer mortality. The most significant change between cancer cells and normal cells is the variation of cell nucleus. Therefore, accurately detecting the biochemical changes in nucleus between cancer cells and normal cells has great potential to explore diagnostic molecular markers for NPC. Highly sensitive surface-enhanced Raman scattering (SERS) could reflect the biochemical changes in the process of cell cancerization at the molecular level. However, rapid nuclear targeting SERS detection remains a challenge. ResultsA novel and accurate nuclear-targeting SERS detection method based on electroporation was proposed. With the assistance of electric pulses, nuclear-targeting nanoprobes were rapidly introduced into different NPC cells (including CNE1, CNE2, C666 cell lines) and normal nasopharyngeal epithelial cells (NP69 cell line), respectively. Under the action of nuclear localization signaling peptides (NLS), the nanoprobes entering cells were located to the nucleus, providing high-quality nuclear SERS signals. Hematoxylin and eosin (H&E) staining and in situ cell SERS imaging confirmed the excellent nuclear targeting performance of the nanoprobes developed in this study. The comparison of SERS signals indicated that there were subtle differences in the biochemical components between NPC cells and normal nasopharyngeal cells. Furthermore, SERS spectra combined with principal component analysis (PCA) and linear discriminant analysis (LDA) were employed to diagnose and distinguish NPC cell samples, and high sensitivity, specificity, and accuracy were obtained in the screening of NPC cells from normal nasopharyngeal epithelial cells. SignificanceTo the best of our knowledge, this is the first study that employing nuclear-targeting SERS testing to screen nasopharyngeal carcinoma cells. Based on the electroporation technology, nanoprobes can be rapidly introduced into living cells for intracellular biochemical detection. Nuclear-targeting SERS detection can analyze the biochemical changes in the nucleus of cancer cells at the molecular level, which has great potential for early cancer screening and cytotoxicity analysis of anticancer drugs.