Search Results (382)

Aminoglycoside antibiotics are critical in clinical use for treating severe infections, but they can occasionally cause irreversible sensorineural hearing loss. To establish a rational pathway for otoprotectant discovery, we provide an integrated, three-tier methodology—comprising cell-model selection, transcriptomic analysis, and a gentamicin–Texas Red (GTTR) uptake assay—to guide the development of otoprotective strategies. We first utilized two murine auditory cell lines—UB/OC-2 and HEI-OC1. We focused on TMC1 and OCT2 and further explored the underlying mechanisms of ototoxicity. UB/OC-2 exhibited a higher sensitivity to gentamicin, which correlated with elevated OCT2 expression confirmed via RT-PCR and Western blot. Transcriptomic analysis revealed upregulation of PI3K-Akt, calcium, and GPCR-related stress pathways in gentamicin-treated HEI-OC1 cells. Protein-level analysis further confirmed that gentamicin suppressed phosphorylated Akt while upregulating ER stress markers (GRP78, CHOP) and apoptotic proteins (cleaved caspase 3, PARP). Co-treatment with PI3K inhibitors (LY294002, wortmannin) further suppressed Akt phosphorylation, supporting the role of PI3K-Akt signaling in auditory cells. To visualize drug entry, we used GTTR to evaluate its applicability as a fluorescence-based uptake assay in these cell lines, which were previously employed mainly in cochlear explants. Sodium thiosulfate (STS) and N-acetylcysteine (NAC) significantly decreased GTTR uptake, suggesting a protective effect against gentamicin-induced hair cell damage. In conclusion, our findings showed a complex ototoxic cascade involving OCT2- and TMC1-mediated drug uptake, calcium imbalance, ER stress, and disruption of PI3K-Akt survival signaling. We believe that UB/OC-2 cells serve as a practical in vitro model for mechanistic investigations and screening of otoprotective compounds. Additionally, GTTR may be a simple, effective method for evaluating protective interventions in auditory cell lines. Overall, this study provides molecular-level insights into aminoglycoside-induced ototoxicity and introduces a platform for protective strategies. Full article

Hypertension and metabolic dysfunction-associated fatty liver disease (MAFLD) are both common chronic diseases globally. Nearly half of patients with hypertension are complicated by MAFLD. The mechanisms of the bidirectional promotion between the two remain unclear. The (pro) renin receptor ((P)RR) is one of the classic members of the renin–angiotensin system (RAS) and serves as the receptor for prorenin. Although the role of (P)RR in the induction and progression of hypertension has been extensively studied, its role and underlying mechanisms in MAFLD remain underreported. In this study, we aim to investigate the role of (P)RR in the pathogenesis of hypertension combined with MAFLD. In this study, SHRs were used for the model for hypertension combined with MAFLD. Liver lipid content analysis, liver H&E staining, the detection of (P)RR, ERK and downstream proteins related to fatty acid synthesis and transport, and RNA sequencing and data analysis were performed. In the in vitro experiments, we activated (P)RR using renin and established the lipid deposition model of HepG2 cells induced by renin for the first time. (P)RR was specifically blocked using handle region peptide (HRP), and Nile red fluorescence staining, (P)RR/ERK/PPARγ protein expression analysis, and immunofluorescence were performed to further verify the role of (P)RR in the pathogenesis of hypertension combined with MAFLD. Our results demonstrate that (P)RR plays a role in the development and progression of hypertension combined with MAFLD. The hepatic TG and FFA levels in the SHRs were increased, and the protein expression of the (P)RR/ERK/PPARγ pathway and downstream proteins related to fatty acid synthesis and transport were upregulated. HRP reversed the activation of these proteins and reduced intracellular lipid accumulation. In conclusion, our study first reveals that (P)RR is a potential therapeutic target for hypertension combined with MAFLD. And we found the (P)RR/ERK/PPARγ axis for the first time, which plays an important role in the progression of spontaneous hypertension combined with MAFLD. Full article

Conditional transgenic systems and multi-copy target transgenes were used to produce transient fluorescent protein expression in adult Drosophila melanogaster, with the goal of developing an in vivo assay of protein turnover. Free-moving flies were assayed at multiple time points using video, and decay in fluorescence was used to calculate protein half-life. Additional experiments involved image capture of anesthetized flies. The half-life of eGFP was increased by the proteasome inhibitor bortezomib, both in vivo and in vitro, indicating proteasomal degradation of eGFP. The accumulation of eGFP in vivo was decreased by the protein synthesis inhibitor cycloheximide, without affecting half-life. The half-lives of several fluorescent proteins were determined, using both tissue-general and tissue-specific expression, in flies of both sexes and varying ages. Typical half-life values varied by fluorescent protein. DsRED showed a greater half-life than eGFP, and little if any degradation was detected for mCherry. Half-life also varied by tissue, with greater eGFP half-life observed in muscle relative to other tissues. Increased half-life with age was detected for DsRED but not for eGFP. Limited effects were observed for sex and female mating status. Taken together, the data indicate the in vivo assays are promising tools for the study of protein degradation regulated by protein sequence, subcellular compartment, tissue and small molecules. Full article

Blood samples and testing are routine in healthcare. Presently, there is a growing interest in using tear samples in place of blood. Tear samples can be obtained non-invasively and collection does not require the skills of a trained phlebotomist. Red blood cells and other cells are not present in tears, which avoids centrifugation. Importantly, basal tear samples contain most of the biomarkers present in blood. The difficulty is the small volume of basal tears, which is about 7 μL in each eye. Any contact with the eye results in additional reflex tears with a different chemical composition. The small tear samples are collected with capillary tubes and then sent out for amplified assays, such as enzyme-linked immunosorbent assay (ELISA) or polymerase chain reaction (PCR). The results are not available for several days or a week and, therefore, are less useful in an ophthalmology office. We propose the use of a contact lens that contains bound antibodies for fluorescence immunoassays. The lenses could be removed from the patient for point-of-care measurements at the bedside. To prove that this concept is possible, we performed a three-layer protein capture assay that mimics an immunoassay. For convenience, we used lysozyme (Lys), which spontaneously coats silicon hydrogel (SiHG) contact lenses (CL). Anti-lysozyme IgG was the second layer captured, with anti-lysozyme considered to be the target biomarker. The third layer was rhodamine or Alexa Fluor-labeled Ab against the IgG Fc region, considered to be the detection antibody. The multiple protein layers were stable and did not wash off the SiHG lenses. These results strongly suggest the contact lens can be used for capture immunoassays for a wide variety of biomarkers. Full article

K. alvarezii is a red macroalgae cultivated on a large scale in Asian countries. In Brazil, it is cultivated in states such as Piaui, Rio de Janeiro, and São Paulo due to the high economic value for the food industry given the high concentrations of carrageenan, a hydrocolloid formed mainly by carbohydrates, used as a gelling agent and emulsifier. Therefore, to aggregate value to its protein content, the goal was to identify the proteins from K. alvarezii and biotechnological potentials against human pathogens. The protein extract produced Na⁺-acetate buffer was the most efficient in inhibiting the growth of C. parapsilosis and C. krusei. The analysis of the mechanism of action revealed that proteins from K. alvarezii cause severe damage to cellular morphology, including the effect on the cell wall and membrane, as indicated by scanning electron microscopy (SEM). Fluorescence microscopy agreed with the SEM results, revealing an increase in membrane permeabilization and pore formation, in addition to high levels of ROS, followed by apoptosis triggered by caspase 3/7. Regarding the characterization of proteins, biochemical analysis revealed the presence of proteolytic enzymes and those involved in ROS metabolism. Proteomic analysis by LC-ESI-MS/MS identified 336 proteins involved in processes such as energetic and nucleotide metabolism, defense against (a)biotic stress, and protein folding. Our results revealed that K. alvarezii proteins presented potential against C. parapsilosis and C. krusei. Full article

Enzyme catalysis represents a promising approach for sustainable chemical synthesis, yet its industrial applications face limitations due to the inefficient regeneration and high cost of essential cofactors, such as adenosine-5′-triphosphate (ATP) and nicotinamide adenine dinucleotide phosphate (NADPH). While natural metabolic systems efficiently recycle cofactors through spatially organized enzymes, replicating this efficiency in vitro remains challenging. Here, we prepare a five-enzyme condensate system using liquid–liquid phase separation (LLPS) mediated by intrinsically disordered proteins (IDPs). By colocalizing a carboxylic acid reductase from Norcadia iowensis (NiCAR) with a reductive aminase from Aspergillus oryzae (AspRedAm) and three cofactor-regenerating enzymes, we generated a phase-separated catalytic condensate that enhanced ATP and NADPH recycling efficiency by 4.7-fold and 1.9-fold relative to free enzymes, respectively. Catalytic performance was correlated with the extent of phase separation, as confirmed by fluorescence microscopy, which revealed clear enrichment of ATP and NADPH within the condensates. This proximity effect enabled efficient cofactor turnover in the one-step reaction, achieving substrate conversion above 90% within 6 h and enhancing the space–time yield (STY) of the chiral imines 1.6-fold, with only one-fifth of the standard cofactor load. This approach creates a scalable and economic tool for performing multienzyme cascade reactions in vitro that are driven by the efficient recycling of multiple cofactors. Full article

Background/Objectives: Metabolic dysfunction-associated fatty liver disease (MAFLD) is a chronic hepatic condition marked by lipid buildup, lipotoxicity, and inflammation. Prior research indicates that 3,3′-Diindolemethane (DIM), a natural indole-type phytochemical that is abundant in brassicaceae vegetables, has been reported to reduce body weight and improve lipid metabolism in mice subjected to a high-fat diet (HFD). The aryl hydrocarbon receptor (AhR), a nuclear receptor implicated in lipid metabolism and immune regulation, serves as a functional receptor for DIM. However, the underlying signaling pathways that regulate MAFLD remain elusive. Our objective is to ascertain the beneficial impact of DIM on MAFLD and the associated mechanisms. Methods: Hematoxylin and eosin staining, together with Oil Red O staining, were utilized to assess the pathological changes and lipid deposition in the liver. Biochemical analysis was employed to measure levels of triglyceride (TG), total cholesterol (TC), free fatty acid (FFA), aspartate transaminase (AST), alanine transaminase (ALT), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C). The cell survival rate of HepG2 cells treated with palmitic acid (PA) and DIM was assessed using the CCK-8 assay. Flow cytometry was employed to measure the fluorescence intensity emitted by lipid droplets within cells. Western blotting analysis was performed to assess AhR pathway and fatty acid transporter expression levels in hepatic tissue. Results: Our results showed that DIM significantly attenuated body weight gain and hepatic injury brought on by HFD, decreased lipid droplet accumulation in HepG2 cells, and effectively suppressed the phosphorylation of p38 MAPK and the protein expression levels of fatty acid transporters CD36 and FATP4. Conclusions: DIM reduced lipid accumulation by activating AhR and suppressing p38 MAPK phosphorylation, thereby inhibiting fatty acid transport and inflammatory responses. These findings suggest that DIM may represent a promising therapeutic candidate for MAFLD, warranting further exploration for clinical applications. Full article

In this study, we systematically investigated the mechanisms of the antioxidation and anti-lipid accumulation effects of antioxidant peptides from Litsea cubeba on a free fatty acid (FFA)-induced NAFLD model of HepG2 cells. The NAFLD cell model was constructed by inducing the HepG2 hepatocellular carcinoma cell line with 0.5 mmol/L FFAs, and AQRDAGLL, QEGPFVR, and DVPPPRGPL were given to the culture to study their lipid-lowering and antioxidant activities on NAFLD cells. The lipid-lowering activities of the three antioxidant peptides were evaluated by Oil Red O staining and TG and TC content assays, and the results showed that all three peptides had strong ameliorating effects on FFA-induced lipid accumulation in NAFLD cells. The intracellular antioxidant protease (CAT, GSH, and SOD) activity levels and lipid peroxidation (MDA) content were measured and intracellular ROS levels were detected. The results showed that after intervention with the antioxidant peptides, the intracellular ROS levels in the NAFLD model cells were significantly reduced, the SOD and CAT activities were increased, the GSH content was elevated, and the MDA content was reduced, which indicated that AQRDAGLL, QEGPFVR, and DVPPPRGPL were able to inhibit the oxidative stress of the cells effectively and to achieve the effect of intervening in NAFLD. JC-1 fluorescence staining experiments showed that the mitochondrial membrane potential function of NAFLD cells was restored under the effect of the antioxidant peptides. Molecular dynamics simulations revealed that the main driving force between QEGPFVR and Keap1 protein was van der Waals forces, ΔG = −62.11 kcal/mol, which indicated that QEGPFVR was capable of spontaneously binding to Keap1 protein. Full article

Fluorescence-lifetime imaging microscopy (FLIM) is a powerful technique for highly multiplexed imaging in live cells. In this work, we present a genetically encoded FLIM multiplexing platform based on a combination of fluorogen-activating protein FAST and red-shifted fluorogen N871b from the arylidene–imidazolone family. We showed that a series of FAST protein mutants exhibit similar steady-state optical properties in complex with N871b fluorogen but have different fluorescence lifetimes. The similar brightness and binding strength of pairs of these FAST protein variants with N871b allows them to be successfully used for multiplexing up to three intracellular structures of living cells simultaneously. Full article

Background: Blueberry anthocyanin such as Cyanidin-3-O-glucoside may help prevent Alzheimer’s disease. We aimed to investigate the preventive and therapeutic effects of Cyanidin-3-O-glucoside against Aβ_1–42-induced apoptosis of SH-SY5Y cells as well as the underlying mechanisms. Methods: Cell viability and intracellular and mitochondrial reactive oxygen species were detected by MTT, a reactive oxygen species detection kit, and a MitoSOX red mitochondrial superoxide indicator. The mitochondrial membrane potential, intracellular calcium ion content, and adenotriphophate (ATP) were identified via a mitochondrial membrane potential detection kit, calcium ion detection kit, and ATP detection kit, and apoptosis was detected via flow cytometry. Transcription of apoptosis-related genes was detected using real-time fluorescence quantitative polymerase chain reaction, and expression of apoptosis-related proteins was identified using Western blot. Results: We found that Cyanidin-3-O-glucoside could downregulate the expression of cytochrome c, caspase 9, caspase 3, and other genes and proteins, which consequently reduced the rate of apoptosis. Additionally, it could upregulate Bcl-2 gene and protein expression, downregulate Bax gene and protein expression, regulate mitochondrial membrane permeability and calcium-release channels, reduce calcium influx into mitochondria, maintain intracellular calcium ion levels, reduce intracellular levels of reactive oxygen species and increase ATP levels, maintain the mitochondrial membrane potential at a normal level, maintain normal mitochondrial functioning, and prevent apoptosis. Discussion: Taken together, Cyanidin-3-O-glucoside showed dose-dependent preventive and therapeutic effects against Aβ_1–42-induced apoptosis of SH-SY5Y cells. Conclusions: Cyanidin 3-O-glucoside showed a better preventive effect than therapeutic effect against Aβ_1–42-induced apoptosis in SH-SY5Y cells. Full article

The cytotoxic and apoptotic properties of four bioactive natural compounds, the prenylated α-pyronephloroglucinol heterodimer arzanol (ARZ), the methoxylated flavones eupatilin (EUP) and xanthomicrol (XAN), and the sesquiterpene zerumbone (ZER), were compared in SH-SY5Y human neuroblastoma cells to assess their potential as neuroblastoma-specific therapeutics. EUP, XAN, and ZER (2.5–100 μM) exerted marked significant cytotoxicity (MTT assay) and morphological changes after 24 h of incubation, following the order XAN > ZER > EUP > ARZ (no toxic effect). The propidium iodide fluorescence assay (PI, red fluorescence) and NucView^® 488 assay (NV, green fluorescence) evidenced a significant increase in the apoptotic cell number, vs. controls, in SH-SY5Y cells pre-incubated for 2 h with the compounds, in the following order of apoptotic potency: XAN > EUP > ZER > ARZ. The PubChem database and freely accessible web tools SwissADME, pkCSM-pharmacokinetics, and SwissTargetPrediction were used to assess the physicochemical/pharmacokinetic properties and potential protein targets of the compounds. At 50 μM, a positive correlation (r = 0.917) between values of % viability reduction and % human intestinal absorption (bioavailability) was observed, indicating a marked contribution of compound membrane permeability to cytotoxicity in SH-SY5Y cells. The capacity of compounds to induce apoptosis emerged as inversely correlated to the computed lipophilicity (r = −0.885). Full article

Fluorescent proteins (FPs) are widely used as optical probes in molecular and cell biology. tKeima is a tetrameric, large Stokes shift red fluorescent protein and the ancestral protein of mt-Keima, which is widely applied as a pH-sensitive fluorescent probe. While the pH sensitivity of mt-Keima is well characterized, the pH-dependent properties of the ancestral tKeima have not been comprehensively elucidated. To obtain a better understanding of the effects of pH on tKeima, its fluorescent emission intensity at various pH levels was measured, and its crystal structure at pH 4.0 was determined at a resolution of 2.2 Å. The fluorescence emission intensity of tKeima at pH 4.0 decreased by approximately 65% compared with its peak emission at pH 10.0. The crystal structure of tKeima at pH 4.0 revealed both cis and trans conformations of the chromophore, in contrast to previously determined structures at pH 8.0, which showed only the cis conformation. This indicates that pH induces a conformational change of the chromophore in tKeima. Both the cis and trans conformations in tKeima were stabilized by hydrogen bonds with neighboring residues. A comparison of tKeima at pH 4.0 with tKeima at basic pH, as well as with mKeima, highlights its unique structural properties. These results provide a deeper understanding of the structural basis for the pH-induced fluorescence emission changes in the Keima family. Full article

Background: Besides conventional norms that recognize synovial fluid (SF) as a joint lubricant, nutritional channel, and a diagnostic tool in knee osteoarthritis (kOA), based on the authors previous studies, this study aims to define functional role of SF in kOA. Methods: U937, a monocytic, human myeloid cell line, was induced with progressive grades of kOA SF, and the induction response was assessed on various pro-inflammatory parameters. This ‘SF challenge test model’ was further extended to determine the impact of SF on U937 differentiation using macrophage-specific markers and associated transcription factor genes. Mitochondrial membrane potential changes in SF-treated cells were evaluated with fluorescent JC-1 probe. Results: a significant increase in nitric oxide, matrix metalloproteinase (MMP) 1, 13, and vascular endothelial growth factor (VEGF)-1 was noted in the induced cells. A marked increase was seen in CD68, CD86, and the transcription factors –activator protein (AP)-1, interferon regulatory factor (IRF)-1, and signal transducer and activator of transcription (STAT)-6 in the SF-treated cells indicating active monocytes to macrophage differentiation. Reduced mitochondrial membrane potential was reflected by a reduced red-to-green ratio in JC-1 staining. Conclusions: these results underline the active role of OA SF in stimulating and maintaining inflammation in joint cells, fostering monocyte differentiation into pro-inflammatory macrophages. The decline in the membrane potential suggestive of additional inflammatory pathway in OA via the release of pro-apoptotic factors and damaged associated molecular patterns (DAMPs) within the cells. Overall, biochemical modulation of SF warrants a potential approach to intervene inflammatory cascade in OA and mitigate its progression. Full article

Background: Fibroblast growth factor 21 (FGF21), a secreted protein, plays a crucial role in regulating metabolism and energy homeostasis. Nevertheless, the expression pattern of Fgf21 across diverse tissues and its responsiveness to various dietary regimens remain incompletely understood. Methods: In this study, we developed a Fgf21-enhanced green fluorescent protein (EGFP) reporter mouse model to explore the expression of endogenous Fgf21 in different tissues under four dietary conditions: normal chow, low-protein diet, fasting, and fasting-refeeding. Results: A low-protein diet was found to induce Fgf21 expression in both the liver and skeletal muscle. Notably, Fgf21 was predominantly expressed in the periportal region of the liver. In the pancreas, Fgf21 exhibited a patchy expression pattern in the exocrine portion, but was absent in the endocrine part, regardless of the dietary regimens. Regarding the spleen, fasting triggered the expression of Fgf21, which was mainly localized in the red pulp area. Moreover, under fasting conditions, Fgf21 showed a scattered expression pattern in the small intestine. Conclusions: The Fgf21-EGFP reporter mouse model serves as a valuable tool for dissecting the expression of endogenous Fgf21 in different tissues under various dietary and stress conditions. Further investigations using this model may contribute to uncovering the hitherto unrecognized functions of locally produced FGF21. Full article

We established a real-time Förster resonance energy transfer (FRET) based assay to evaluate targeted drug delivery using polymeric micelles. Red fluorescent protein (RFP)-expressing E. coli cells were used as a test system to monitor the delivery of drug-fluorophore such as curcumin and umbelliferones (MUmb and AMC) encapsulated in the polymeric micellar formulations. The efficiency of the drug delivery was quantified using the FRET efficiency, measured as the degree of energy transfer from the drug to the RFP. FRET efficiency directly provides the determination of the delivery efficacy, offering a versatile platform adaptable to various drugs and cell types. We used polymer micelles as a carrier for targeted delivery of fluorescent drugs to bacterial cells expressing RFP. The physicochemical characterization of the interaction between the drugs and the micelles including spectral properties, and the solubility and binding constants, were determined. We revealed a stronger affinity of MUmb for heparin-based micelles (K_d~10⁻⁵ M) compared to chitosan-based micelles (K_d~10⁻⁴ M), underscoring the influence of polymer composition on drug loading efficiency. For micelles containing MUmb, a FRET efficiency significantly exceeds (by three times) the efficiency for non-micellar MUmb, which have minimal penetration into bacterial cells. The most noticeable effect was observed with the use of the micellar curcumin providing pronounced activation of the RPF fluorescence signal, due to the interaction with curcumins (fluorophore-donor). Curcumin delivery using Chit5-OA micelle resulted in a 115% increase in RFP fluorescence intensity, and Hep-LA showed a significant seven-fold increase. These results highlight the significant effect of micellar composition on the effectiveness of drug delivery. In addition, we have developed a visual platform designed to evaluate the effectiveness of a pharmaceutical product through the visualization of the fluorescence of a bacterial culture on a Petri dish. This method allows us to quickly and accurately assess the penetration of a drug into bacteria, or those located inside other cells, such as macrophages, where the intercellular latent forms of the infection are located. Micellar formulations show enhanced antibacterial activity compared to free drugs, and formulations with Hep-OA micelles demonstrate the most significant reduction in E. coli viability. Synergistic effects were observed when combining curcumin and MUmb with moxifloxacin, resulting in a remarkable 40–50% increase in efficacy. The presented approach, based on the FRET test system with RFP expressed in the bacterial cells, establishes a powerful platform for development and optimizing targeted drug delivery systems. Full article