Search Results (373)

Natural therapeutic alternatives are increasingly explored in endometriosis, a highly prevalent gynecological disorder with limited therapeutic options. Rosmarinus officinalis (rosemary) has attracted increasing scientific interest due to its biological activity. This study aimed to characterize a hydroethanolic rosemary extract (RE) and evaluate its effects on key cellular processes involved in endometriosis pathophysiology. Major phenolic compounds in RE were quantified by RP-HPLC, and antioxidant activity was assessed using DPPH, ABTS, and FRAP assays. After RE treatment, cell viability (WST-1), migration (wound healing assay), cell cycle distribution (DAPI staining), apoptosis (Annexin V/PI), p21 and cyclin A expression (Western blot), and intracellular ROS levels (DCFH-DA) were evaluated in endometrial stromal (t-HESC, St-T1b) and endometriotic epithelial (12-Z) cells. Phytochemical analysis revealed rosmarinic acid (RA) at 4.2%, while carnosic acid (CA) and carnosol (CS) together accounted for 23.7% of the extract. RE reduced cell viability and cell migration in 12-Z and t-HESC cells (p < 0.05). S-phase accumulation with a concomitant reduction in the G1 phase was observed across all evaluated cell lines (p < 0.05), along with increased p21 and cyclin A expression in stromal cells (p < 0.05). RE induced cell death in both 12-Z (p < 0.05) and St-T1b cells (p < 0.0001). In t-HESC cells, RE reduced both basal and H₂O₂-induced ROS levels (p < 0.01). These findings indicate that RE modulates key mechanisms involved in endometriosis pathophysiology, supporting its multi-target therapeutic potential as a nutraceutical approach for endometriosis management. Full article

Immunohistochemistry (IHC) is a widely used method for detecting specific proteins in tissue samples, helping diagnose diseases such as cancer. Traditional analysis methods rely heavily on human interpretation, which can lead to inconsistencies. In this study, we propose DeepFlare, a weakly supervised deep learning framework for cross-modality translation and segmentation of immunofluorescence and immunohistochemistry images. The proposed method utilizes multiplex immunofluorescence (mpIF) and co-registered IHC images, combined with preprocessing techniques such as affine transformation, stain normalization, noise reduction, and artifact removal. Multiple imaging channels, including hematoxylin, DAPI, Lap2, and nuclear envelope signals, are leveraged to generate segmentation masks using a U-Net++ architecture. The final segmentation mask is obtained through weighted fusion of modality-specific outputs. A generative adversarial network (GAN) is employed to measure translation fidelity between generated and real images. Weakly supervised learning techniques, including image-level supervision and consistency constraints, are applied to enhance performance under limited annotation scenarios. Pretrained pathology foundation encoders such as UNI and Virchow are integrated to extract multi-scale morphological and contextual features. Explainable AI techniques are incorporated to highlight critical regions and refine model attention. Experimental results demonstrate strong performance, achieving an SSIM of 0.7077 for image translation and a Dice score of 0.7424 for segmentation. The integration of the UNI encoder provides marginal improvement over the baseline (0.72 Dice score), indicating limited domain adaptation without fine-tuning on the dataset of 1264 training samples. Full article

An undescribed seco-kaurane diterpenoid, benincaside A (BA), was isolated from the seeds of Benincasa hispida. The seeds of B. hispida have been traditionally used in folk medicine and previous studies have reported anti-tumor potential in B. hispida seed extracts. Accordingly, we investigated the cytotoxicity and underlying mechanisms of BA in colorectal cancer cells. BA inhibited growth in HT29, Colo205, HCT116, and CT26 colorectal cancer cells, as determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, while showing no toxicity toward normal human umbilical vein endothelial cells (HUVEC) and human fibroblast WS-1 cells. In HCT116 cells, BA-induced deoxyribonucleic acid (DNA) damage and apoptosis, as evidenced by morphological changes, 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining, and assays of caspase-8 and caspase-3 activities. BA triggered apoptotic cell death via the extrinsic pathway, as indicated by elevated caspase-8 and caspase-3 activities. Intracellular reactive oxygen species (ROS) generation was observed in BA-treated HCT116 cells. The growth-inhibitory effects were significantly attenuated by pretreatment with N-acetylcysteine (NAC, an antioxidant), caffeine (an ATM kinase inhibitor), z-VAD-fmk (pan-caspase inhibitor), or z-IETD-fmk (caspase-8-specific inhibitor). Colorimetric assays confirmed increased caspase-8 and caspase-3 activities in BA-treated cells. This study is the first to report ROS-dependent signaling as a key mechanism underlying BA-induced cell death in HCT116 human colorectal cancer cells. Full article

Baikal skullcap (Scutellaria baicalensis) is one of the most important herbs used for centuries in traditional Chinese medicine. The main objective of the research was to micropropagate and introduce the most vigorous lines into garden cultivation. Eleven lines representing individual genotypes were selected and propagated using nodal segments. 6-Benzylaminopurine (BAP) at 1.0 mg L⁻¹, alone or with indole-3-acetic acid (IAA) at 0.1 mg L⁻¹, was the most effective for shoot regeneration. Significant variability in multiplication rate (2.5–6.4 shoots per explant) was observed among the lines. Shoots were successfully rooted (97.9%) and then acclimatized. After six months of cultivation, 113 well-developed plants representing all genotypes were obtained. Depending on the genotype, the final survival rates ranged from 26.9 to 80.0%. Flow cytometry (FCM-DAPI) analysis confirmed the ploidy stability of the micropropagated plants and ISSR markers revealed notable variability among the lines. Moreover, a polymorphism level of 36.4% was detected within line SB_6, whereas the other two analyzed lines (SB_3 and SB_7) maintained in vitro for four years showed no somaclonal variation. Therefore, genetic stability must be monitored, particularly in long-term cultures. In subsequent studies, the acclimatized lines will be evaluated for root yield and phytochemical content under garden conditions. Full article

Although calcium overload dysregulation has been implicated in cellular senescence, its role in ionizing radiation (IR)-induced osteocyte senescence, a key pathogenic mechanism underlying radiotherapy-associated bone injury, remains poorly explored. This study investigated whether IR-induced osteocyte senescence is mediated through the Ca²⁺-NFATc1-ATF3 pathway. Exposure to 2 Gy X-rays impaired osteocyte homeostasis, manifesting as reduced viability and proliferation, G2/M phase arrest, and dendritic retraction. IR also induced persistent DNA damage response and senescence-associated phenotypes, including increased γ-H2AX foci, SA-β-gal activity, condensed punctate DAPI-dense nuclear foci, p16/p21 expression, and pro-inflammatory SASP profile. Intracellular Ca²⁺ levels surged within 6 h post-irradiation and remained elevated for at least 72 h in a dose-dependent manner. Pharmacological Ca²⁺ modulation with BAPTA-AM or verapamil attenuated IR-induced intracellular Ca²⁺ accumulation, G2/M arrest, SA-β-gal positivity, p21/p53 upregulation, and SASP secretion. Conditioned medium from irradiated osteocytes inhibited BMSC-mediated osteogenesis and enhanced BMM-driven osteoclastogenesis, whereas Ca²⁺ modulation partially mitigated these paracrine effects. Mechanistically, IR promoted NFATc1 nuclear translocation and ATF3 upregulation. Collectively, these findings support an important role for pathological intracellular Ca²⁺ elevation in IR-induced osteocyte senescence and suggest that the Ca²⁺-NFATc1-ATF3 axis may represent a potential therapeutic target for mitigating radiation-associated disruption of bone homeostasis. Full article

This study identified the fertility alteration characteristics and cytological mechanisms of the thermo-photo-sensitive genic male sterile (TPSGMS) wheat line K64S. The fertility-sensitive stage of K64S extends from pollen mother cell formation to the tetrad development stage, with critical fertility alteration thresholds of 14–14.5 °C for temperature and 9–9.5 h for daylength. Under low-temperature and short-day conditions, K64S exhibits complete male sterility, whereas it returns to fertility under high-temperature and long-day conditions. Cytological analysis shows that K64S undergoes normal meiosis and successfully forms normal uninucleate microspores. 4′,6-diamidino-2-phenylindole (DAPI) staining revealed the uninucleate microspores failed to form binucleate microspores, with abortion occurring during the late uninucleate stage. Transmission electron microscopy indicates the pollen abortion in sterile K64S arises primarily from premature tapetal degeneration (a form of programmed cell death, PCD), initiated at the pollen mother cell stage, which disrupts nutrient supply and leads to abnormal nuclear division during subsequent microspore development. These findings provide insights into the cytological mechanism of pollen abortion in TPSGMS wheat and may guide hybrid wheat breeding and application. Full article

Green synthesis of silver nanoparticles (CP-AgNPs) using Calotropis procera (CP) offers a sustainable approach to producing multifunctional therapeutic nanomaterials. This study aimed to synthesize CP-AgNPs and evaluate their antimicrobial, antioxidant, anti-inflammatory, and anticancer potential, with a focus on Helicobacter pylori and gastric cancer cells. CP-AgNPs were prepared by phytochemical reduction using CP leaf extract and characterized by UV–Vis, XRD, FTIR, SEM, EDX, TEM, and Zeta. Antibacterial activity against H. pylori, time-kill kinetics, and SEM imaging of membrane damage were performed. Antioxidant (DPPH, ABTS) and anti-inflammatory assays, together with cytotoxicity studies in AGS cells (DAPI, AO/EtBr, and SEM), were also conducted. CP-AgNPs exhibited an SPR peak at 432 nm, face-centered cubic crystallinity, and spherical morphology (8–32 nm). They showed strong, dose-dependent antibacterial activity against H. pylori, surpassing metronidazole at higher doses. Time-kill assays and SEM confirmed membrane disruption. Antioxidant activity was notable (IC₅₀: 40 µg/mL for DPPH; 60 µg/mL for ABTS). CP-AgNPs demonstrated significant anti-inflammatory effects and dose-dependent cytotoxicity in AGS cells, inducing apoptosis and morphological alterations. The broad biological activity of CP-AgNPs likely arises from the synergy between silver ions and CP phytochemicals. Their superior antibacterial effects, combined with antioxidant and anti-inflammatory properties, indicate strong therapeutic potential for gastric diseases. Anticancer activity in AGS cells suggests additional biomedical relevance, which may involve ROS-associated and apoptosis-related pathways, as suggested by previous studies. CP-AgNPs represent a promising natural nanoplatform for managing H. pylori infection, oxidative stress, inflammation, and gastric cancer, warranting further mechanistic and in vivo studies. Full article

Background/Objectives: Chromosome research is essential for advancing our understanding of cytogenetics, gene regulation and numerous aspects of organismal health. Staining chromosomes with 4′,6-diamidino-2-phenylindole (DAPI) and applying Fluorescence Lifetime Imaging Microscopy (FLIM) enables the assessment of structural changes in pericentromeric and heterochromatin-rich region of chromosomes 1, with a shorter fluorescence lifetime (FLT) in the pericentromeric regions compared to the arms. Methods: We used FLIM to optimise sample preparation conditions for more robust imaging and furthermore to measure the impact of low-dose X-ray ionising radiation on chromosome structure when labelled with DAPI. Results: We applied this method to different DNA stains bound to chromosomes where only DAPI led to a clear FLT difference between the chromosome arms (p,q) with 2.98 ± 0.12 ns and 2.65 ± 0.07 ns at the pericentromeric region, while similar stains, such as Hoechst 33258 and NucBlue^TM did not highlight these regions as clearly following FLIM analysis. Our data showed that chromosomes of cells irradiated with 0.1 Gy and 1 Gy did not show a significant change in FLTs (2.94 ± 0.09 ns on the arms and 2.60 ± 0.06 ns on the pericentromeric region) of chromosome 1. Whilst irradiation with 0.5 Gy led to a noticeable and significant reduction in FLT with 2.42 ± 0.13 ns on the arms and 2.12 ± 0.06 ns on the pericentromeric region of HeLa chromosomes. The same pattern could also be seen on X-ray-irradiated T-cell chromosomes. Conclusions: These findings indicate that DAPI FLT may be a useful tool to measure chromosomal structural changes and further suggests that chromosomes undergo distinct structural changes at the pericentromeric region following low-dose irradiation. Full article

Three-dimensional (3D) bioreactor systems are essential for vascular tissue engineering as they provide controlled environments that better mimic physiological conditions compared to static culture systems. In this study, an IoT-enabled modular rotating 3D bioreactor platform was designed, fabricated using Fused Deposition Modeling (FDM), and biologically validated. The system integrates a Wi-Fi-supported ESP8266 controller and a touchscreen human–machine interface (HMI), enabling real-time monitoring and remote operation. Agarose-chitosan-based tubular hydrogel constructs were seeded with human aortic smooth muscle cells (HASMCs) and cultured under dynamic conditions for 14 days. Biocompatibility was assessed using a lactate dehydrogenase (LDH) assay, while cellular distribution and mitochondrial activity were evaluated by confocal microscopy using DAPI and MitoTracker staining. Fluorescence intensity was further quantified using ImageJ, and 3D surface plots were generated to visualize spatial signal distribution. The results demonstrated sustained cell viability with decreasing cytotoxicity over time. Confocal analysis confirmed a homogeneous distribution of cells within the hydrogel matrix, and quantitative fluorescence analysis showed significantly higher MitoTracker intensity compared to DAPI, indicating increased metabolic activity under dynamic conditions. These findings suggest that the developed bioreactor provides a stable, controllable, and effective platform for vascular tissue engineering applications. Full article

Colorectal cancer (CRC) is the third most prevalent cancer globally. TASK-1, encoded by the KCNK3 gene, is emerging as a putative target in cancer; it regulates resting membrane potential, cell proliferation, and apoptosis. A series of 27 novel xanthene derivatives, modified at position 9, were synthesized via azide coupling of 2-(9H-xanthen-9-yl)acetohydrazide with selected amines and amino acids, followed by hydrazine-mediated conversion to the corresponding hydrazides. The cytotoxic activity of selected compounds (5a–5g, 6a–6h, 7b, 7f–7h) was evaluated against the HCT-116 cell line in vitro. In addition, molecular docking and molecular dynamics simulations were performed to investigate binding interactions and assess the stability of the protein–ligand complexes. Several compounds (5f, 5g, 6c, 6d, 6f, 6g, 7b, 7f, and 7h) exhibited moderate cytotoxic activity against HCT-116 cells (IC₅₀: 66.97–99.62 µM), compared to cisplatin (IC₅₀: 18.25 µM). Compound 7h demonstrated pronounced antiproliferative effects, evidenced by DAPI staining showing chromatin condensation and apoptotic body formation, along with a marked reduction in cell count and coverage. Molecular docking indicated favorable binding within the TASK-1 potassium channel, and molecular dynamics simulations confirmed the stability of the protein–ligand complex, with consistent interactions, including a key hydrogen bond with Asn240. These findings support 7h as a promising lead candidate. These findings identify xanthene-based derivatives as promising lead compounds for further optimization as TASK-1-targeted therapeutic candidates in colorectal cancer. Full article

Cadmium (Cd) is a pervasive environmental contaminant with potent cytotoxic effects in a wide range of organisms. Although autophagy and apoptosis are recognized as major cellular responses to heavy metal stress, the molecular basis of Cd-induced cell death in insects remains insufficiently understood. In this study, we used fifth-instar day-4 (5L4D) larvae of Bombyx mori and the silkworm-derived Bm-12 cell line to investigate the involvement of three core autophagy-related proteins, Bombyx mori Autophagy-related protein 5(BmATG5), Bombyx mori Autophagy-related protein 6(BmATG6), and Autophagy-related protein 8(BmATG8), in Cd-induced autophagy and apoptosis. Exposure to CdCl₂ markedly induced autophagic and apoptotic responses in both larval midgut tissue and Bm-12 cells, as demonstrated by monodansylcadaverine(MDC) staining, Lyso-Tracker Red staining, DAPI and Hoechst 33258 staining, and DNA fragmentation assays. qPCR and Western blot analyses showed significant upregulation of BmATG5, BmATG6, and BmATG8 following Cd exposure. Notably, the cleaved forms tBmATG5-N (24 kDa) and tBmATG6-C (35 and 37 kDa), as well as the lipidated form BmATG8-PE (12 kDa), accumulated substantially under Cd stress. In parallel, intracellular Ca²⁺ levels and calpain activity were significantly increased, suggesting activation of a calcium-dependent regulatory pathway. Pharmacological inhibition experiments further indicated that autophagy and apoptosis are functionally interconnected during the Cd response. Collectively, these findings demonstrate that BmATG5, BmATG6, and BmATG8, together with their processed forms, play central roles in coordinating autophagy–apoptosis crosstalk during Cd-induced cytotoxicity in Bombyx mori. This study provides new mechanistic insight into heavy metal toxicity in insects and expands our understanding of stress-induced programmed cell death during silkworm metamorphosis. Full article

Kiwifruit soft rot is a major cause of postharvest loss owing to rapid fruit decay during storage. This study focused on kiwifruit soft rot during the postharvest storage stage, when fungal development may be promoted by room temperature and high humidity. Soft rot symptoms were observed in the pericarp and fruit flesh. In this study, carvacrol-loaded nanoliposomes (CAR@NL) were prepared by an O/W emulsification–solvent evaporation method to control kiwifruit soft rot. The physicochemical properties of CAR@NL were characterized by laser particle size analysis, Fourier transform infrared spectroscopy (FTIR), and transmission electron microscopy (TEM). Their antifungal activity and preservation efficacy were evaluated by in vitro antifungal assays and fruit storage experiments. The prepared CAR@NL showed an average particle size of approximately 280 nm, an encapsulation efficiency of 85.75%, and a drug loading capacity of 20.14%, along with favorable sustained-release properties. CAR@NL exhibited strong antifungal activity, with an EC₅₀ value of 41.76 mg/L. DAPI staining indicated no obvious effect on fungal DNA, whereas propidium iodide (PI) staining revealed increased fluorescence intensity with increasing concentration and treatment time, indicating disruption of hyphal membrane integrity and severe structural damage. Flow cytometric analysis further showed that, at 50 mg/L, the total apoptosis rate was 2.96% in the untreated control group, 5.22% in the CAR@NL-treated group, and 33.6% in the carbendazim-treated group, demonstrating the lower cytotoxicity of CAR@NL toward mammalian cells. In addition, CAR@NL showed good stability and preservation performance during fruit storage. Overall, CAR@NL may serve as a safe and effective postharvest agent for the control of kiwifruit soft rot. Full article

Pulmonary bioengineering holds significant promise for the development of functional lungs suitable for transplantation in patients with terminal lung diseases; however, it encounters considerable challenges. The inherent structural complexity, diverse cellular composition, and the intricate process of re-endothelialization the pulmonary vasculature complicate efforts to reconstruct viable lungs for transplantation. This study aimed to establish an innovative re-endothelialization technique utilizing decellularized scaffolds, integrating canine yolk sac-derived endothelial precursor cells with mechanical respiratory stimuli within a bioreactor framework. Wistar rat lungs were subjected to a decellularization protocol employing SDS + Triton X-100 0.5% and subsequently assessed for cytocompatibility with murine fibroblasts (3T3) and yolk sac (YS) cells in fragments. Following this, the recellularization of the whole-lung scaffold was evaluated under constant mechanical respiratory stimulation with YS cells. Each stage of the process was rigorously analyzed using histological staining, DAPI, scanning electron microscopy (SEM), and genomic DNA quantification. The findings reveal that the implemented alternating decellularization protocol resulted in a structured scaffold conducive to the culture of various cell types in fragments. When subjected to the complete scaffold recellularization model, the results indicated that YS cells are advantageous for the re-endothelialization process. Moreover, when employed in conjunction with the bioreactor model incorporating respiratory stimulation, these cells demonstrated enhanced cellular diffusion capacity and facilitated more homogeneous recellularization of the entire organ. These results signify a notable advancement in the reconstruction of new tissues for pulmonary transplantation. Full article

Optogenetic modulation employs light-sensitive proteins known as opsins to regulate cellular activity. A unique therapeutic application of this technique involves modulating pain perception by selectively targeting neural pathways within the spinal cord. Multi-Characteristic Opsin (MCO) represents an innovative optogenetic actuator capable of activation across a broad spectrum of light wavelengths, exhibiting a slow depolarizing phase that resembles natural photoreceptors. This study examines the current advancements in spinal optogenetic modulation utilizing MCO for pain management. Due to its high sensitivity, MCO facilitates minimally invasive, remotely controlled optogenetic modulation of spinal neurons. This approach enables the regulation of extensive spatial regions, provided the MCO channel receives sufficient light intensity to surpass the activation threshold. Nano-enhanced optical delivery (NOD) successfully transfected spinal neurons with the GAD67-MCO2-mCherry construct, as confirmed by membrane-localized mCherry fluorescence with DAPI-labeled nuclei. Using this platform, 5 Hz spinal optogenetic stimulation produced a significant reduction in formalin-evoked pain behaviors, demonstrating frequency-specific modulation of spinal pain circuits. Neither 2 Hz nor 10 Hz stimulation yielded comparable analgesic effects, underscoring the importance of precise stimulation parameters. The therapeutic impact also depended on transfection efficiency: reducing the fGNR–plasmid concentration diminished MCO expression and weakened the analgesic response. Together, these results show that effective spinal optogenetic pain modulation requires both optimal stimulation frequency and robust gene delivery. Full article

Although medicinal plants possess vast biological properties, crude medicinal plant extracts often show limited therapeutic efficacy due to poor aqueous solubility, instability, and inadequate bioavailability, which restricts efficient intracellular delivery. As cancer is a genetic disease requiring intracellular and nuclear targeting, improved delivery systems are essential. Warburgia salutaris is traditionally used in Southern Africa and possesses reported anticancer and anti-inflammatory properties; however, its crude extracts exhibit suboptimal delivery characteristics. This study comparatively evaluated the anticancer effects of unencapsulated (WSN) and liposomal-encapsulated (WSE) crude leaf extracts, with emphasis on apoptotic mechanisms. Liposomal formulation was confirmed by FTIR, PXRD, and DLS, yielding stable nanoparticles (159.4 nm; PDI 0.114; +79.3 mV). Both WSN and WSE demonstrated efficacy and concentration-dependent cytotoxicity against MCF-7 breast cancer cells (IC₅₀ < 0.0195 mg/mL) with minimal toxicity toward Vero kidney cells and RAW 264.7 macrophages. Mechanistically, WSN induced rapid cytotoxicity with necrotic features, whereas WSE promoted regulated apoptosis. Apoptosis was validated by DAPI/PI staining, Annexin V/PI flow cytometry, mRNA expression levels of Bax, Bcl-2, and caspase-3 measured with RT-PCR and proteome profiling array, confirming activation of intrinsic and extrinsic pathways. Both extracts also reduced LPS-induced ROS production. LC-MS identified multiple bioactive phytochemicals. Overall, liposomal encapsulation enhanced therapeutic precision, stability, and selectivity cytotoxicity, supporting its development as a nanomedicine-based anticancer strategy. Full article