Confocal Microscopy Analysis Research Articles

Cyclodextrin Derivatives as Modulators for Enhanced Drug Delivery from Niosome Membrane: A Fluorescence Correlation Spectroscopy and Isothermal Titration Calorimetry Approach.

Designing efficient drug delivery systems for optimum therapeutic outcomes and minimum adverse effects remains a pivotal focus in pharmaceutical research. Understanding the nature of interactions between drugs and drug carriers and the drug-release mechanism are the key aspects for the development of effective delivery systems. This work presents a detailed investigation into the intricate interactions between niosomes and the drug Phenosafranin (PSF), and the subsequent release induced by a variety of cyclodextrins (CDs) employing a multifaceted approach. Ensemble average spectroscopic and single molecular level investigations based on fluorescence correlation spectroscopy (FCS), are employed to explore the binding interactions of PSF with the niosome membrane. Subsequently, the release of the drug was studied by disrupting the niosome structure using various CDs, and their efficacy was accessed through steady-state and time-resolved photophysical responses. FCS experiments provided precise insights into the binding and drug release process at the single-molecule level through the variation in translational and diffusion characteristics of the drug. Additionally, isothermal titration calorimetric (ITC) investigations further revealed the thermodynamics governing the CD-niosome host:guest interactions and the varying potential of different CDs in disrupting the niosome to release the drug which were further validated by electron microscopy and confocal fluorescence microscopy analyses. A broader analysis of niosomes prepared with various nonionic surfactants highlighted the influence of cavitand size and structure on the interaction with different niosome constituents. This comprehensive analysis sheds light on the complex interplay of these components and their interactions, providing insights into drug delivery systems and their potential therapeutic applications.

A Bioinspired Virus-Like Mechano-Bactericidal Nanomotor for Ocular Multidrug-Resistant Bacterial Infection Treatment.

Multidrug-resistant (MDR) bacteria and their associated biofilms are major causative factors in eye infections, often resulting in blindness and presenting considerable global health challenges. Presently, mechano-bactericidal systems, which combine distinct topological geometries with mechanical forces to physically induce bacterial apoptosis, show promising potential. However, the physical interaction process between current mechano-bactericidal systems and bacteria is generally based on passive diffusion or Brownian motion and lacks the force required for biofilm penetration; thus, featuring low antibacterial efficacy. Here, a biomimetic mechano-bactericidal nanomotor (VMSNT) is synthesized by functionalizing COOH-PEG-phenylboronic acid (PBA) on virus-like mesoporous silica, with subsequent partial coating of Au caps. Enhanced by self-thermophoresis capabilities and virus-like topological shapes, VMSNT significantly improves mechanical antibacterial effects and biofilm penetration. In addition, scanning electron microscope (SEM) and confocal laser scanning microscope (CLSM)analyses demonstrate that VMSNT can precisely target bacteria within the infection microenvironment, facilitated by PBA's ability to recognize and bind to the peptidoglycan on bacterial surfaces. Remarkably, VMSNT is also effective in eliminating MDR bacteria and reducing inflammation in mice models of methicillin-resistant Staphylococcus aureus (MRSA)-infected keratitis and endophthalmitis, with minimal adverse effects. Overall, such a nanomotor presents a promising approach for addressing the challenges of ocular MDR bacterial infections.

Assessment of the Effects of Newly Fabricated CaO, CuO, ZnO Nanoparticles on Callus Formation Maintenance of Alfalfa (Medicago sativa L.) Under In Vitro Salt Stress

Nanoparticles play an important role in plant response to abiotic stresses including salt stress. In this study, the physiological and histological responses of CuO, ZnO, and CaO nanoparticle (NP) applications on callus tissues developed from two alfalfa lines (Erzurum and Muş) exposed to salt (NaCl) stress were evaluated. The NPs were synthesized from the extracts obtained from healthy walnut shells using the green synthesis approach and then characterized by Scanning Electron Microscopy (SEM) and X-ray diffraction analysis (XRD). The leaf explants were placed in an MS medium containing 4 mg L−1 2,4-D (2,4-dichlorophenoxyacetic acid), 50 mM NaCl, and 0.8 ppm of NPs for 1 month in the dark. CaO NP is determined to be more effective than CuO and ZnO in callus induction from leaf explants. Malondialdehyde (MDA) content was higher in the callus treated with 0.8 ppm CuO NP + 50 mM NaCl compared to other treatments. The callus induction stage, without salt treatments, showed the best results with 0.8 ppm CaO NPs for both H2O2 levels and peroxidase (POX) activity compared to the other NPs. The highest protein rate was obtained from the callus induction stage and callus formation stage after 50 mM treatment NaCl with 0.8 ppm CuO. The LCSM results displayed, under in vitro conditions, that the treatment of NPs can greatly suppress the negative effects of salt stress on calli samples. SEM analysis supported the results obtained by laser scanning confocal microscopy (LSCM) analysis. Our findings suggest that CuO, CaO, and ZnO NPs can offer a simple and effective method to protect alfalfa callus from NaCl stress severity. Furthermore, these NPs, particularly CaO, hold potential for broader application and should be evaluated under various abiotic conditions beyond salt stress.

Exploring the Anticancer Potential of MonoHER (7-Mono-O-(β-Hydroxyethyl)-Rutoside): Mitochondrial-Dependent Apoptosis in HepG2 Cells.

Flavonoids are a group of polyphenols, abundantly present in our diet. Although, based on their chemoprotective effects, intake of flavonoids is associated with a high anticancer potential as evidenced in in vitro and in vivo models, the molecular mechanism is still elusive. This study explores the antiproliferative and cytotoxic effects of the semi-synthetic flavonoid MonoHER (7-mono-O-(β-hydroxyethyl)-rutoside) in vitro on cancer cells. HepG2 liver, MCF7 breast, and H1299 lung cancer cells were grown under ambient conditions with or without MonoHER exposure. CCK8 assay was used to assess cell viability. Apoptosis, JC-1, and mitochondrial mass were determined using flow cytometry and confocal analysis. The effects of monoHER on apoptosis proteins were detected by confocal microscopy analysis and Western blot. It was found that MonoHER can reduce HepG2 cells' and MCF7 cells' viability, but not H1299 cells', and induced apoptosis only in HepG2 cells. MonoHER has the potential to enhance the expression of caspase-9 and caspase-3, to damage mitochondria, and to provoke the release of cytochrome C from the mitochondria. MonoHER can inhibit cell growth and induce apoptosis especially in HepG2 human liver cancer cells by triggering the mitochondrial signal transduction pathway, leading to the release of cytochrome C in the cytoplasm and the subsequent activation of caspase-9 and caspase-3. Future research should further explore MonoHER's mechanism of action, efficacy, and potential for clinical translation.

Topical review: Ocular surface abnormalities in neurodegenerative disorders.

In an aging population, the number of people living with neurodegenerative disease is projected to increase. It is vital to develop reliable, noninvasive biomarkers to detect disease onset and monitor progression, and there is a growing body of research into the ocular surface as a potential source of such biomarkers. This article reviews the potential of in vivo corneal confocal microscopy and tear fluid analysis as tools for biomarker development. Corneal confocal microscopy, traditionally used for studying corneal health, offers high-resolution imaging of corneal nerves and has shown promise for examining systemic diseases such as Alzheimer disease and Parkinson's disease. Complementarily, tear fluid analysis, known for its ease of collection, reflects systemic changes in neurodegenerative conditions. Together, these noninvasive techniques provide insights into disease onset and progression and hold potential for advancing diagnostic and treatment strategies.

TREM1 interferes with macrophage mitophagy via the E2F1-mediated TOMM40 transcription axis in rheumatoid arthritis.

Elevated synovial expression of the triggering receptor expressed on myeloid cells 1 (TREM1) has been identified as a significant biomarker for assessing disease activity in rheumatoid arthritis (RA). The upregulated expression of TREM1, induced by inflammatory mediators in infiltrating macrophages, plays a critical role in synovitis and joint destruction in RA. Our previous sequencing data linked TREM1 activation to aberrant mitophagy. Thus, we explored the efficacy of targeting TREM1 in treating experimental arthritis and its regulatory effect on mitophagy. TREM1 signalling activation was assessed via TREM1, DAP12, and p-SYK levels, and mitophagy was measured through PINK1, PARKIN, and LC3A/B levels. In vitro, TREM1-overexpressing RAW264.7cells were generated, and the differences in expression and pathways were analyzed via RNA-seq. Changes in the number and morphology of mitochondria and mitophagy in TREM1-overexpressing RAW264.7cells and normal control were observed via transmission electron microscopy, MitoTracker confocal microscopy and mitochondrial membrane potential analysis. The promotion of TOMM40 gene transcription by TREM1-activated E2F1 was determined via ChIP-PCR and E2F1 siRNA. We found that TREM1 was highly expressed and activated in the synovial tissues of CIA mice concomitant with abnormal mitophagy. The mitochondrial outer membrane transporter TOMM40 was upregulated in experimental arthritis, and the protein levels of PINK1 and LC3B were decreased. RNA-seq analysis indicated that mitophagy-related proteins were extensively downregulated and that the transcription factor E2F1 and the mitochondrial outer membrane transporter TOMM40 were significantly upregulated in TREM1-overexpressing cells. ChIP-PCR revealed that TREM1 overexpression significantly promoted the interaction between E2F1 and TOMM40 gene in RAW264.7cells. E2F1 knockdown markedly reversed TOMM40 upregulation, mitophagy injury and ROS production in TREM1-overexpressing macrophages but not in control cells. Our study provides preliminary evidence that E2F1 regulates TOMM40 transcription and disrupts mitophagy flux in TREM1-activated macrophages. Inhibiting TREM1 effectively mitigated experimental arthritis by restoring macrophage mitophagy and reducing intracellular ROS levels.

Dietary limonin alleviates Salmonella Typhimurium-induced colitis via dual targeting virulence SopB and SopE2 and inhibiting RAC1/CDC42/Arp2/3 pathway and regulating gut microbiota.

Salmonella enterica serovar Typhimurium (STM) causes severe colitis, necessitating the development of effective drugs. Here, the dockings of limonin with the STM T3SS-1 virulence factor SopB or SopE2 showed strong binding activity in silico and was verified by CETSA and DARTS assays in vitro. Limonin inhibited the enzyme activities and expression of SopB and SopE2 in vitro. Furthermore, we found that limonin treatment significantly reduced the number of STM colony-forming units (CFUs) in infected HeLa and Raw264.7 cells, which resulted in a decrease in the rate of membrane ruffling mediated by SopB-regulated Arf6/Cyth2/Arf1-, RAC1-, and CDC42-driven Arp2/3-dependent actin polymerization and the SopE2-regulated CDC42/Arp2/3 pathway, and the confocal laser scanning microscopy analysis revealed that limonin treatment repressed the recruitment of the Salmonella-containing vacuole (SCV) biomarkers LC3, Rab7, GAL8 and NDP52. Furthermore, limonin treatment ameliorated STM-induced colitis by reducing the disease activity index (DAI), colon shortening, and MPO and EPO activities; mitigating the severity of S. Typhimurium-induced colitis damage; and influencing the levels of inflammatory factors (IL-1β, IL-6, IL-10, TNF-α and IFN-γ) while increasing the levels of colonic epithelial barrier and tight junction genes (Mucin 1, Mucin 2, Occludin, Claudin-3 and ZO-1). A gut microbiota analysis revealed that limonin treatment influenced α- and β-diversity of the flora and increased the counts of the beneficial bacteria Muribaculum and Faecalibaculum to regulate gut microbiota dysbiosis. Finally, colon SCFA measurements revealed that limonin treatment significantly increased acetate, butyrate, propionate and valerate concentrations. Thus, this study is an important reference for the anti-STM effects of limonin on induced colitis.

Seaweeds: Nature's super therapeutics? Immunomodulatory and anti-viral properties of sulfated rhamnoxyloglucuronan isolated from Ulva fasciata Delile.

Green seaweeds, which make up a major population of total seaweed worldwide, possess various therapeutic properties. The aim of the study directed at isolating a (1 → 4) linked sulfated rhamno xyloglucuronan, designated as UFP-2, from the edible green seaweed Ulva fasciata Delile, and to evaluate its efficacy in modulating immune responses and inhibiting SARS-CoV-2 (Delta variant) infection. Anti-inflammatory potential of UFP-2 was demonstrated through the regulation of key cytokines involved in inflammatory responses triggered by viral infections, including interferons (IFN-α/γ), interleukin (IL-1β/12/33), and tumor necrosis factor (TNF-α). Confocal microscopy and flow cytometry analyses indicated downregulation of IFN-α and IL-1β, while TNF-α expression reduced from 29.28 % in lipopolysaccharide (LPS)-induced CALU-1 cells to 1.6-5.4 % upon UFP-2 treatment. Treatment with UFP-2 at a concentration of 125 μg/mL significantly downregulated the overexpression of IL-1β in SARS-CoV-2-infected CALU-1 cells. Administering UFP-2 to SARS-CoV-2 (delta variant) induced cells led to increased cell viability and higher cycle threshold (Ct) values (20.34), indicating reduced viral load, with viral copy numbers decreasing from over 6.5 × 107 to <2.5 × 107 per cell. Structure-activity relationship analysis suggested that sulfate groups and overall hydrophilicity of UFP-2 contribute to its binding affinity with target receptors, potentially disrupting viral entry and replication processes.

Effect of EGCG-Methacrylate-Functionalized Resin Infiltrant on White Spot Lesions: An In Vitro Study.

This study evaluated the color change (ΔE00) and penetration depth (PD) of white spot lesions (WSLs) infiltrated with the resin infiltrant (Icon®) functionalized with methacrylate epigallocatechin-3-gallate (EGCG). To introduce polymerizable double bonds, EGCG was reacted with methacryloyl chloride (EM). Subsequently, the Icon resin infiltrant (I) was loaded with neat EGCG (IE) or EGCG-methacrylate (IEM) at 2 wt% each. WSLs were created on bovine enamel blocks and treated with I, IE, or IEM. Sound and untreated enamel surfaces were used as controls (C). Infiltrant PD (%) was determined by Confocal Laser Scanning Microscopy (CLSM, n = 12) analysis. For color change (ΔE00) determination (n = 14), ΔL, Δa, and Δb, half of each sample was kept sound as a reference area. The color was determined with a spectrophotometer. Data were statistically evaluated (p = 0.05). Surface morphology was obtained as a qualitative response variable using 3D CLSM. PD (%) did not differ statistically for I, IE, and IEM (p = 0.780). Groups I and IEM showed similar performance on color change (ΔE00) compared to the control group, while IE exhibited intermediate results, with no significant difference observed between the untreated, I, and IEM groups (p < 0.001). IEM promoted the masking of the WSL color without interfering with the PD.

Nanotransethosomal dual-drug loaded gel of methotrexate and mangiferin as a potent synergistic intervention for rheumatoid arthritis via transdermal delivery

The goal of this study is to assess the potential advantages of utilising methotrexate (MTH), and mangiferin (MFR), in nanoparticulate configuration which is transethosomes (TRS), which could result in increased stability and solubility, as well as improved infiltration into the arthritic tissues under investigation. The synthesised MTH-MFR-TRS demonstrated a particle size of 151.7 nm and a PDI of 0.1199. Additionally, the zeta potential was observed to be favourable at -30.43 mV. Supplementary evaluations were performed, comprising transmission electron microscopy (TEM), confocal microscopy and skin permeation analysis. The CLSM study revealed that the MTH-MFR-TRS gel formulation demonstrated enhanced permeation of MTH and MFR through the skin layers in comparison with MTH-MFR suspension gel. The MTT assay conducted on Raw 264.7 and SW982 cell lines demonstrated a significant decrease (P < 0.05) in the IC50 value of the MTH-MFR-TRS formulation (9.0 mM and 60.8 mM, respectively) when compared to the drug suspension. The results of the in vivo investigation indicate that the MTH-MFR-TRS gel displays favourable anti-arthritic characteristics compared to the diclofenac standard gel. The aforementioned phenomenon was evidenced by means of histopathological investigations and radiographic scrutiny. The study at hand has validated the utility of TRS vesicles as a carrier for the transdermal administration of MTH and MFR, thereby offering a promising therapeutic approach for the management of rheumatoid arthritis.

High-temperature confocal laser scanning microscopy analysis of phase transformation of steels: a review

High-temperature confocal laser scanning microscopy analysis of phase transformation of steels: a review

Synergistic Effects and Mechanisms of Action of Rutin with Conventional Antibiotics Against Escherichia coli.

Rutin is a widely known plant secondary metabolite that exhibits multiple physiological functions. The present study focused on screening for synergistic antibacterial combinations containing rutin, and further explored the mechanisms behind this synergy. In vitro antibacterial test results of rutin showed that the ranges of minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC) are 0.125-1 and 0.125-2 mg/mL, respectively. However, rutin and amikacin have a significant synergistic effect, with a fractional inhibitory concentration index (FICI) range of 0.1875-0.5. The time bactericidal curve proved that the combination of rutin and amikacin inhibited bacterial growth within 8 h. Scanning electron microscopy (SEM) revealed that a low-dose combination treatment could disrupt the cell membrane of Escherichia coli (E. coli). A comprehensive analysis using alkaline phosphatase (AKP), K+, and a protein leakage assay revealed that co-treatment destroyed the cell membrane of E. coli, resulting in the significant leakage of AKP, intracellular K+, and proteins. Moreover, confocal laser scanning microscopy (CLSM) and red-green cell ratio analysis indicated severe damage to the E. coli cell membrane following the co-treatment of rutin and amikacin. This study indicates the remarkable potential of strategically selecting antibacterial agents with maximum synergistic effect, which could significantly control antibiotic resistance.

Formation and Characterization of Mycelium-Potato Protein Hybrid Materials for Application in Meat Analogs or Substitutes.

There is increasing interest in the development of meat analogs due to growing concerns about the environmental, ethical, and health impacts of livestock production and consumption. Among non-meat protein sources, mycoproteins derived from fungal fermentation are emerging as promising meat alternatives because of their natural fibrous structure, high nutritional content, and low environmental impact. However, their poor gelling properties limit their application in creating meat analogs. This study investigated the potential of creating meat analogs by combining mycoprotein (MCP), a mycelium-based protein, with potato protein (PP), a plant-based protein, to create hybrid products with meat-like structures and textures. The PP-MCP composites were evaluated for their physicochemical, rheological, textural, and microstructural properties using electrophoresis, differential scanning calorimetry, dynamic shear rheology, texture profile analysis, confocal fluorescence microscopy, and scanning electron microscopy analyses. The PP-MCP hybrid gels were stronger and had more fibrous structures than simple PP gels, which was mainly attributed to the presence of hyphae fibers in mycelia. Dynamic shear rheology showed that the PP-MCP hybrids formed irreversible heat-set gels with a setting temperature of around 70 °C during heating, which was attributed to the unfolding and aggregation of the potato proteins. Confocal and electron microscopy analyses showed that the hybrid gels contained a network of mycelia fibers embedded within a potato protein matrix. The hardness of the PP-MCP composites could be increased by raising the potato protein content. These findings suggest that PP-MCP composites may be useful for the development of meat analogs with more meat-like structures and textures.

Pre-Adipocytes in 3D Co-Culture Underwent Self-Differentiation: New Perspectives for an Old Model

Adipogenesis is a complex process influenced by various cellular interactions within adipose tissue, which plays a critical role in metabolic homeostasis. This study aimed to develop a novel in vitro three-dimensional (3D) co-culture model using murine 3T3-L1 pre-adipocytes, J774 macrophages, and NIH-3T3 fibroblasts to investigate adipogenic differentiation and inflammatory pathways. We first validated an adipogenic differentiation protocol in a two-dimensional (2D) model, where 3T3-L1 pre-adipocytes were subjected to a hormonal medium containing 3-isobutyl-1-methylxanthine, dexamethasone and insulin. After 7 days, differentiated cells were analyzed using Oil Red O and Nile Red staining, confirming lipid accumulation. Subsequently, spheroids were formed in 3D cultures, with monospheroids and heterospheroids maintained in either control medium or MDI for 11 days. Size measurements indicated significant growth in heterospheroids, particularly in the 3T3-L1:J774 combination, underscoring the importance of cellular interactions. Confocal microscopy and flow cytometry analyses demonstrated that even in the absence of hormonal stimuli, control spheroids exhibited adipogenic differentiation, evidenced by a notable proportion of Nile Red-positive cells (75.7 ± 1.7%). Inflammatory profiling revealed that the heterospheroid 3:J produced the highest levels of nitric oxide (NO), with no significant differences observed between control and MDI conditions. This study highlights the potential of 3D co-culture systems for elucidating the intricate interactions among adipocytes, macrophages, and fibroblasts. The findings may provide valuable insights into novel therapeutic targets for metabolic disorders.

The Role of the Bactericidal Mechanism of Copper Elements and Its Effect on the Corrosion Resistance of Steel.

In this paper, two kinds of copper-containing steels with copper contents of 2.31 and 6.01 wt.% were designed. By comparing with commercial Q355, the bactericidal properties of copper in seawater containing sulfate-reducing bacteria (SRB) and its influence on the corrosion process of steel were revealed. The corrosion rate, morphology of products, and bactericidal action of copper were tracked by scanning electron microscopy, X-ray diffraction, confocal microscopy, and electrochemical analysis techniques. It was found that the resistance of copper-containing steel to bacterial corrosion was obviously better than that of non-copper-containing steel. At 28 days, the weight loss rates in the SRB environment for 0Ni2Cu6 samples increased by merely 5.43%, which was nearly half that of Q355 of 9.75%. Cu-containing steels exhibited potent antibacterial action, with the ε-Cu phase altering the corrosion byproduct composition from brittle flakes to robust particles and inhibiting the production of H2S. The killed bacteria adhered to the surface of the steel and slowed down the corrosion of the steel. The confocal laser scanning microscope and electrochemical experiments showed that a dense CuFeO4 film formed on the substrate, impeding corrosive ion penetration, and an upsurge in Cu content markedly enhanced the material's anti-corrosion and antimicrobial attributes.

Antimicrobial Efficacy of Ampicillin With Ceftriaxone in Comparison With Diclofenac Sodium, Modified Triple Antibiotic Paste, and Calcium Hydroxide Against Enterococcus faecalis: An In-Vitro Study.

Objective This in vitro study aimed to assess and compare the antimicrobial effectiveness of ampicillin with ceftriaxone (AC), diclofenac sodium (DS), modified triple antibiotic paste (MTAP), and calcium hydroxide (CH) against Enterococcus faecalis in root canal systems. Materials and methods The antimicrobial activity of the medicaments was assessed by determining the minimum inhibitory concentrations (MIC) via the agar well diffusion method. A total of 40 extracted permanent teeth underwent root canal treatment, and Enterococcus faecalis was introduced into the canal preparations. Intracanal medicaments, including AC, DS, MTAP, and CH, were applied, and their antimicrobial effects were quantified by colony-forming unit (CFU) counts before and after application. The impact on bacterial viability was further examined using confocal laser microscopy on root canal sections treated with different medicaments. Results The MIC results indicated lower concentrations of AC and MTAP, suggesting higher efficacy. AC demonstrated a more significant reduction in CFU counts than DS, MTAP, and CH. Confocal laser microscopy further supported the superior antimicrobial activity of AC. Conclusion The MIC results revealed thatACandMTAPrequired lower concentrations to inhibit Enterococcus faecalis, indicating higher antimicrobial efficacy. Among the medicaments tested, AC significantly reduced CFU counts more than DS, MTAP, and CH. Confocal laser microscopy analysis further confirmed the superior antimicrobial activity of AC by showing a greater reduction in bacterial viability in the treated root canal sections.

Bacterial biofilm inactivation by plasma activated nanobubble water

This research developed novel plasma activated nanobubble water (PANBW) by integrating atmospheric cold plasma and nanobubble water (NBW) technologies. Mixing of plasma reactive species with NBW to generate the PANBW makes it an effective solution for microbial biofilm inactivation and water treatment, possibly by leveraging the benefits of both technologies. Selected properties of PANBW, including the concentrations of reactive oxygen and nitrogen species (RONS) were characterized, and the stability of RONS during storage for 7 days were evaluated. The combination of argon and air as feed gases was used to determine the influence of feed gas type on RONS production and the effect of the generated PANBW on biofilm reduction. The effectiveness of PANBW in inactivating mixed-species bacterial biofilms was assessed against NBW, plasma activated water (PAW), and their combinations. This comparison involved treating biofilms of Salmonella enterica Typhimurium ATCC 13311 and Aeromonas australiensis, that were grown on stainless steel coupons by these solutions. The PANBW treatment was most effective in the inactivation of the tested mixed species biofilms with a reduction of >2 log CFU/cm2 in the biofilm population. The confocal laser scanning microscopy analysis was consistent with the bacterial inactivation results. This study highlights the potential of atmospheric cold plasma when combined with nanobubble technology, as a novel and efficient method for biofilm control and food safety applications.

1,8-Cineole reduces pulmonary vascular remodelling in pulmonary arterial hypertension by restoring intercellular communication and inhibiting angiogenesis.

Pulmonary Arterial Hypertension (PAH) is characterized by pulmonary vascular remodelling, often associated with disruption of BMPR2/Smad1/5 and BMPR2/PPAR-γ signalling pathways that ultimately lead to right ventricle failure. Disruption of intercellular junctions and communication and a pro-angiogenic environment are also characteristic features of PAH. Although, current therapies improve pulmonary vascular tone, they fail to tackle other key pathological features that could prevent disease progression. In this scenario, aromatic plants emerge as promising sources of bioactive compounds, with 1,8-cineole standing out due to its hypotensive properties and cardioprotective effect in PAH. The present study aims to explore for the first time the effect of 1,8-cineole in pulmonary vascular remodelling associated with PAH. Resorting to the monocrotaline (MCT)-induced PAH animal model, the effect of 1,8-cineole on vascular remodelling including interstitial collagen accumulation, smooth muscle cell proliferation and protein levels of BMPR2 pathway-related proteins, was assessed by microscopy and western blot (WB) analysis. The integrity of gap junctions, pulmonary surfactant, mitochondrial structure and endothelial cell barrier were evaluated by transmission electron microscopy, confocal microscopy and WB analysis. Furthermore, the effect of 1,8-cineole on angiogenesis was determined on pulmonary artery endothelial cells (PAEC) submitted to hypoxia using the scratch wound and Matrigel angiogenesis assays, and the number of sprouts on isolated healthy and diseased pulmonary artery rings, treated with the compound, enabled the validation of these effects. 1,8-Cineole mitigated PAH-associated derailment of both BMPR2/Smad1/5 and BMPR2/PPAR-γ pathways and concomitantly reduced interstitial fibrosis and the arterial medial layer thickness in pulmonary arteries. The compound restored gap junction, lung surfactant and mitochondrial integrity and preserved endothelial barrier integrity. Furthermore, 1,8-cineole exerted an anti-angiogenic effect, by impairing the formation of vessel-like structures in PAEC and sprouting formation in isolated pulmonary arteries. The present study brings new insights about the mechanisms whereby 1,8-cineole impacts pulmonary vascular remodelling and demonstrates the potential of 1,8-cineole as a therapeutic strategy to hamper PAH progression.

Chondroitin Sulfate-Coated Heteroduplex-Molecular Spherical Nucleic Acids.

Molecular Spherical Nucleic Acids (MSNAs) are atomically uniform dendritic nanostructures and potential delivery vehicles for oligonucleotides. The radial formulation combined with covalent conjugation may hide the oligonucleotide content and simultaneously enhance the role of appropriate conjugate groups on the outer sphere. The conjugate halo may be modulated to affect the delivery properties of the MSNAs. In the present study, [60]fullerene-based molecular spherical nucleic acids, consisting of a 2'-deoxyribonucleotide and a ribonucleotide sequence, were used as hybridization-mediated carriers ("DNA and RNA-carriers") for an antisense oligonucleotide, suppressing Tau protein, (i. e. Tau-ASO) and its conjugates with chondroitin sulfate tetrasaccharides (CS) with different sulfation patterns. The impact of the MSNA carriers, CS-moieties on the conjugates and the CS-decorations on the MSNAs on cellular uptake and - activity (Tau-suppression) of the Tau-ASO was studied with hippocampal neurons in vitro. The formation and stability of these heteroduplex ASO-MSNAs were evaluated by UV melting profile analysis, polyacrylamide gel electrophoresis (PAGE), dynamic light scattering (DLS) and size exclusion chromatography equipped with a multi angle light scattering detector (SEC-MALS). The cellular uptake and - activity were studied by confocal microscopy and Western blot analysis, respectively.

Nutritionally physiological cell culture medium and 3D culture influence breast tumour proteomics and anti-cancer drug effectiveness

Many drugs have been discontinued during phase II/III breast cancer clinical trials due to lack of clinical efficacy, indicating shortcomings in predictive value of preclinical data. Nutrient availability in the tumour cell microenvironment and the dimensionality of in vitro tumour cells likely impact on drug responsiveness. Global proteomics experiments were conducted to assess the impact of nutrient availability and dimensionality of culture. Protein set enrichment analyses identified “pathways in cancer”, “focal adhesion” and “ECM receptor in interaction” related to cell culture dimensionality in MDA-MB-231 cells. In MCF-7 cells, 4 pathways were influenced by medium composition, and 2 pathways were influenced by cell culture dimensionality (2D vs. 3D). These pathways were also identified using KEGG analyses. Eight drugs were selected for investigation according to the differential expression of their putative or known target proteins. The influence of medium composition on drug effectiveness was explored using the “Melbourne Medium” (MM), developed to have nutritionally physiological levels of metabolites as compared with conventional (hyper-nutritional) cell culture medium (CM). The influence of dimensionality on drug effectiveness was also explored, using an innovative 3D viability assessment combining automated confocal microscopy and image analysis. Dimensionality of culture appeared to have a greater influence on the proteome and drug effects than variation in nutrient levels. The number of differentially expressed proteins in the different media was greater in 2D than 3D. We conclude that the risk of qualifying inactive compounds in preclinical assessment may be mitigated using additional models incorporating physiological media and 3-dimensionality.