Search Results (63)

Background/Objectives: Non-syndromic cleft lip with or without palate (NSCL/P) is a common, multifactorial congenital anomaly. As genetic associations can be population-specific, this study aimed to investigate single-nucleotide polymorphisms (SNPs) in the VAX1, MAFB, and WNT3 genes for association with NSCL/P in a Japanese cohort. Methods: A case–control study was conducted with 310 Japanese patients with NSCL/P and 308 ethnically matched healthy controls from Aichi Gakuin Dental Hospital. We genotyped SNPs rs7078160 (VAX1), rs13041247 (MAFB), and rs3809857 (WNT3) using TaqMan assays. Associations were assessed using chi-squared tests, with results stratified by sex and corrected for multiple comparisons using the Bonferroni method. Results: The VAX1 rs7078160 A allele was significantly associated with an increased risk for NSCL/P (OR = 1.67, p < 0.00001). The association was particularly strong in females (OR = 1.93, p < 0.00001) but not significant in males after correction. The MAFB rs13041247 variant showed a nominal protective association with the NSCLO subtype that was not significant after Bonferroni correction. No significant association was found for WNT3. A notable gene–gene interaction was observed, where carrying risk alleles for both VAX1 and MAFB significantly increased overall NSCL/P risk (OR = 2.65, p = 0.00008). Conclusions: VAX1 rs7078160 is a significant risk factor for NSCL/P in the Japanese population, with a pronounced female-specific effect. A synergistic interaction between VAX1 and MAFB elevates disease risk, whereas WNT3 was not implicated in this cohort. These findings underscore the population-specific genetic architecture of NSCL/P. Full article

The optimization of bioactive compound mixtures is critical for enhancing pharmacological efficacy. This study investigates, for the first time, the combined effects of eugenol, camphor, and terpineol, focusing on their half-maximal inhibitory concentrations (IC₅₀) across multiple biological responses related to diabetes management. Using a mixture design approach, the objective was to determine the optimal formulation that maximizes bioactivity and validate the findings experimentally. A simplex-centroid design was applied to evaluate the combined effects of eugenol, camphor, and terpineol on AAI _IC50, AGI _IC50, LIP _IC50, and ALR _IC50 responses. The desirability function was used to determine the ideal composition. The optimized formulation was experimentally validated using in vitro assays, and IC50 values were measured for each response using standard protocols. Results: The optimal formulation identified was 44% eugenol, 0.19% camphor, and 37% terpineol, yielding IC₅₀ values of 10.38 µg/mL (AAI), 62.22 µg/mL (AGI), 3.42 µg/mL (LIP), and 49.58 µg/mL (ALR). The desirability score (0.99) confirmed the effectiveness of the optimized blend. Experimental validation of the optimal mixture resulted in IC₅₀ values of 11.02 µg/mL (AAI), 60.85 µg/mL (AGI), 3.75 µg/mL (LIP), and 50.12 µg/mL (ALR), showing less than 10% deviation from predicted values, indicating high model accuracy. This study confirms the combined potential of eugenol, camphor, and terpineol, with eugenol and terpineol significantly enhancing bioactivity. The validated formulation demonstrates potential for pharmaceutical and cosmeceutical applications. Future research should explore mechanistic interactions, bioavailability, and in vivo efficacy to support the development of optimized natural compound-based therapies. Full article

Lipases are important industrial enzymes with a wide range of applications across various sectors. Cold-active lipases are particularly well suited for industrial processes that operate at low temperatures (such as food processing and environmental remediation) due to their high catalytic efficiency and energy-saving benefits. In this study, a novel lipase—LipU (GenBank accession: PV094892)—was heterologously expressed from Bacillus cereus U2 and characterized for its low-temperature adaptability and alkaline resistance. LipU belongs to the lipase Subfamily I.5 and shares the highest amino acid sequence identity (53.32%) with known homologs. Enzymatic assays revealed that LipU exhibits optimal activity at 20 °C and pH 11. It retained 95% of its initial activity after 24 h of incubation at 4 °C and pH 11.0. Furthermore, the activity of LipU was enhanced by Ca²⁺, Na⁺, Tween 20, and Tween 80, whereas it was inhibited by Cu²⁺, Zn²⁺, Mn²⁺, and sodium dodecyl sulfate (SDS). LipU demonstrated tolerance to various organic solvents of differing polarity; after 1 h of exposure to 15% (v/v) ethanol, n-butanol, isoamyl alcohol, dimethyl sulfoxide, or glycerol, it retained over 78.6% of its activity. These properties make LipU a promising candidate for industrial applications, including for leather degreasing, alkaline wastewater treatment, and low-temperature biocatalysis. Full article

Objectives: Nasal mucosa wound healing faces challenges such as acidic microenvironments and bacterial proliferation. Persistent mucosal defects predispose to complications such as nasal septal perforation. Conventional drug delivery systems suffer from nonspecific release and short-term efficacy. This study aimed to develop a pH-responsive liposome-hydrogel composite (HYD-Lip/DXMS@HG) to integrate pH-triggered dexamethasone (DXMS) delivery, antifouling properties, and mechanical support for refractory injuries. Methods: The composite combined acylhydrazone-modified liposomes with a hydrogel synthesized from hydroxyethylacrylamide (HEAA) and diethylacrylamide (DEAA). In vitro assays evaluated DXMS release kinetics, RPMI 2650 cell migration/proliferation, and antibacterial properties. In vivo rabbit nasal mucosal injury models assessed healing efficacy via histology analyses. RNA sequencing was performed to identify key signaling pathways. Results: HYD-Lip/DXMS@HG exhibited sustained DXMS release in acidic conditions, accelerating cell migration/proliferation in vitro. In rabbits, the composite reduced TNF-α expression and CD45+ leukocyte infiltration, while enhancing collagen alignment and epithelial thickness. RNA sequencing identified upregulated ECM receptor interaction, Hippo, TGF-β, and PI3K-Akt pathways, linked to collagen remodeling, anti-apoptosis, and angiogenesis. Conclusions: This multifunctional platform synergizes pH-triggered drug delivery, mechanical support, and antibacterial activity, offering a promising therapeutic strategy for refractory nasal mucosal injuries and postoperative recovery. Full article

Background/Objectives: Iron overload is a serious condition that can increase the production of reactive oxygen species (ROS), leading to oxidative tissue damage and organ dysfunction. While current pharmaceutical drugs for iron chelation have limitations, the search for natural herbs with iron-chelating properties is crucial. This study aimed to explore the various biological functions of the Perilla frutescens seed, regarding antioxidant activity and hepatoprotective and iron-chelating properties. Methods:Perilla frutescens seeds were subjected to extraction using a solvent-partitioning technique. Each fraction was evaluated for total phenolic content (TPC), total flavonoid content (TFC), and rosmarinic acid (RA) content by Folin–Ciocalteu assay, aluminum chloride colorimetric assay, and ultra-high-performance liquid chromatography (UHPLC), respectively. Antioxidant activity was assessed using DPPH, ABTS, and FRAP assays. The inhibition of lipid peroxidation was evaluated using the TBARS assay in HepG2 cells and an egg yolk model. The iron-chelating activity was examined using a ferric nitrilotriacetate (Fe³⁺-NTA)-binding assay, labile iron pool (LIP) level assessment, and the transferrin receptor (TfR) expression in HepG2 cells. Results: Phytochemical analysis indicated that the ethyl acetate (EtOAc) fraction had the highest TPC, TFC, and RA. This fraction demonstrated strong antioxidant properties and attenuated lipid peroxidation in HepG2 cells and egg yolk. In addition, this fraction exhibited iron-binding activity, decreased LIP levels, and induced TfR expression in iron-loaded HepG2 cells similar to the rosmarinic acid standard. Conclusions: These findings suggest that the EtOAc fraction of the Perilla frutescens seed possesses promising potential as a therapeutic agent for treating iron overload. Full article

Objectives: This work aimed to develop and investigate liposomes incorporating Rhein (Lip-Rh) into the liposomal membrane to enhance the compound’s water solubility and oral bioavailability. Methods: Liposomes were produced by the thin lipid film technique, with a phosphatidylcholine-to-cholesterol molar ratio of 5:1, dissolved in chloroform and methanol, and thereafter hydrated with ultrapure water and subjected to sonication. The resultant liposomes were studied from a physicochemical perspective using DLS, zeta potential, STEM, UV–Vis, and fluorescence spectroscopies, while oral bioavailability was assessed by fluorescence imaging. Additionally, cell viability assays were performed on tumour cells (MCF-7) in comparison to normal cells (HGFs). Results: The resultant nanoparticles exhibited relatively uniform sizes and narrow size distribution. In vivo fluorescence imaging studies performed on Wistar rats demonstrated significantly enhanced oral bioavailability for Lip-Rh, with rapid absorption into the bloodstream observed one hour after administration, in contrast to the free compound dissolved in vegetable oil. Cell viability assays demonstrated higher cytotoxicity of Lip-Rh towards MCF-7 cells compared to HGF cells, highlighting the selective therapeutic potential of the product. Moreover, we determined that the optimal dose of Rhein per kilogram of body weight, when encapsulated in liposomes, is approximately 2.5 times less than when Rhein is delivered in its unencapsulated form. Conclusions: Lip-Rh is a promising candidate for oncological treatments, presenting three key advantages: increased cytotoxicity towards tumour cells, protection of normal tissues, and the practicality of oral delivery. Additional investigation is required to explore its application in anticancer therapy, whether as monotherapy or as a complementary treatment. Full article

Background: The emergence of multidrug-resistant bacteria presents a significant global health threat. Liposomal antibiotics have shown a potential to improve antibiotic delivery and efficacy. This study aimed to develop liposomes encapsulating tobramycin (TOB) and methylglyoxal (MGO) to enhance TOB activity while reducing bacterial adhesion and biofilm formation. Methods: Clinical isolates of Pseudomonas aeruginosa and Klebsiella pneumoniae were characterized using whole-genome sequencing. Liposomes (Lip-MGO-TOB) were formulated using Manuka honey as a surfactant and loaded with MGO and TOB. Antibacterial activity, biofilm formation, and bacterial cell adhesion assays were performed to compare the efficacy of Lip-MGO-TOB against free TOB. Liposome characterization included analyses of morphology, zeta potential, TOB encapsulation efficiency, and stability under various biological conditions. Results: The Lip-MGO-TOB formulation, at a minimum inhibitory concentration (MIC) of 32 µg/mL, reduced the biofilm formation of the P. aeruginosa isolate (PA85) by 68%. Conversely, free TOB, at a MIC of 64 µg/mL, achieved only a 21% reduction. For the K. pneumoniae isolate (KP57), Lip-MGO-TOB inhibited bacterial adhesion to A549 cells at a lower concentration (256 µg/mL) compared to free TOB (512 µg/mL). Lip-MGO-TOB demonstrated sustained drug release over 24 h under tested conditions and retained over 99% of TOB. Conclusions: The Lip-MGO-TOB formulation significantly enhanced TOB activity against resistant bacteria compared to free TOB. Additionally, it provided a stable drug delivery system with controlled drug release. Liposomal TOB represents a promising advancement in combating antibiotic resistance by improving the efficacy and delivery of conventional antibiotics. Full article

Integrated pest management based on the use of biopesticides is largely applied. Experimental bioassays are critical to assess biopesticide biosafety at the ecotoxicological level. In this study, we investigated the effects of the new Bacillus thuringiensis subsp. kurstaki (Btk)-formulated-based biopesticides BLB1 and Lip, efficiently tested in field assays (IPM-4-CITRUS EC project no. 734921) on two aquatic non-target organisms, precisely the water flea Daphnia magna and the bioluminescent bacteria Aliivibrio fischeri. Acute toxicity studies, carried out in a comparative manner with Delfin^® as the reference bioproduct and the lactose-based Blank formulation, show that no significant toxicity was observed up to 1 g/L. Our results indicated that BLB1- and Lip-formulated new bioproducts are far less toxic than the Delfin^® reference bioproduct. Full article

The expanded newborn screening (NBS) program in the Russian Federation was initiated in 2023, among which severe combined immunodeficiency (SCID) is screened using TREC/KREC assays. Here, we report a rare case of a TP63-associated disease identified through this NBS program. Dried blood spots from newborns were initially screened for TREC/KREC levels, and those with values below the cut-off underwent confirmatory testing and further genetic analysis, including whole-exome sequencing (WES). A male newborn was identified with significantly reduced TREC values, indicative of T cell lymphopenia. Genetic analysis revealed a heterozygous NM_003722.5:c.1027C>T variant in TP63, leading to the p.(Arg343Trp) substitution within the DNA binding domain. This mutation has been previously associated with Ectrodactyly–Ectodermal Dysplasia–Cleft lip/palate syndrome (EEC) syndrome and shown to reduce the transactivation activity of TP63 in a dominant-negative manner. This case represents one of the few instances of immune system involvement in a patient with a TP63 mutation, highlighting the need for further investigation into the immunological aspects of TP63-associated disorders. Our findings suggest that comprehensive immunological evaluation should be considered for patients with TP63 mutations to better understand and manage potential immune dysfunctions. Full article

Background: This study aimed to determine, at the phenotypic and molecular levels, resistance and virulence markers in Candida spp. isolated from community-acquired infections in Bucharest outpatients during 2021, and to demonstrate the efficiency of alternative solutions against them based on silver nanoparticles (AgNPs). Methods: A total of 62 Candida spp. strains were isolated from dermatomycoses and identified using chromogenic culture media and MALDI-TOF MS, and then investigated for their antimicrobial resistance and virulence markers (VMs), as well as for metabolic enzymes using enzymatic tests for the expression of soluble virulence factors, their biofilm formation and adherence capacity on HeLa cells, and PCR assays for the detection of virulence markers and the antimicrobial activity of alternative solutions based on AgNPs. Results: Of the total of 62 strains, 45.16% were Candida parapsilosis; 29.03% Candida albicans; 9.67% Candida guilliermondii; 3.22% Candida lusitaniae, Candia pararugosa, and Candida tropicalis; and 1.66% Candida kefyr, Candida famata, Candida haemulonii, and Candida metapsilosis. Aesculin hydrolysis, caseinase, and amylase production were detected in the analyzed strains. The strains exhibited different indices of adherence to HeLa cells and were positive in decreasing frequency order for the LIP1, HWP1, and ALS1,3 genes (C. tropicalis/C. albicans). An inhibitory effect on microbial growth, adherence capacity, and on the production of virulence factors was obtained using AgNPs. Conclusions: The obtained results in C. albicans and Candida non-albicans circulating in Bucharest outpatients were characterized by moderate-to-high potential to produce VMs, necessitating epidemiological surveillance measures to minimize the chances of severe invasive infections. Full article

Betulinic acid is a lupane-type pentacyclic triterpene mostly found in birch bark and thoroughly explored for its wide range of pharmacological activities. Despite its impressive biological potential, its low bioavailability has challenged many researchers to develop different formulations for achieving better in vitro and in vivo effects. We previously reported the synthesis of fatty acid esters of betulinic acid using butyric, stearic, and palmitic acids (But-BA, St-BA, and Pal-BA) and included them in surfaced-modified liposomes (But-BA-Lip, St-BA-Lip, Pal-BA-Lip). In the current study, we evaluated the cytotoxic effects of both fatty acid esters and their respective liposomal formulations against MCF-7, HT-29, and NCI-H460 cell line. The cytotoxic assessment of BA derivatives revealed that both the fatty esters and their liposomal formulations acted as cytotoxic agents in a dose- and time-dependent manner. But-BA-Lip exerted stronger cytotoxic effects than the parent compound, BA and its liposomal formulation, and even stronger effects than 5-FU against HT-29 cells (IC₅₀ of 30.57 μM) and NCI-H460 cells (IC₅₀ of 30.74 μM). BA’s fatty esters and their respective liposomal formulations facilitated apoptosis in cancer cells by inducing nuclear morphological changes and increasing caspase-3/-7 activity. The HET-CAM assay proved that none of the tested compounds induced any irritative effect, suggesting that they can be used safely for local applications. Full article

Background: Hepatocellular carcinoma (HCC), currently ranking as the third most lethal malignancy, poses a grave threat to human health. Ferroptosis, a form of programmed cell demise, has emerged as a promising therapeutic target in HCC treatment. In this study, we investigated the impact of ginsenoside RK1 on ferroptosis induction in HCC cells and elucidated the underlying mechanisms. Methods: The HCC cell line HepG2 was utilized to evaluate the effects of ginsenoside RK1. Distinct dosages of ginsenoside RK1 (25 μM, 50 μM, and 100 μM) were selected based on half-maximal inhibitory concentration (IC50) values. Cellular viability was assessed using a CCK8 assay, cytotoxicity was measured via lactate dehydrogenase (LDH) release assay, and colony-forming ability was evaluated using the clone formation assay. Various inhibitors targeting apoptosis (Z-VAD-FMK 20 μM), necrosis (Nec-1, 10 μM), and ferroptosis (Fer-1, 10 μM; Lip-1, 1 μM) were employed to assess ginsenoside RK1’s impact on cell demise. Intracellular levels of key ions, including glutathione (GSH), malondialdehyde (MDA), and iron ions, were quantified, and the protein expression levels of ferroptosis-related genes were evaluated. The sensitivity of HCC cells to ferroptosis induction by ginsenoside RK1 was examined following the overexpression and silencing of the aforementioned target genes. Results: Ginsenoside RK1 exhibited an inhibitory effect on HCC cells with an IC50 value of approximately 20 μM. It attenuated cellular viability and colony-forming capacity in a dose-dependent manner, concurrently reducing intracellular GSH levels and increasing intracellular Malondialdehyde (MDA) and iron ion contents. Importantly, cell demise induced by ginsenoside RK1 was specifically counteracted by ferroptosis inhibitors. Furthermore, the modulation of Ferroptosis suppressor protein 1 (FSP1) expression influenced the ability of ginsenoside RK1 to induce ferroptosis. FSP1 overexpression or silencing enhanced or inhibited ferroptosis induction by ginsenoside RK1, respectively. Conclusions: Ginsenoside RK1 enhances ferroptosis in hepatocellular carcinoma through an FSP1-dependent pathway. Full article

Background: Nanoparticles conjugated with fluorescent probes have versatile applications, serving not only for targeted fluorescent imaging but also for evaluating the in vivo profiles of designed nanoparticles. However, the relationship between fluorophore density and nanoparticle behavior remains unexplored. Methods: The IR783-modified liposomes (IR783-sLip) were prepared through a modified ethanol injection and extrusion method. The cellular uptake efficiency of IR783-sLip was characterized by flow cytometry and fluorescence microscope imaging. The effects of IR783 density on liposomal in vivo behavior were investigated by pharmacokinetic studies, biodistribution studies, and in vivo imaging. The constitution of protein corona was analyzed by the Western blot assay. Results: Dense IR783 modification improved cellular uptake of liposomes in vitro but hindered their blood retention and tumor imaging performance in vivo. We found a correlation between IR783 density and protein corona absorption, particularly IgM, which significantly impacted the liposome performance. Meanwhile, we observed that increasing IR783 density did not consistently improve the effectiveness of tumor imaging. Conclusions: Increasing the density of modified IR783 on liposomes is not always beneficial for tumor near-infrared (NIR) imaging yield. It is not advisable to prematurely evaluate novel nanomaterials through fluorescence dye conjugation without carefully optimizing the density of the modifications. Full article

Cutaneous leishmaniasis (CL) is a neglected tropical disease. The treatment is restricted to drugs, such as meglumine antimoniate and amphotericin B, that exhibit toxic effects, high cost, long-term treatment, and limited efficacy. The development of new alternative therapies, including the identification of effective drugs for the topical and oral treatment of CL, is of great interest. In this sense, a combination of topical photodynamic therapy (PDT) with chloroaluminum phthalocyanine liposomes (Lip-ClAlPc) and the oral administration of a self-emulsifying drug delivery system containing fexinidazole (SEDDS-FEX) emerges as a new strategy. The aim of the present study was to prepare, characterize, and evaluate the efficacy of combined therapy with Lip-ClAlPc and SEDDS-FEX in the experimental treatment of Leishmania (Leishmania) major. Lip-ClAlPc and SEDDS-FEX were prepared, and the antileishmanial efficacy study was conducted with the following groups: 1. Lip-ClAlPc (0.05 mL); 2. SEDDS-FEX (50 mg/kg/day); 3. Lip-ClAlPc (0.05 mL)+SEDDS-FEX (50 mg/kg/day) combination; 4. FEX suspension (50 mg/kg/day); and 5. control (untreated). BALB/c mice received 10 sessions of topical Lip-ClAlPc on alternate days and 20 consecutive days of SEDDS-FEX or FEX oral suspension. Therapeutical efficacy was evaluated via the parasite burden (limiting-dilution assay), lesion size (mm), healing of the lesion, and histological analyses. Lip-ClAlPc and SEDDS-FEX presented physicochemical characteristics that are compatible with the administration routes used in the treatments. Lip-ClAlPc+SEDDS-FEX led to a significant reduction in the parasitic burden in the lesion and spleen when compared to the control group (p < 0.05) and the complete healing of the lesion in 43% of animals. The Lip-ClAlPc+SEDDS-FEX combination may be promising for the treatment of CL caused by L. major. Full article

Aeromonas veronii is widespread in aquatic environments and is responsible for infecting various aquatic animals. In this study, a dominant strain was isolated from the hepatopancreas of diseased Macrobrachium rosenbergii and was named JDM1-1. According to its morphological, physiological, and biochemical characteristics and molecular identification, isolate JDM1-1 was identified as A. veronii. The results of artificial challenge showed isolate JDM1-1 had high pathogenicity to M. rosenbergii with an LD₅₀ value of 8.35 × 10⁵ CFU/mL during the challenge test. Histopathological analysis revealed severe damage in the hepatopancreas and gills of the diseased prawns, characterized by the enlargement of the hepatic tubule lumen and gaps between the tubules as well as clubbing and degeneration observed at the distal end of the gill filament. Eight virulence-related genes, namely aer, ompA, lip, tapA, hlyA, flgA, flgM, and flgN, were screened by PCR assay. In addition, virulence factor detection showed that the JDM1-1 isolate produced lipase, lecithinase, gelatinase, and hemolysin. Furthermore, the mRNA expression profiles of immune-related genes of M. rosenbergii following A. veronii infection, including ALF1, ALF2, Crustin, C-lectin, and Lysozyme, were assessed, and the results revealed a significant upregulation in the hepatopancreas and intestines at different hours post infection. This study demonstrates that A. veronii is a causative agent associated with massive die-offs of M. rosenbergii and contributes valuable insights into the pathogenesis and host defense mechanisms of A. veronii invasion. Full article