Search Results (344)

Lipid nanoparticle (LNP)-based delivery systems are promising tools for advancing RNA-based therapies. However, there are underlying challenges for the oral delivery of LNPs. In this study, we optimized an LNP formulation, which we encapsulated in a pH-sensitive Eudragit^® S 100 (Eu) coating. LNPs were prepared using the DLin-MC3-DMA ionizable lipid, cholesterol, DMG-PEG, and DSPC at a molar ratio of 50:38.5:10:1.5. LNPs were coated with 1% Eu solution via nanoprecipitation using 0.25% acetic acid to get Eu-coated LNPs (Eu-LNPs). Particle characteristics of LNPs were determined by using dynamic light scattering (DLS). Ribogreen and agarose gel retardation assays were used to evaluate nucleic acid entrapment and stability. LNPs and Eu-LNPs were ~120 nm and 4.5 μm in size, respectively. Eu-LNPs decrease to an average size of ~191 ± 22.9 nm at a pH of 8. Phosphate buffer (PB)-treated and untreated Eu-LNPs and uncoated LNPs were transfected in HEK-293 cells. PB-treated Eu-LNPs showed significant transfection capability compared to their non-PB-treated counterparts. Eu-LNPs protected their nucleic acid payloads in the presence of a simulated gastric fluid (SGF) with pepsin and maintained transfection capacity following SGF or simulated intestinal fluid. Hence, Eu coating is a potentially promising approach for the oral administration of LNPs. Full article

Background/Objectives: Genetic polymorphisms in the BRCA2 gene have been implicated in breast cancer susceptibility. While numerous studies have investigated this polymorphism, its precise role in breast cancer development remains unclear. Furthermore, to the best of our knowledge, no related studies have been conducted in Azerbaijan. The aim of this study was to determine the distribution of the BRCA2 Met1915Thr polymorphism (rs4987117) in the Azerbaijani population and to evaluate its potential association with breast cancer risk. Methods: A total of 144 breast cancer patients and 152 healthy controls were recruited from the Oncology Clinic of Azerbaijan Medical University between 2021 and 2024. The Met1915Thr polymorphism was genotyped using PCR-RFLP and visualized on a 2% agarose gel. Results: A statistically significant association with increased breast cancer susceptibility was observed for the heterozygous Met/Thr genotype (OR = 1.83, 95%CI = 1.08–3.11, p = 0.02), the Thr allele (OR = 1.57, 95%CI = 1.12–2.20, p = 0.008), and under the dominant inheritance model (OR = 1.83, 95%CI = 1.15–2.90, p = 0.01). Notably, this association was more evident among individuals aged over 58 years, in whom the Met/Thr genotype conferred a significantly elevated risk (OR = 2.35, 95%CI = 1.17–4.73, p = 0.02). Conclusions: The BRCA2 Met1915Thr polymorphism is associated with an increased risk of breast cancer in the Azerbaijani population. These findings suggest a potential role of this polymorphism in breast cancer susceptibility and highlight the need for further studies in larger cohorts to validate these associations. Full article

Background: This study is the first to investigate the association between colorectal cancer (CRC) risk and the hMLH1 −93G>A and hMSH2 1032G>A polymorphisms of mismatch repair (MMR) genes in the Azerbaijani population. Methods: Peripheral blood samples containing EDTA were collected from the study subjects (134 patients and 137 controls), and genomic DNA was extracted using the non-enzymatic salting-out method. Genotypes were determined by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP), and the results were visualized through agarose gel electrophoresis. Results: Overall, no statistically significant correlation was observed between CRC risk and the hMLH1 −93G>A polymorphism in the heterozygous GA (OR = 0.760; 95% CI = 0.374–1.542; p = 0.446), the mutant AA (OR = 1.474; 95% CI = 0.738–2.945; p = 0.270), or the A allele (OR = 1.400; 95% CI = 0.984–1.995; p = 0.062). However, in contrast to the dominant model, a statistically significant association was found between the recessive model and an increased CRC risk, with an odds ratio of 1.788 (95% CI = 1.102–2.900; p = 0.018). The hMLH1 −93G>A polymorphism was identified at a significantly higher frequency across the TNM stages, with the distribution showing statistical significance (p < 0.05). Additionally, no statistically significant association was observed between the hMSH2 1032G>A polymorphism and CRC risk. Conclusions: Although no overall association was observed for hMLH1 −93G>A, our findings suggest a potential link with increased colorectal cancer risk under the recessive model in the Azerbaijani population. Further studies are warranted to confirm this model-specific association and investigate the underlying biological mechanisms. Full article

The synthesis of novel biodegradable polymers as non-viral vectors remains one of the challenging tasks in the field of gene delivery. In this study, the synthesis of the polysaccharide-g-polypeptide copolymers, namely, hyaluronic acid-g-polylysine (HA-g-PLys), using a copper-free strain-promoted azide-alkyne cycloaddition reaction was proposed. For this purpose, hyaluronic acid was modified with dibenzocyclooctyne moieties, and poly-L-lysine with a terminal azido group was obtained using ring-opening polymerization of N-carboxyanhydride of the corresponding protected amino acid, initiated with the amino group azido-PEG₃-amine. Two HA-g-PLys samples with different degrees of grafting were synthesized, and the structures of all modified and synthesized polymers were confirmed using ¹H NMR and FTIR spectroscopy. The HA-g-PLys samples obtained were able to form nanoparticles in aqueous media due to self-assembly driven by electrostatic interactions. The binding of DNA and model siRNA by copolymers to form polyplexes was analyzed using ethidium bromide, agarose gel electrophoresis, and SybrGreen I assays. The hydrodynamic diameter of polyplexes was ˂300 nm (polydispersity index, PDI ˂ 0.3). The release of a model fluorescently-labeled oligonucleotide in the complex biological medium was significantly higher in the case of HA-g-PLys as compared to that in the case of PLys-based polyplexes. In addition, the cytotoxicity in normal and cancer cells, as well as the ability of HA-g-PLys to facilitate intracellular delivery of anti-GFP siRNA to NIH-3T3/GFP+ cells, were evaluated. Full article

Localized treatment has emerged as an excellent alternative to minimize the side effects associated with the systemic dispersion of therapeutic agents, which can damage healthy tissues. Injectable hydrogels offer a promising solution because they can encapsulate and release therapeutic agents in a controlled manner. In this context, this study focuses on the development and characterization of an injectable hydrogel based on carboxymethyl chitosan (CMCh) and oxidized agarose (OA), in which chemical crosslinking through imine bond formation avoids the use of external crosslinking agents. Several polymer ratios were evaluated to obtain hydrogels (OA:CMCh), and stable gels were formed at physiological temperatures in all cases. The hydrogels were injectable through a 21 G needle with forces below 30 N, formed porous structures, and exhibited a self-healing capacity after 48 h. Additionally, the hydrogels displayed compressive strengths ranging from 26 to 71 kPa and elastic moduli similar to those of human tissues (6–20 kPa). Swelling percentages of up to 3090% were achieved owing to the high hydrophilicity of CMCh and OA, and strong chemical crosslinking maintained the gel stability for two weeks with low mass loss rates (<21%). Furthermore, polymer ratio variation and storage at 4 °C were observed to affect the hydrogel characteristics, allowing for property modulation according to the application needs. These results indicate that the proposed polymeric combination enables the formation of hydrogels with the potential for localized drug delivery. Full article

Tomato spotted wilt virus (TSWV) is a highly destructive plant pathogen and transmitted by several thrips including the western flower thrips, Frankliniella occidentalis. A structural N protein encoded in the viral genome represents the nucleocapsid protein by binding to the viral RNA genome. However, it remains unknown how the RNA-binding protein specifically interacts with the viral RNA from host RNAs in the target cells. To study the molecular basis of N function, we produced the protein in Escherichia coli and the resulting purified recombinant protein was used to investigate the protein–RNA interactions. The recombinant N protein migrated on agarose gel to the anode in the electric field due to its high basic isoelectric point. This electrostatic property led N protein to bind to DNA as well as RNA. It also bound to both single-stranded (ssRNA) and double-stranded RNA (dsRNA). However, when the total RNA was extracted from plant tissues collected from TSWV-infected host, the RNA extract using the recombinant N protein was much richer in the TSWV genome compared to that without the protein. To investigate the specificity of N protein to ssRNA, the three-dimensional structure was predicted using the AlphaFold program and showed its trimeric oligomerization with the binding pocket for ssRNA. This was supported by the differential susceptibility of N protein with ssRNA and dsRNA against RNase attack. Furthermore, a thermal shift assay to analyze the RNA and protein interaction showed that ssRNA strongly interacted with N protein compared to dsRNA. In addition, the N gene was expressed along with the multiplication of the viral RNA genome segments from the segment-specific fluorescence in situ hybridization analysis in different tissues during different developmental stages of the virus-infected F. occidentalis. These results suggest that the functional trimeric N proteins bind to the viral RNA to form a basic nucleocapsid structure at a specific virus-replicating compartment within the host cells. Full article

Constructing stable and flexible neuronal networks with multi-neurite wiring is essential for the in vitro modeling of brain function, connectivity, and neuroplasticity. However, most existing neuroengineering platforms rely on static microfabrication techniques, which limit the ability to dynamically control circuit architecture during cultivation. In this study, we developed a modifiable agarose gel-based platform that enables real-time microstructure fabrication using an infrared (IR) laser system under live-cell conditions. This approach allows for the stepwise construction of directional neurite paths, including sequential microchannel formation, cell chamber fabrication, and controlled neurite–neurite crossings. To support long-term neuronal health and network integrity in agarose microstructures, we incorporated direct glial co-culture into the system. A comparative analysis showed that co-culture significantly enhanced neuronal adhesion, neurite outgrowth, and survival over several weeks. The feeder layer configuration provided localized trophic support while maintaining a clear separation between glial and neuronal populations. Dynamic wiring experiments further confirmed the platform’s precision and compatibility. Neurites extended through newly fabricated channels and crossed pre-existing neurites without morphological damage, even when laser fabrication occurred after initial outgrowth. Time-lapse imaging showed a temporary growth cone stalling at crossing points, followed by successful elongation in all tested samples. Furthermore, the direct laser irradiation of extending neurites during microstructure modification did not visibly impair neurite elongation, suggesting minimal morphological damage under the applied conditions. However, potential effects on molecular signaling and electrophysiological function remain to be evaluated in future studies. Together, these findings establish a powerful, flexible system for constructive neuroengineering. The platform supports long-term culture, real-time modification, and multidirectional wiring, offering new opportunities for studying neural development, synaptic integration, and regeneration in vitro. Full article

Thyroid eye disease (TED) is characterized by autoimmune inflammation and structural remodelling of orbital tissues, which is a consequence of the activation of orbital fibroblasts (OFs). As a result of this activation, the production of hyaluronan (HA) and the proliferation and adipocyte differentiation of OFs are enhanced. Adipogenesis leads to additional accumulation of HA. The aim of this study was to elucidate the molecular weight distribution of HA produced by OFs under basic conditions and after adipogenic stimuli. The concentration and the molecular weight distribution of HA were examined using ELISA and agarose gel electrophoresis, respectively, in TED (n = 3) and non-TED (n = 3) OF cultures. Under adipogenic stimuli, HA production is increased in OFs. In TED OF cultures, which, unlike non-TED OFs, can differentiate into adipocytes, the enhanced proportion of high-molecular-weight (HMW) HA of more than 2000 kDa is responsible for the increased HA concentration in the culture media. In non-TED OF cultures, which contain a negligible number of differentiating cells after adipogenic stimulation, the medium-molecular-weight (MMW) HA fragments from 50 to 1000 kDa also contribute to the enhanced HA content. Increased production of HMW-HA during adipocyte differentiation of TED OFs is responsible for the elevated HA content in the culture media, which may be an important contributor to both connective tissue matrix expansion and edema in the pathogenesis of TED. Full article

Bovine anaplasmosis, caused by Anaplasma marginale, is a major tick-borne disease in tropical and subtropical regions of the world, leading to significant production losses. Prolonged convalescence periods are common and surviving animals often become subclinical carriers. This study aimed to detect and characterise A. marginale in bovines in smallholder dairy farms across diverse climatic zones of Pakistan using molecular methods. In total, 321 blood DNA samples from apparently healthy cattle (n = 174) and buffaloes (n = 147) from six districts in Pakistan were tested for A. marginale using a nested PCR assay, targeting part of the major surface protein B gene (msp1β) as a genetic marker, followed by agarose gel electrophoresis and selective sequencing of amplicons from test-positive samples. Of the 321 DNA samples tested, 135 (42.1%) were test-positive for A. marginale. Prevalence was significantly higher in cattle (64.4%; 112/174) than in buffaloes (15.6%; 23/147), and female bovines (43.5%; 108/248) were more frequently infected than males (37%; 27/73). Phylogenetic analysis of the msp1β sequence data (n = 42) revealed that A. marginale from Pakistan clustered with those from Brazil, Thailand, South Africa, and the USA. This study represents the first comprehensive investigation of A. marginale from bovines from diverse agroecological zones of Pakistan and will further stimulate population genetic studies of A. marginale and investigations into the economic impact of subclinical infections in bovines in smallholder farming systems. Full article

Rice (Oryza sativa L.) is essential for global food security and sustains billions worldwide, emphasizing the need to improve production and quality. One key challenge in rice breeding is the inheritance and environmental sensitivity of amylose content, a starch component that influences the texture, water absorption, and firmness after cooking, which are crucial for market acceptance. While international markets prefer low-amylose varieties for their softness, intermediate- and high-amylose varieties are favored in Latin America for their firmness. The objective of this study was to develop a molecular quality assessment methodology that, combined with morphological and culinary evaluations, helps in the selection of rice varieties during the breeding process. First, ten Ecuadorian rice materials were evaluated for milling and culinary quality characteristics, revealing significant grain size, sterility, milling yield, cooking time, and texture variations. Amylose content (AC) is genetically regulated by the waxy gene and its allelic variants, affecting granule-bound starch synthase (GBSS) enzyme expression. Secondly, to classify rice varieties molecularly based on AC, the testing ten genotypes plus nine control varieties were analyzed using microsatellite (SSR) markers. The waxy molecular marker, combined with metaphor agarose gel electrophoresis (MAGE), proved effective for early-stage AC analysis, reducing variety selection costs and improving breeding efficiency. Additionally, a restriction enzyme protocol assay facilitated variety differentiation by selectively cleaving the waxy gene sequence at a specific single-nucleotide polymorphism (SNP) site, allowing for precise AC genetic classification. By integrating molecular techniques with traditional assessments, this study reveals that using marker-assisted selection in breeding programs, as well as supporting the identification and development of high-quality local rice varieties to meet market demands, improves production efficiency and optimizes the assessment of developing varieties under diverse environmental conditions. Full article

The increasing prevalence of antimicrobial-resistant (AMR) bacteria in humans, animals, and the environment underscores the necessity for a rapid, sensitive, and specific method to identify resistance genes. Objectives: This study aims to develop a reliable detection tool for identifying the tetracycline-resistant gene tet(M) in Enterococcus species using a real-time loop-mediated isothermal amplification (RT-LAMP) assay. Real-time visualization through a turbidimeter enabled precise estimation of time-to-positivity for gene detection. Methodology: Six primers were designed using PrimerExplorer v.5, and the assay was optimized across different temperatures and incubation times. Validation was conducted by testing 52 tet(M)-positive clinical enterococci isolates and spiking urine samples from a healthy volunteer and a cow with tet(M)-positive Enterococcus species. Results: The tet(M) gene was detected as early as 33 min, with optimal amplification occurring within 60 min at 60 °C. The assay demonstrated 100% specificity with the established primers. The sigmoidal graphs were corroborated with visual confirmation methods, including a green color change (visible to the naked eye), green fluorescence (under UV light), and a 200 bp PCR product observed via agarose gel electrophoresis. Notably, the tet(M) RT-LAMP assay exhibited a detection limit of 0.001 pg/μL, significantly surpassing conventional PCR, which had a detection limit of 0.1 pg/μL. Conclusions: This rapid, cost-effective, highly sensitive, and specific tet(M) RT-LAMP assay holds significant promise as a surveillance tool for antimicrobial resistance monitoring within a One Health framework, particularly in low-resource countries. Full article

Background/Objectives: PURE (Protein synthesis Using Recombinant Elements), an ideal system for ribosome display, has been successfully used for nanobody selection. However, its limitations in nanobody selection, especially for synthetic nanobody libraries, have not been clearly elucidated, thereby restricting its utilization. Methods: The PURE ribosome display selection process was closely monitored using RNA agarose gel electrophoresis to assess the presence of mRNA molecules in each fraction, including the flow-through, washing, and elution fractions. Additionally, a real-time validation method for monitoring each biopanning round was implemented, ensuring the successful enrichment of target protein-specific binders. The selection process was further optimized by introducing a target protein elution step prior to the EDTA-mediated disassembly, as well as by altering the immobilization surfaces. Finally, the efficiency of PURE ribosome display was enhanced by replacing the spacer gene. Results: The efficiency of PURE ribosome display was merely 4% with an unfavourable spacer gene. Using this spacer gene, EGFP- and human fatty acid-binding protein 4-specific nanobodies from a synthetic nanobody library were we successfully identified through optimizing the selection process. Choosing a spacer gene less prone to secondary structure formation increased significantly its efficiency in displaying synthetic nanobody libraries. Conclusions: Implementing a target protein elution step prior to EDTA-mediated disassembly and modifying the immobilization surfaces effectively increase selection efficiency. For PURE ribosome display, efficiency was further improved using a suitable spacer gene, enabling the display of large libraries. Full article

This study presents an innovative approach to the sustainable valorization of industrial sweet cherry (Prunus avium L.) waste from the Vignola Region, Italy, transforming what is typically discarded into a high-value bioactive resource. Unlike conventional extractions, our hydroethanolic extract (VCE) was obtained from the entire cherry waste, including the pericarp, pulp, and stone, as generated by industrial processing. This full-fruit extraction strategy represents a novel and efficient use of agricultural by-products, aligning with circular bioeconomy principles. Sweet cherries are known for their phenolic richness, and spectrophotometric assays (TPC, TFC, reducing power, DPPH, and ABTS) confirmed the extract’s antioxidant capacity. In vitro studies using RAW 264.7 macrophages revealed no cytotoxic effects (MTT assay), along with significant anti-inflammatory activity, evidenced by reduced ROS and NO production and downregulation of iNOS and COX-2. Western blotting showed inhibition of NF-κB nuclear translocation and MAPK pathway signaling. Additionally, agarose gel electrophoresis showed protection against oxidative DNA damage. UPLC-MS/MS analysis identified sakuranetin, aequinetin, and dihydrowogonin as the most representative compounds in VCE. Molecular docking simulations revealed strong and specific binding affinities of these compounds to NF-κB p65 and key MAPK targets. These findings highlight whole sweet cherry waste—including the pit—as a potent and sustainable source of bioactive compounds with promising nutraceutical and pharmaceutical applications. Full article

A great interest in agarose, with many health-promoting and gel properties, has been registered, especially in the field of bone regeneration and repair. Agarose and its major bioactive compounds are involved in biological activities such as inflammation, cell adhesion and proliferation, and the promotion of tissue repair. Due to its unique physical properties like gelation and solubility, agarose is increasingly utilized in the medical industry. The aim of this review is to present an overview of the applications of agarose hydrogels in bone tissue engineering, introducing agarose and its modified products as innovative solutions for bone regeneration. Additionally, the injectability of agarose hydrogels and their applications in bioprinting are also summarized. Data indicate that agarose will play an increasing role in current and future global medical sectors. Full article

Hellenic Open University has developed Onlabs, a virtual biology laboratory designed to safely and effectively prepare its students for hands-on work in the university’s on-site labs. This platform simulates key experimental processes, such as 10X TBE solution preparation, agarose gel preparation and electrophoresis, which involve liquid transfers between bottles. However, accurately depicting liquid volumes and their flow within complex-shaped laboratory vessels, such as Erlenmeyer flasks and burettes, remains a challenge. This paper addresses this limitation by introducing a unified parametric framework for modeling circular cross-section pipes, including straight pipes with a constant diameter, curved pipes with a constant diameter and straight conical pipes. Analytical expressions are developed to define the position and orientation of points along a pipe’s central axis, as well as the surface geometry of composite pipes formed by combining these elements in planar configurations. Moreover, the process of surface discretization with finite triangular elements is analyzed with the aim of optimizing their representation during the algorithmic implementation. The functions of the current length with respect to the volume of each considered container shape are developed. Finally, the methodology for handling and combining the analytical expressions during the filling of a composite pipe is explained, the filling of certain characteristic bottles is implemented and the results of the implementations are presented. The primary goal is to enable the precise algorithmic generation of 3D graphics representing the surfaces of liquids within various laboratory vessels and, subsequently, the simulation of their flow. By leveraging these parametric models, liquid volumes can be accurately visualized, reflecting the vessels’ geometries and improving the realism of simulations and the filling of various vessels can be realistically simulated. Full article