Search Results (69)

Background: Pharmacological treatment under conditions of slow fibrinolysis/thrombolysis requires the targeted delivery of plasminogen-activating activity. Echogenic liposomal formulations (regular TELIP) of single-chain tissue plasminogen activator (sctPA), while possessing high affinity to fibrin, contain free/loosely bound sctPA. We hypothesized that removal of free sctPA, which competes with liposomes and plasmin for fibrin, enhances unique features of the TELIP. Methods: Optimized and regular TELIP were assessed for the distribution of active sctPA (loosely bound, tightly bound, encapsulated), stability, binding to fibrin, initiating fibrinolysis in vitro and ex vivo using a battery of biochemical methods. Results: One milligram of the regular TELIP consists of 2.0–5.0 × 10⁹ echogenic liposomes (700–900 nm diameter). Non-specifically bound sctPA readily dissociates at the physiological ionic strength and pH. While up to 60% of sctPA in the regular TELIP is loosely bound with 6–15% encapsulated, and the rest is tightly bound to the liposomes; in the optimized TELIP, more than 80% of active sctPA is tightly bound with up to 40% of encapsulated. The latter is protected from high-molecular-weight ligands and could be released by an ultrasound pulse. Optimized TELIP shows low competition with plasmin for fibrin and effectively supports fibrinolysis in vitro and ex vivo. The optimized TELIP with maximal load of sctPA 3% (w/w) retains integrity at 37 °C for 5 h in vitro and up to 2 h ex vivo. Conclusions: The optimized TELIP is stable in vitro and ex vivo, does not interfere with fibrinolysis and retains a high level of encapsulated sctPA delivered precisely to the thrombus/fibrin clot. Full article

Sumizyme PEG, a glucanase/cellulase enzyme preparation produced by Talaromyces versatilis PF8, was investigated to characterize its systemic and genotoxic toxicity profile to support its intended use in food processing applications. A comprehensive toxicological program was conducted in accordance with OECD guidelines, comprising a bacterial reverse mutation (Ames) test, an in vitro chromosomal aberration assay, an in vivo micronucleus test, and a 90-day repeated-dose oral toxicity study in male and female Crl:CD(SD) rats. In the subchronic study, Sumizyme PEG was administered by oral gavage at doses of 107, 1070, and 10,700 U/kg/day. No treatment-related adverse effects were observed across clinical, hematological, biochemical, urinalysis, organ weight, or histopathological endpoints, and the highest dose was identified as the NOAEL. Genotoxic testing showed no consistent mutagenic or clastogenic response across the test battery. A positive in vitro signal was observed in CHL/IU cells; however, this was not reproduced in a human TK6 cell assay or in vivo micronucleus testing, indicating assay-dependent sensitivity within a weight-of-evidence framework. Overall, the integrated dataset does not indicate a consistent treatment-related systemic or genotoxic effect under the conditions of the studies conducted. Full article

Heat generated during photopolymerization of resin-based composites from both the exothermic reaction of the material and the irradiance of light-curing units poses a risk to pulp vitality, especially in deep restorations. This study aimed to evaluate temperature variation (ΔT) during the photopolymerization of different resin composites, considering material type, shade, increment thickness, and light-curing unit output. An in vitro experimental study with a factorial design was conducted. Specimens were prepared using 2.0 mm and 4.0 mm increments from conventional (nanohybrid), bulk-fill, and flowable resin composites in different shades (BW, A1, A3, A4, and XB) and different light-curing unit output (100% and 50% battery charge). ΔT was measured using a type K thermocouple (Omega Engineering, Norwalk, CT, USA) positioned at the center of each increment. Data were analyzed using four-way analysis of variance (ANOVA) (α = 0.05). All groups demonstrated a statistically significant temperature increase (p < 0.05), with ΔT values ranging from 3.24 °C to 18.18 °C. Composite type significantly influenced ΔT (p < 0.001), with flowable composites showing the highest temperature rise, followed by bulk-fill and conventional composites. Increment thickness also had a significant effect (p = 0.008), with 4.0 mm increments producing greater temperature increases. Shade significantly affected ΔT (p < 0.001), with the XB shade exhibiting the highest values. Additionally, higher light-curing output (100%) resulted in significantly greater temperature increases compared to 50% output (p < 0.001). Photopolymerization temperature rise is influenced by multiple interacting factors. The combination of flowable composites, darker shades, thicker increments, and higher curing output may increase thermal risk. These findings should be considered when optimizing clinical protocols to minimize potential pulpal damage. Full article

People suffering from chronic diseases like diabetes, heart disease, and Parkinson’s disease are reliant on their implantable devices to improve their quality of life and to manage their chronic conditions. Despite their advantages, some systems are battery-powered, which can lead to battery failure, resulting in prophylactic surgery. One solution to this issue is an implantable antenna that provides an adequate link margin across various skin sites. In this study, we introduce an implantable antenna design optimized using an open-source PSO algorithm. The antenna is a tunable WMTS-motivated design fabricated on a Rogers 6010.2 substrate and evaluated by simulation and in vitro testing using phantom tissues. Validation measurements are performed to evaluate the effects of implantation depth across various adipose thicknesses. Full article

Clinoptilolite is a zeolite with a microporous structure that enables ion exchange, molecular sieving, and adsorption, conferring detoxifying, antioxidant, and anti-inflammatory properties. These properties have applications in food, medicine, catalysis, and environmental remediation. This study evaluated the safety of the lab-made Clinoptilolite as a potential food ingredient through a 90-day repeated-dose toxicity study in male and female Sprague Dawley rats. The test substance was administered via oral gavage at doses of 0, 5, 10, and 15 mg/kg bw/day, followed by a 28-day recovery period. In addition, genotoxicity was assessed using the Ames test, in vitro chromosomal aberration assay, and an in vivo micronucleus test. All studies were conducted in accordance with OECD and FDA guidelines. Results showed no adverse systemic, genotoxic, or irreversible effects at any dose, with minor clinical variations being incidental and reversible. Genotoxicity tests confirmed no mutagenic or clastogenic potential. Overall, the lab-made Clinoptilolite evaluated in this investigation was well tolerated, non-toxic, and showed no evidence of treatment-related toxicity at the doses tested. These findings provide supportive evidence for its consideration toward a Generally Recognized as Safe (GRAS) determination. Full article

Oxidative stress plays a central role in the pathogenesis of neurodegenerative disorders, including Parkinson’s disease. Therefore, compounds with antioxidant and neuroprotective properties represent promising candidates for therapeutic development. N-pyrrolyl hydrazide-hydrazones, a class of pyrrole-based derivatives, have shown promising potential due to their diverse biological activities, including monoamine oxidase-B (MAO-B) inhibition. This study investigated the neuroprotective properties of 10 N-pyrrolyl hydrazide-hydrazones using experimental in vitro and in vivo models of neurodegeneration. The compounds were tested on SH-SY5Y neuroblastoma cells subjected to oxidative stress induced by 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpyridinium (MPP⁺). A battery of in vitro and in vivo experimental methods was used, including cell viability assay, reactive oxygen species (ROS) production, and apoptosis evaluation by quantifying the sub-G0/G1 cell population. In vivo neuroprotective efficacy was further tested in a rotenone-induced Parkinsonism mouse model by analyzing oxidative biomarkers and brain histopathology. Compounds 2, 4, 5, 6, and 10 significantly preserved cell viability in the 6-OHDA-induced toxicity model, while no protection was observed in the MPP⁺ model. Particularly compound 2 reduced ROS levels and apoptosis in SH-SY5Y cells. In vivo, compound 2 demonstrated strong antioxidant activity by maintaining glutathione levels and reducing lipid peroxidation. Histological analysis confirmed its protective effect against rotenone-induced neuronal damage. These results suggest that N-pyrrolyl hydrazide-hydrazones, especially compound 2, possess significant antioxidant and MAO-B inhibitory properties, supporting their potential as neuroprotective agents. Full article

Human cell-based assays for neurotoxicity (NT) and developmental neurotoxicity (DNT) have reached a high level of readiness, but some tests require improvements in the specificity and sensitivity at which mitochondrial toxicants are detected. This study aimed to optimize the PeriTox assay, which uses peripheral neurons (PNs) and predicts the potential of chemicals to trigger peripheral neuropathies. By introducing a glucose-to-galactose switch in the medium composition, cells were forced to rely on mitochondrial respiration. Using pre-differentiated PNs cultured in either glucose (Glc) or galactose (Gal), we observed no major differences in baseline phenotype, gene expression, neurite outgrowth, or total ATP content. However, a marked metabolic shift was confirmed by the increased oxygen consumption in Gal conditions. Based on measurements of neurite growth and ATP levels, Gal-adapted neurons showed a heightened sensitivity, up to 7500-fold, to a range of mitochondrial respiratory chain (MRC) inhibitors. The sensitivity shift was high for inhibitors of MRC complexes I and III and modest or absent for unrelated compounds such as proteasome inhibitors or cytoskeletal poisons. For complex I-III inhibitors, the enhanced detection of mitochondrial neurotoxicants was coupled with a more accurate distinction between cytotoxic and neurite-specific effects, i.e., an improved assay specificity. In conclusion, our study on 39 compounds suggests that running the PeriTox assay in galactose increases its sensitivity and specificity for several mitochondrial toxicants, while no general disadvantages or shortcomings were observed. The modified version (PeriTox-M) may increase the performance of in vitro test batteries for scientific and regulatory applications. Full article

Compounds, which rely on metabolism to exhibit toxicity, pose a challenge for next-generation risk assessment (NGRA). Since many of the currently available non-animal new approach methods (NAMs) lack metabolic activity, their use may lead to an underestimation of the true hazard to humans (false negative predictions). We explored here strategies to deal with metabolite-mediated toxicity in assays for developmental neurotoxicity. First, we present an overview of substances that may serve as potential positive controls for metabolite-related neurotoxicity. Then, we demonstrate, using the MitoMet (UKN4b) assay, which assesses the adverse effects of chemicals on neurites of human neurons, that some metabolites have a higher toxic potency than their parent compound. Next, we designed a strategy to integrate elements of xenobiotic metabolism into assays used for (developmental) neurotoxicity testing. In the first step of this approach, hepatic post-mitochondrial fractions (S9) were used to generate metabolite mixtures (“metabolisation module”). In the second step, these were applied to a NAM (exemplified by the UKN4b assay) to identify metabolite-mediated toxicity. We demonstrate the applicability and transferability of these approaches to other assays, by an exemplary study on the basis of the cMINC (UKN2) assay, another NAM of the developmental neurotoxicity in vitro battery. Based on the experience gained from these experiments, we discuss key issues to be addressed if this approach is to be used more broadly for NAM in the NGRA context. Full article

Background: Within the solar ultraviolet (UV) spectrum, ultraviolet A rays (UVA, 320–400 nm), although less energetic than ultraviolet B rays (UVB, 280–320 nm), constitute at least 95% of solar UV radiation that penetrates deep into the skin The UV rays are associated with both epidermal and dermal damage resulting from the generation of reactive oxygen species (ROS). Among them, the longest UVA wavelengths (UVA1, 340–400 nm) can represent up to 75% of the total UV energy. Therefore, UVA radiation is linked to various acute and chronic conditions, including increased skin pigmentation and photoaging. Despite many advances in the skin photoprotection category, there is still a growing demand for natural daily photoprotection active ingredients that offer broad protection against skin damage caused by UVA exposure. In our quest to discover new, disruptive, next generation of photoprotective ingredients, we were drawn to pomegranate, based on its diverse polyphenolic profile. We investigated the pericarp of the fruit, so far considered as byproducts of the pomegranate supply chain, to design a novel patented extract “POMAOX” with a desired spectrum of phenolic components comprising of αβ-punicalagins, αβ-punicalins and ellagic acid. Methods: Antioxidant properties of POMAOX were measured using in-tubo standard tests capable of revealing a battery of radical oxygen species (ROS): peroxyl radical (ORAC), singlet oxygen (SOAC), superoxide anion (SORAC), peroxynitrite (NORAC), and hydroxyl radical (HORAC). In vitro, confirmation of antioxidant properties was first performed by evaluating protection against UVA-induced lipid peroxidation in human dermal fibroblasts (HDF), via the release of 8 iso-prostanes. The protection offered by POMAOX was further validated in a 3D in vitro reconstructed T-Skin^TM model, by analyzing tissue viability/morphology and measuring the release of Matrix Metallopeptidase 1 (MMP-1) & pro-inflammatory mediators (IL-1α, IL-1ra, IL-6, IL-8, GM-CSF, and TNF-α) after UVA1 exposure. Results: POMAOX displayed strong antioxidant activity against peroxynitrite (NORAC) at 1.0–3.0 ppm, comparable to the reference vitaminC, as well as singlet oxygen (SOAC) at 220 ppm, and superoxide radicals with a SORAC value of 500 ppm. Additionally, POMAOX demonstrated strong photoprotection benefit at 0.001% concentration, offering up to 74% protection against UVA-induced lipid peroxidation on HDF, in a similar range as the positive reference, Vitamin E at 0.002% (50 µM), and with higher efficacy than ellagic acid alone at 5 µM. Moreover, our pomegranate-derived extract delivered photoprotection at 0.001%, mitigating dermal damages induced by UVA1, through inhibition of MMP-1 and significant inhibition of pro-inflammatory mediators release (including IL-1α, IL-1ra, IL-6, IL-8, GM-CSF, and TNFα) on an in vitro reconstructed full-thickness human skin model with a similar level of protection to that of Vitamin C tested at 0.035% (200 µM). Conclusions: Overall, the novel pomegranate-derived extract “POMAOX” significantly reduced the impact of UVA on human skin, due to its broad-spectrum antioxidant profile. These findings suggest that POMAOX could offer enhanced protection against the detrimental effects of UV exposure, addressing the growing consumer demand for strong photoprotection with skincare benefits. Full article

Background: The nuclear-encoded enzyme polymerase gamma (Pol-γ) is crucial in the replication of the mitochondrial genome (mtDNA), which in turn is vital for mitochondria and hence numerous metabolic processes and energy production in eukaryotic cells. Variants in the POLG gene, which encodes the catalytic subunit of Pol-γ, can significantly impair Pol-γ enzyme function. Pol-γ-associated disorders are referred to as POLG-spectrum disorders (POLG-SDs) and are mainly autosomal-recessively inherited. Clinical manifestations include muscle weakness and fatigue, and severe forms of the disease can lead to premature death in infancy, childhood, and early adulthood, often associated with seizures, liver failure, or intractable epilepsy. Here, we analyzed fibroblasts from a compound heterozygous patient with the established pathogenic variant c.2419C>T; p.(Arg807Cys) and a previously undescribed variant c.678G>C; p.(Gln226His) with a clinical manifestation compatible with POLG-SDs, sensory ataxic neuropathy, and infantile muscular atrophy. We conducted a battery of functional studies for Pol-γ and mitochondrial dysfunction on the patient’s fibroblasts, to test whether the novel variant c.678G>C; p.(Gln226His) may be causative in human disease. Aims/Methods: We analyzed skin-derived fibroblasts in comparison to a first-degree relative (the mother of the patient), an asymptomatic carrier harboring only the established c.2419C>T; p.(Arg807Cys) mutation. Assessments of mitochondrial function included measurements of mtDNA content, mRNA levels of mitochondrial genes, mitochondrial mass, and mitochondrial morphology. Case Presentation and Results: A 13-year-old male presented with symptoms starting at three years of age, including muscle weakness and atrophy in the lower extremities and facial muscles, which later extended to the upper limbs, voice, and back muscles, without further progression. The patient also reported fatigue and muscle pain after physical activity, with no sensory deficits. Extensive diagnostic tests such as electromyography, nerve conduction studies, muscle biopsy, and MRI were unremarkable. Exome sequencing revealed that he carried the compound heterozygous variants in POLG c.678G>C; p.(Gln226His) and c.2419C>T; p.(Arg807Cys), but no other potential genetic pathogenic causes. In comparison to a first-degree relative (his mother) who only carried the c.2419C>T; p.(Arg807Cys) pathogenic mutation, in vitro analyses revealed a significant reduction in mtDNA content (~50%) and mRNA levels of mtDNA-encoded proteins. Mitochondrial mass was reduced by approximately 20%, and mitochondrial interconnectivity within cells was impaired, as determined by fluorescence microscopy and mitochondrial staining. Conclusions: Our findings suggest that the c.678G>C; p.(Gln226His) variant, in conjunction with the c.2419C>T; p.(Arg807Cys) mutation, may compromise mtDNA replication and mitochondrial function and could result in clinically significant mitochondriopathy. As this study is based on one patient compared to a first-degree relative (but with an identical mitochondrial genome), the pathogenicity of c.678G>C; p.(Gln226His) of POLG should be confirmed in future studies, in particular, in conjunction with other POLG-variants. Full article

An alkaline pH in soils reduces Fe availability, limiting Fe uptake, compromising plant growth, and showing chlorosis due to a decrease in chlorophyll content. To achieve proper Fe homeostasis, dicotyledonous plants activate a battery of strategies involving not only Fe absorption mechanisms, but also releasing phyto-siderophores and recruiting siderophore-producing bacterial strains. A screening for siderophore-producing bacterial isolates from the rhizosphere of Pinus pinea was carried out, resulting in two Pseudomonas strains, Z8.8 and Z10.4, with an outstanding in vitro potential to solubilize Fe, Mn, and Co. The delivery of each strain to 4-week-old iron-starved tomatoes reverted chlorosis, consistent with enhanced Fe contents up to 40%. Photosynthesis performance was improved, revealing different strategies. While Z8.8 increased energy absorption together with enhanced chlorophyll “a” content, followed by enhanced energy dissipation, Z10.4 lowered pigment contents, indicating a better use of absorbed energy, leading to a better survival rate. The systemic reprogramming induced by both strains reveals a lower expression of Fe uptake-related genes, suggesting that both strains have activated plant metabolism to accelerate Fe absorption faster than controls, consistent with increased Fe content in leaves (47% by Z8.8 and 42% by Z10.4), with the difference probably due to the ability of Z8.8 to produce auxins affecting root structure. In view of these results, both strains are effective candidates to develop biofertilizers. Full article

Helicobacter pylori infection constitutes a silent pandemic of global concern. In the last decades, the alarming increase in multidrug resistance evolved by this pathogen has led to a marked drop in the eradication rates of traditional therapies worldwide. By using a high-throughput screening strategy, in combination with in vitro DNA binding assays and antibacterial activity testing, we identified a battery of novel drug-like HsrA inhibitors with MIC values ranging from 0.031 to 4 mg/L against several antibiotic-resistant strains of H. pylori, and minor effects against both Gram-negative and Gram-positive species of human microbiota. The most potent anti-H. pylori candidate demonstrated a high therapeutic index, an additive effect in combination with metronidazole and clarithromycin as well as a strong antimicrobial action against Campylobacter jejuni, another clinically relevant pathogen of phylum Campylobacterota. Transcriptomic analysis suggests that the in vivo inhibition of HsrA triggers lethal global disturbances in H. pylori physiology including the arrest of protein biosynthesis, malfunction of respiratory chain, detriment in ATP generation, and oxidative stress. The novel drug-like HsrA inhibitors described here constitute valuable candidates to a new family of narrow-spectrum antibiotics that allow overcoming the current resistome, protecting from dysbiosis, and increasing therapeutic options for novel personalized treatments against H. pylori. Full article

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are widely used for disease modeling and pharmacological screening. However, their application has mainly focused on inherited cardiopathies affecting ventricular cardiomyocytes, leading to extensive knowledge on generating ventricular-like hiPSC-CMs. Electronic pacemakers, despite their utility, have significant disadvantages, including lack of hormonal responsiveness, infection risk, limited battery life, and inability to adapt to changes in heart size. Therefore, developing an in vitro multiscale model of the human sinoatrial node (SAN) pacemaker using hiPSC-CM and SAN-like cardiomyocyte differentiation protocols is essential. This would enhance the understanding of SAN-related pathologies and support targeted therapies. Generating SAN-like cardiomyocytes offers the potential for biological pacemakers and specialized conduction tissues, promising significant benefits for patients with conduction system defects. This review focuses on arrythmias related to pacemaker dysfunction, examining protocols’ advantages and drawbacks for generating SAN-like cardiomyocytes from hESCs/hiPSCs, and discussing therapeutic approaches involving their engraftment in animal models. Full article

Congestive heart failure (CHF) is a fatal disease with progressive severity and no cure; the heart’s inability to adequately pump blood leads to fluid accumulation and frequent hospital readmissions after initial treatments. Therefore, it is imperative to continuously monitor CHF patients during its early stages to slow its progression and enable timely medical interventions for optimal treatment. An increase in interstitial fluid pressure (IFP) is indicative of acute CHF exacerbation, making IFP a viable biomarker for predicting upcoming CHF if continuously monitored. In this paper, we present an inductor-capacitor (LC) sensor for subcutaneous wireless and continuous IFP monitoring. The sensor is composed of inexpensive planar copper coils defined by a simple craft cutter, which serves as both the inductor and capacitor. Because of its sensing mechanism, the sensor does not require batteries and can wirelessly transmit pressure information. The sensor has a low-profile form factor for subcutaneous implantation and can communicate with a readout device through 4 layers of skin (12.7 mm thick in total). With a soft silicone rubber as the dielectric material between the copper coils, the sensor demonstrates an average sensitivity as high as –8.03 MHz/mmHg during in vitro simulations. Full article

The immune system is affected by the dietary products humans intake. Immune system regulation by nutrition has uses in the clinical context, but it can also benefit healthy populations by delaying or preventing the emergence of immune-mediated chronic illnesses. In this study, the purpose was to describe and compare the modulator effects on the immune system of the routine ingestion of fresh vs. pasteurized yogurt. A unicentral, prospective, randomized, double-blind, parallel group 8-week nutritional study was carried out comparing the ingestion of 125 g of the products in healthy adults three times a day. A complete battery of in vitro tests on the activity of the immune system, processes and phenomena was performed. Exclusive immune-modulatory effects of fresh yogurt with respect to base line were found in terms of increased systemic IgM (primary immune responses), increased synthesis of IFN-gamma upon stimulation (Th1) and increased peripheral T cells (mainly “naive” CD4s). In the three interventions, we observed an increased phagocytic activity and burst test in granulocytes, together with increased secretion of IL-6, IL-1 β and IL-8 (pro-inflammatory) and increased CD16 expression (FcR favoring phagocytosis) in granulocytes. Overall, it is concluded that regardless of bacteria being alive or thermally inactivated, yogurt has common effects on the innate system, but the presence of live bacteria is necessary to achieve a potentiating effect on the specific immune response. Full article