DNA Methylation Machinery in Gastropod Mollusks.

The role of DNA methylation in mollusks is just beginning to be understood. This review synthesizes current knowledge on this potent molecular hallmark of epigenetic control in gastropods-the largest class of mollusks and ubiquitous inhabitants of diverse habitats. Their DNA methylation machinery shows a high degree of conservation in CG maintenance methylation mechanisms, driven mainly by DNMT1 homologues, and the presence of MBD2 and MBD2/3 proteins as DNA methylation readers. The mosaic-like DNA methylation landscape occurs mainly in a CG context and is primarily confined to gene bodies and housekeeping genes. DNA methylation emerges as a critical regulator of reproduction, development, and adaptation, with tissue-specific patterns being observed in gonadal structures. Its dynamics also serve as an important regulatory mechanism underlying learning and memory processes. DNA methylation can be affected by various environmental stimuli, including as pathogens and abiotic stresses, potentially impacting phenotypic variation and population diversity. Overall, the features of DNA methylation in gastropods are complex, being an essential part of their epigenome. However, comprehensive studies integrating developmental stages, tissues, and environmental conditions, functional annotation of methylated regions, and integrated genomic-epigenomic analyses are lacking. Addressing these knowledge gaps will advance our understanding of gastropod biology, ecology, and evolution.

Biosynthesis of Iron Oxide Nanoparticles: Physico-Chemical Characterization and Their In Vitro Cytotoxicity on Healthy and Tumorigenic Cell Lines.

Iron oxide nanoparticles were synthesized starting from two aqueous extracts based on Artemisia absinthium L. leaf and stems, employing a simplest, eco-friendliness and low toxicity method-green synthesis. The nanoparticles were characterized by powder X-ray diffraction (XRD), Fourier transformed infrared spectroscopy (FT-IR), X-ray fluorescence analysis (XRF), thermal analysis (TG/DSC), and scanning electron microscopy (SEM). Lack of magnetic properties and the reddish-brown color of all the samples confirms the presence of hematite as majority phase. The FTIR bands located at 435 cm-1 and 590 cm-1, are assigned to Fe-O stretching vibration from hematite, confirming the formation of α-Fe2O3 nanoparticles (NPs). The in vitro screening of the samples revealed that the healthy cell line (HaCaT) presents a good viability (above 80%) after exposure to iron oxide NPs and lack of apoptotic features, while the tumorigenic cell lines manifested a higher sensitivity, especially the melanoma cells (A375) when exposed to concentration of 500 µg/mL iron oxide NPs for 72 h. Moreover, A375 cells elicited significant apoptotic markers under these parameters (concentration of 500 µg/mL iron oxide NPs for a contact time of 72 h).

Effects of Cadmium Sulfate on the Brown Garden Snail Cornu aspersum: Implications for DNA Methylation.

An extensive literature exists regarding the cellular, physiological, and genetic effects of cadmium (Cd)-A highly toxic, but commonly used trace metal in modern industry. However, limited data are available on its epigenetic effects, especially for terrestrial sentinel invertebrates. We determined Cd retention, total DNA methylation, and the methylation status of 5' end of the Cd-MT gene in the hepatopancreas of the brown garden snail, Cornu aspersum, fed Cd sulfate for four weeks. Bodyweight changes and survival were also measured. Hepatopancreas cadmium increased in a dose-dependent manner from the third-lowest dose onward, with very large amounts being found for the highest treatment group. However, no mortalities occurred, irrespective of dietary Cd dose. We identified significant genome-wide hypermethylation in specimens given the highest dose, which overlapped with a significant bodyweight decrease. The Cd-MT gene showed an unmethylated 5' end of the Cd-MT gene and this status was not affected by cadmium exposure. Hepatopancreas DNA methylation is as sensitive as bodyweight to non-lethal concentrations of dietary Cd given as cadmium sulfate but less responsive than tissue accumulation. Such an exposure event, by contrast, does not affect the methylation status of the Cd-MT gene 5' end.

Space Maintainers Used in Pediatric Dentistry: An Insight of Their Biosecurity Profile by Applying In Vitro Methods.

Space maintainers have presented an increased interest due to their chemical composition which influences the electrochemical and electrolytic processes of the oral cavity, leading to important biological activity. The present study was purported to evaluate the biological in vitro activity of three types of space maintainers (S1, S2, and S3, differing from each other in terms of metal composition) used in pediatric dentistry, in terms of their antimicrobial effect and biosecurity profile using two types of keratinocytes (PGK: primary gingival keratinocytes, and HaCaT: human immortalized keratinocytes) by assessing the morphology, viability, cytotoxicity, and gene expression of the cells. Statistical differences were calculated by the one-way ANOVA test, followed by Tukey's post-test. Antimicrobial screening highlighted a dilution-dependent influence that, in the case of all strains tested, did not show inhibition or stimulation of bacterial growth. The in vitro evaluations revealed that the test samples did not induce important cytotoxic potential on both keratinocyte cell lines (HaCaT and PGK), with the cells manifesting no morphological alteration, a good viability rate (above 90%: PGK-S1, * p < 0.05), and a low cytotoxic activity (less than 11%: PGK, S1 *** p < 0.001 and S3 * p < 0.05; HaCaT, S1 ** p < 0.01). The data obtained in this study highlight the fact that the samples analyzed are biocompatible and do not develop the growth of the studied bacteria or encode the gene expression of primary and immortalized keratinocytes.

Oleogel Formulations for the Topical Delivery of Betulin and Lupeol in Skin Injuries-Preparation, Physicochemical Characterization, and Pharmaco-Toxicological Evaluation.

The skin integrity is essential due to its pivotal role as a biological barrier against external noxious factors. Pentacyclic triterpenes stand as valuable plant-derived natural compounds in the treatment of skin injuries due to their anti-inflammatory, antioxidant, antimicrobial, and healing properties. Consequently, the primary aim of the current investigation was the development as well as the physicochemical and pharmaco-toxicological characterization of betulin- and lupeol-based oleogels (Bet OG and Lup OG) for topical application in skin injuries. The results revealed suitable pH as well as organoleptic, rheological, and textural properties. The penetration and permeation of Bet and Lup oleogels through porcine ear skin as well as the retention of both oleogels in the skin were demonstrated through ex vivo studies. In vitro, Bet OG and Lup OG showed good biocompatibility on HaCaT human immortalized cells. Moreover, Bet OG exerted a potent wound-healing property by stimulating the migration of the HaCaT cells. The in ovo results demonstrated the non-irritative potential of the developed formulations. Additionally, the undertaken in vivo investigation indicated a positive effect of oleogels treatment on skin parameters by increasing skin hydration and decreasing erythema. In conclusion, oleogel formulations are ideal for the local delivery of betulin and lupeol in skin disorders.

Insights into the Behavior of Triple-Negative MDA-MB-231 Breast Carcinoma Cells Following the Treatment with 17ß-Ethinylestradiol and Levonorgestrel.

Oral contraceptives (OCs) are widely used due to their efficiency in preventing unplanned pregnancies and treating several human illnesses. Despite their medical value, the toxicity of OCs remains a public concern. Previous studies indicate the carcinogenic potential of synthetic sex hormones and their link to the development and progression of hormone-dependent malignancies such as breast cancer. However, little is known about their influence on the evolution of triple-negative breast carcinoma (TNBC), a malignancy defined by the absence of estrogen, progesterone, and HER2 receptors. This study reveals that the active ingredients of modern OCs, 17ß-Ethinylestradiol, Levonorgestrel, and their combination induce differential effects in MDA-MB-231 TNBC cells. The most relevant behavioral changes occurred after the 24 h treatment with 17ß-Ethinylestradiol, summarized as follows: (i) decreased cell viability (64.32% at 10 µM); (ii) cell roundness and loss of confluence; (iii) apoptotic aspect of cell nuclei (fragmentation, membrane blebbing); and (iv) inhibited cell migration, suggesting a potential anticancer effect. Conversely, Levonorgestrel was generally associated with a proliferative activity. The association of the two OCs exerted similar effects as 17ß-Ethinylestradiol but was less effective. Further studies are necessary to elucidate the hormones' cytotoxic mechanism of action on TNBC cells.

The "Sandwich" System: A Potential Solution for Protecting Overwintering Cornu aspersum Snails Reared in Semi-Intensive Heliciculture Farms in Colder Climates.

(1) Background: Hibernation in pens covered with LFC was associated with high mortality of C. aspersum snails in Romanian snail farms. This three-year study aimed to develop a simple, but effective system for protecting breeders in colder climates. (2) Methods: The first phase investigated the (pre)hibernal burrowing behavior and the overwintering habitat choice. Protective structures based on straw, LFC, and/or HDPE were tested at pilot level (no snails). The most suitable system was applied under farm conditions. (3) Results: Wood and ridge-tile micro shelters were significantly preferred to corrugated iron micro shelters. Burrowing specimens acted as shallow-burrowers, and this behaviorwas significantly more common for looser soils. All pilot systems displayed significantly higher thermal protection efficiency compared to the sole use of LFC. The balance between straw moistening and thermal protection favored using structure soil/LFC/straw/10-cm air cushion/HDPE. Its use yielded significantly higher survival compared to the sole use of LFC. Most hibernating snails clustered together in large groups, attached on the lower surface of micro shelters. Predator occurrence appeared to marginally affect overwintering survival. (4) Conclusions: The "sandwich" system could be an effective solution for overwintering mature C. aspersum snails in colder climates.

Assessment of Betulinic Acid Cytotoxicity and Mitochondrial Metabolism Impairment in a Human Melanoma Cell Line.

Melanoma represents one of the most aggressive and drug resistant skin cancers with poor prognosis in its advanced stages. Despite the increasing number of targeted therapies, novel approaches are needed to counteract both therapeutic resistance and the side effects of classic therapy. Betulinic acid (BA) is a bioactive phytocompound that has been reported to induce apoptosis in several types of cancers including melanomas; however, its effects on mitochondrial bioenergetics are less investigated. The present study performed in A375 human melanoma cells was aimed to characterize the effects of BA on mitochondrial bioenergetics and cellular behavior. BA demonstrated a dose-dependent inhibitory effect in both mitochondrial respiration and glycolysis in A375 melanoma cells and at sub-toxic concentrations (10 µM) induced mitochondrial dysfunction by eliciting a decrease in the mitochondrial membrane potential and changes in mitochondria morphology and localization. In addition, BA triggered a dose-dependent cytotoxic effect characterized by apoptotic features: morphological alterations (nuclear fragmentation, apoptotic bodies) and the upregulation of pro-apoptotic markers mRNA expression (Bax, Bad and Bak). BA represents a viable therapeutic option via a complex modulatory effect on mitochondrial metabolism that might be useful in advanced melanoma or as reliable strategy to counteract resistance to standard therapy.

Exposure to cadmium and copper triggers cytotoxic effects and epigenetic changes in human colorectal carcinoma HT-29 cells.

Recent scientific evidence suggests a link between epigenetic changes (DNA methylation) and tumorigenesis. Moreover, a potential carcinogenic mechanism of cadmium was associated with changes in DNA methylation. In this study we investigated the impact of CdCl2 and CuSO4 aqueous solutions on DNA methylation in HT-29 cells by quantifying DNA methyltransferase (DNMT1, DNMT3A and DNMT3B) mRNA expression. Furthermore, we also studied the cytotoxic and anti-migratory potential of these substances. The results showed a dose-dependent decrease of viable cell percentage following 24 h of exposure (at concentrations of 0.05; 0.2; 1; 10 and 100 µg/ml), and an inhibitory effect on HT-29 cell migration capacity. In addition, RT-qPCR results showed that cadmium acts as a hypomethylating agent by suppressing DNMT expression, whereas copper acts as a hypermethylating compound by increasing DNMT expression. These findings suggest a cytotoxic potential of both cadmium and copper on HT-29 cells and their capacity to induce epigenetic changes.

Soil copper uptake by land snails: A semi-field experiment with juvenile Cantareus aspersus snails.

There is currently limited scientific evidence linking soil copper and land snails, although these invertebrates are important players in terrestrial ecosystems. In the present study, Cantareus aspersus juveniles, were exposed in two successive phases of 30 days each, to soil spiked with increasing concentrations of copper sulfate. Copper concentrated preferentially and in a dose-dependent manner in the hepatopancreas. In the case of specimens previously exposed to Cu-spiked soils, Cu retention kinetics were independent from the effects of a new exposure event. There was no effect on shell growth, but significant mortality was observed at 60 days. The no observed effect concentration and the lowest observed effect concentration for mortality in snails, were ˜ 41 and 54 mg, respectively, per grams dry weight in the hepatopancreas. The results demonstrate, for the first time, that terrestrial gastropods can accumulate soil Cu autonomously from dietary uptake.

Oleic Acid Double Coated Fe3O4 Nanoparticles as Anti-Melanoma Compounds with a Complex Mechanism of Activity-In Vitro and In Ovo Assessment.

Magnetic iron oxide nanoparticles (MIONPs) were placed in the spotlight lately due to their excellent biocompatibility and the possibility to tailor their magnetic properties making them useful in a plethora of bioapplications, including magnetic resonance imaging and targeted nanoplatforms for anticancer drugs delivery. The aim of the present study consisted in achieving a toxicological profile of the biocompatible colloidal suspension of iron oxide nanoparticles coated with a double layer of oleic acid (Fe3O4 @OA) obtained by combustion method by performing in vitro (on human keratinocytes-HaCaT and human and murine melanoma cells) and in ovo studies on chick chorioallantoic membrane (HET-CAM assay). The colloidal suspension obtained proved to be stable in phosphate buffer saline and the size of the nanoparticles were in the range of 30 nm, an optimum size for biomedical applications. Fe3O4 @OA colloidal suspension reduced viability of human keratinocytes only at concentrations higher than 25 µg/mL, whereas in the case of melanoma cells the effect was observed at lower doses (starting with 10 µg/mL). An interesting phenomena was detected at the highest concentration tested (50 µg/mL) to all cell lines, more precisely, a particular enucleation process associated only with Fe3O4 @OA colloidal suspension stimulation. The irritant potential data evaluated by HET-CAM assay indicated the following hierarchy: Fe3O4 < Fe3O4 @OA < OA. Our results provide relevant information regarding the mechanism of action of Fe3O4 @OA that needs elucidation by future in vitro and in vivo studies.

Impact of soil cadmium on land snails: a two-stage exposure approach under semi-field conditions using bioaccumulative and conchological end-points of exposure.

Land snails are highly tolerant to cadmium exposure and are able to accumulate soil cadmium independently of food ingestion. However, little information exists on the kinetics of cadmium retention in terrestrial gastropods exposed to an increase in the soil cadmium content, over time. There is also little knowledge about how exposure to cadmium-polluted soils influences shell growth and architecture. In this context, we examined cadmium accumulation in the hepatopancreas and shell of juvenile Cantareus aspersus exposed to elevating high levels of cadmium in soil. Also, the toxicity of cadmium to snails was assessed using a range of conchological endpoints, including shell height, width, volume, allometry and integrity. Test snails, aged three months, were reared under semi-field conditions, fed an uncontaminated diet and exposed first, for a period of 30 days, to a series of soil cadmium concentrations, and then, for a second period of 30 days, to soils with higher cadmium content. Cadmium showed a dose-dependent accumulation in both the hepatopancreas and shell. The kinetics of cadmium retention in the hepatopancreas of snails previously exposed to cadmium-spiked soils was significantly influenced by a new exposure event. The shell was not a relevant bioaccumulator for soil cadmium. Under the present experimental conditions, only high cadmium exposure significantly affected either the shell growth or snail survival. There was no consistent effect on shell allometry, but the shell integrity, especially in rapidly growing parts, appeared to be affected by high cadmium exposure. Our results attest to the value of hepatopancreas for describing cadmium retention in land snails and to the difficulty of using conchological parameters in field surveys for estimating the environmental hazard of soil cadmium.