DNA Fragmentation Research Articles

Gene Expression, Oxidative Stress, and Neurotransmitters in Rotenone-induced Parkinson’s Disease in Rats: Role of Naringin from Citrus aurantium via Blocking Adenosine A2A Receptor

Background:: Lack of control in voluntary movements, resting tremor, postural instability, and stiffness are the hallmarks of Parkinson's disease (PD). Objective:: The current work's objective is to assess naringin isolated from Citrus aurantium L. peels as an anti-parkinsonism agent in rats. Methods:: The HPLC and LC-ESI-MS analysis of Citrus aurantium L. peels methanol extract was done. The behavioral, biochemical, genetic, and histopathological analysis were evaluated in parkinsonism rats. Results:: Fourteen phenolics and nine flavonoids were found in the extract, according to the HPLC analysis, while LC-ESI-MS analysis revealed the presence of twenty-six flavonoids. The dominant flavonoid subclasses were 4 aglycones, 11 monoglycosides, 5 diglycosides, and 6 polymethoxy flavonoids, beside 4 coumarines, 4 alkaloids and a limonin triterpene. Adenosine A2A receptor (A2AR) gene expression, malondialdehyde (MDA), interleukin-6 (IL-6), caspase-3 (Cas-3) and DNA fragmentation levels significantly increased in rotenone-treated rats. Dopamine (DA), norepinephrine (NE), serotonin (5-HT), reduced glutathione (GSH), succinate, and lactate dehydrogenase (SDH &LDH) levels all significantly decreased. Treatment with naringin and A2AR antagonists enhanced the animals’ behavior and improved all the selected parameters. The brain hippocampal features confirmed our results. Conclusion:: Naringin could be considered a nutraceutical agent by attenuating the neurodegeneration associated with PD via blocking adenosine A2AR.

Case report: Difficult diagnosis of Mycobacterium tuberculosis infection in patients after allogeneic hematopoietic stem cell transplantation: two case reports and a literature review

BackgroundMycobacterium tuberculosis (MTB) is a relatively infrequent infection encountered during hematopoietic stem-cell transplantation (HSCT). The identification of MTB following HSCT remains a complex task, with delayed detection and misdiagnosis potentially resulting in unfavorable outcomes. Metagenomic next-generation sequencing (mNGS) represents a novel, highly sensitive, and rapid diagnostic tool in clinical settings for discerning intricate infections and detecting exceedingly rare pathogensMethodsWith the aid of mNGS, we diagnosed MTB in the lymph nodes and lungs of two patients with hematological diseases following allogeneic peripheral blood hematopoietic stem cell transplantation. Both patients presented with a fever, localized symptoms, and clinical signs. Following inconclusive results from routine tests, impractical biopsy procedures, and unsuccessful responses to empirical treatments, mNGS was employed as a final recourse, revealing DNA fragments of MTB in blood samples.ResultsThe diagnoses were ultimately confirmed in conjunction with additional clinical evidence. The application of mNGS in MTB cases after allogeneic HSCT has rarely been reported. The mNGS technique can provide a prompt and highly sensitive indication leading to the definitive diagnosis of MTB in complex post-transplant scenarios.

Single-stranded pre-methylated 5mC adapters uncover the methylation profile of plasma ultrashort Single-stranded cell-free DNA.

Whole-genome bisulfite sequencing (BS-Seq) measures cytosine methylation changes at single-base resolution and can be used to profile cell-free DNA (cfDNA). In plasma, ultrashort single-stranded cfDNA (uscfDNA, ∼50 nt) has been identified together with 167 bp double-stranded mononucleosomal cell-free DNA (mncfDNA). However, the methylation profile of uscfDNA has not been described. Conventional BS-Seq workflows may not be helpful because bisulfite conversion degrades larger DNA into smaller fragments, leading to erroneous categorization as uscfDNA. We describe the '5mCAdpBS-Seq' workflow in which pre-methylated 5mC (5-methylcytosine) single-stranded adapters are ligated to heat-denatured cfDNA before bisulfite conversion. This method retains only DNA fragments that are unaltered by bisulfite treatment, resulting in less biased uscfDNA methylation analysis. Using 5mCAdpBS-Seq, uscfDNA had lower levels of DNA methylation (∼15%) compared to mncfDNA and was enriched in promoters and CpG islands. Hypomethylated uscfDNA fragments were enriched in upstream transcription start sites (TSSs), and the intensity of enrichment was correlated with expressedgenesof hemopoietic cells. Using tissue-of-origin deconvolution, we inferred that uscfDNA is derived primarily from eosinophils, neutrophils, and monocytes. As proof-of-principle, we show that characteristics of the methylation profile of uscfDNA can distinguish non-small cell lung carcinoma from non-cancer samples. The 5mCAdpBS-Seq workflow is recommended for any cfDNA methylation-based investigations.

Binding energies and hydrogen bonds effects on DNA-cisplatin interactions: a DFT-xTB study.

A systematic study of hydrogen bonds in base pairs and the interaction of cisplatin with DNA fragments was carried out. Structure, binding energies, and electron density were analyzed. xTB has proven to be an accurate method for obtaining structures and binding energies in DNA structures. Our xTB values for DNA base binding energy were in the same order and in some cases better than CAM-B3LYP values compared to experimental values. Double-stranded DNA-cisplatin structures have been calculated and the hydrogen bonds of water molecules are a decisive factor contributing to the preference for the cisplatin-Guanine interaction. Higher values of the water hydrogen bonding energies were obtained in cisplatin-Guanine structures. Furthermore, the electrostatic potential was used to investigate and improve the analysis of DNA-cisplatin structures. We applied the xTB method and the CAM-B3LYP functional combined with def2-SVP basis set to perform and analyze of the bonding energies of the cisplatin interaction and the effects of the hydrogen bonds. Results were calculated employing the xTB and the ORCA software.

Deep DNAshape webserver: prediction and real-time visualization of DNA shape considering extended k-mers.

Sequence-dependent DNA shape plays an important role in understanding protein-DNA binding mechanisms. High-throughput prediction of DNA shape features has become a valuable tool in the field of protein-DNA recognition, transcription factor-DNA binding specificity, and gene regulation. However, our widely used webserver, DNAshape, relies on statistically summarized pentamer query tables to query DNA shape features. These query tables do not consider flanking regions longer than two base pairs, and acquiring a query table for hexamers or higher-order k-mers is currently still unrealistic due to limitations in achieving sufficient statistical coverage in molecular simulations or structural biology experiments. A recent deep-learning method, Deep DNAshape, can predict DNA shape features at the core of a DNA fragment considering flanking regions of up to seven base pairs, trained on limited simulation data. However, Deep DNAshape is rather complicated to install, and it must run locally compared to the pentamer-based DNAshape webserver, creating a barrier for users. Here, we present the Deep DNAshape webserver, which has the benefits of both methods while being accurate, fast, and accessible to all users. Additional improvements of the webserver include the detection of user input in real time, the ability of interactive visualization tools and different modes of analyses. URL: https://deepdnashape.usc.edu.

Buffered Lugol's iodine preserves DNA fragment lengths

Buffered Lugol's iodine preserves DNA fragment lengths

Human circBOULE RNAs as potential biomarkers for sperm quality and male infertility.

Reliable molecular biomarkers to predict fertility remain scarce. The current study explored the potential of testis-specific circBOULE RNAs as biomarkers for male infertility and sperm quality. Using RT-PCR and RT-qPCR assays, we identified seven circular RNAs from the human BOULE gene in human sperm. We found that sperm circEx3-6 RNA exhibited a significantly decreased expression in asthenozoospermia while circEx2-6 and circEx2-7 expression decreased in teratozoospermia, compared with the controls. Furthermore, circEx2-6 expression exhibited a negative correlation with sperm DNA Fragmentation Index (DFI), and circEx2-7 levels were correlated with both fertilization and cleavage rates involving assisted reproductive technologies. Further functional analyses in a transgenic fly model lent support for the roles of circBOULE RNAs in sperm development and human fertility. Collectively, our findings support that sperm circBOULE RNAs may serve as diagnostic biomarkers for assessing sperm motility and DNA quality. Hence clinical application and significance of sperm circular RNAs in assisted reproductive technologies warrant further investigation.

The impact of kit, environment, and sampling contamination on the observed microbiome of bovine milk.

In low-microbial biomass samples such as bovine milk, contaminants can outnumber endogenous bacteria. Because of this, milk microbiome research suffers from a critical knowledge gap, namely, does non-mastitis bovine milk contain a native microbiome? In this study, we sampled external and internal mammary epithelia and stripped and cisternal milk and used numerous negative controls, including air and sampling controls and extraction and library preparation blanks, to identify the potential sources of contamination. Two algorithms were used to mathematically remove contaminants and track the potential movement of microbes among samples. Results suggest that the majority (i.e., >75%) of sequence data generated from bovine milk and mammary epithelium samples represents contaminating DNA. Contaminants in milk samples were primarily sourced from DNA extraction kits and the internal and external skin of the teat, while teat canal and apex samples were mainly contaminated during the sampling process. After decontamination, the milk microbiome displayed a more dispersed, less diverse, and compositionally distinct bacterial profile compared with epithelial samples. Similar microbial compositions were observed between cisternal and stripped milk samples, as well as between teat apex and canal samples. Staphylococcus and Acinetobacter were the predominant genera detected in milk sample sequences, and bacterial culture showed growth of Staphylococcus and Corynebacterium spp. in 50% (7/14) of stripped milk samples and growth of Staphylococcus spp. in 7% (1/14) of cisternal milk samples. Our study suggests that microbiome data generated from milk samples obtained from clinically healthy bovine udders may be heavily biased by contaminants that enter the sample during sample collection and processing workflows.IMPORTANCEObtaining a non-contaminated sample of bovine milk is challenging due to the nature of the sampling environment and the route by which milk is typically extracted from the mammary gland. Furthermore, the very low bacterial biomass of bovine milk exacerbates the impacts of contaminant sequences in downstream analyses, which can lead to severe biases. Our finding showed that bovine milk contains very low bacterial biomass and each contamination event (including sampling procedure and DNA extraction process) introduces bacteria and/or DNA fragments that easily outnumber the native bacterial cells. This finding has important implications for our ability to draw robust conclusions from milk microbiome data, especially if the data have not been subjected to rigorous decontamination procedures. Based on these findings, we strongly urge researchers to include numerous negative controls into their sampling and sample processing workflows and to utilize several complementary methods for identifying potential contaminants within the resulting sequence data. These measures will improve the accuracy, reliability, reproducibility, and interpretability of milk microbiome data and research.

Micronutrient Antioxidants for Men (Menevit®) Improve Sperm Function by Reducing Oxidative Stress, Resulting in Improved Assisted Reproductive Technology Outcomes

Oxidative stress (OS) affects men’s health and impairs spermatogenesis. Micronutrient antioxidants are available for male infertility as complemental support; however, their efficacy remains debatable. This study aimed to investigate whether antioxidants can help to reduce sperm OS and improve semen analysis and quality. We included 171 male partners of couples planning to undergo assisted reproductive technology (ART). Male partners, aged 29–41 years, of couples intending to conceive were self-selected to take daily antioxidants (n = 84) containing folic acid and zinc, or not to take antioxidants (n = 52) for 6 months. We analyzed the alterations in serum oxidant levels, sperm parameters, OS, and deoxyribonucleic acid fragmentation after 3 and 6 months. Additionally, implantation, clinical pregnancy, and miscarriage rates after vitrified–warmed embryo transfer were compared between those taking antioxidants and those not taking them after 6 months. In men with high static oxidation–reduction potential (sORP), we observed a significant improvement in sperm concentration and sORP. The high-quality blastocyst rate tended to increase, and implantation and clinical pregnancy rates also significantly increased after 6 months of intervention. The micronutrient antioxidants could improve sperm function by reducing OS and improving ART outcomes. Therefore, micronutrient antioxidants may be a viable treatment option for male infertility.

Molecular Characterization of Methicillin-resistant Staphylococcus aureus (MRSA) Isolates from Ruminants

Background: Methicillin-resistant Staphylococcus aureus (MRSA) infection is one of the leading causes of infections in animals as well as human beings and is associated with significant morbidity, mortality, length of stay and cost burden. Methods: In this study, 725 samples of nasal swabs and milk were collected randomly from cattle, buffalo, sheep and goats and these samples were inoculated on Mannitol salt agar mixed with Oxacillin Resistance Selective Supplement for the molecular characterization of MRSA isolates through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and polymerase chain reaction (PCR) test. Result: Out of 725 samples, 171 were found positive for Oxacillin resistant S. aureus. The SDS-PAGE showed different bands of molecular weight 13, 17, 20, 24, 26, 28, 33, 36, 39, 43, 47, 59, 64, 72, 86, 97 and 121 kDa. An amplified mec A DNA fragment (137) of 533 base pairs (bp) and PCR product of fem A (133 isolates) of 510 bp were detected in isolates.

DNA Barcoding Revealed a High Percentage of Mislabeling in Commercial Fish Products: The First Empirical Survey in South Texas

Seafood mislabeling is a widespread problem that have produced a growing distrust of seafood industry. In this study, we examined the prevalence of mislabeling in fish samples from regional markets in the greater Houston area and close-by coastal communities. A total of 63 fish fillet samples were purchased, labeled, and stored at -20˚C in individual packages until DNA extraction. DNA fragments of roughly 700 base pairs encoding cytochrome oxidase I (COI) were PCR-amplified from each DNA sample. With 99.6–100% nucleotide identity in the GenBank and BOLD databases, all samples were correctly identified at the species level. The scientific names identified by DNA barcoding were matched with legally acceptable market names using the US Food and Drug Administration (FDA) Seafood List. Out of the 63 samples examined, 13 samples (20.6%) were mislabeled. Moreover, 24 samples (38.1%) did not use the acceptable market names, indicating that the FDA policy is poorly implemented in seafood industry. The first DNA barcoding survey this area warranted the need of continuous monitoring and the dissemination of the regulation combined with taxonomic knowledge.

Evaluating the possible genotoxicity of nanoaluminum incorporated in human vaccines and the potential protective role of nanocurcumin: an in vivo study

For nearly 90 years, aluminum (Al) salts have been utilized as vaccination adjuvants. Nevertheless, there is a risk of adverse effects associated with the amount of nanoaluminum used in various national pediatric immunization regimens. This study aimed to investigate the possible genotoxic effects of nanoaluminum incorporated in human vaccines on the brains of newborn albino rats and whether nanocurcumin has a potential protective effect against this toxicity. Fifty newborn albino rats were randomly assigned to 5 groups, with 10 in each group. Groups 1 and 2 received "high" and "low" Al injections corresponding to either the American or Scandinavian pediatric immunization schedules, respectively, as opposed to the control rats (group 5) that received saline injections. Groups 3 and 4 received the same regimens as groups 1 and 2 in addition to oral nanocurcumin. The expression of both the cell breakdown gene tumor protein (P53) and the cell stress gene uncoupling protein 2 (UCP2) was significantly greater in groups 1 and 2 than in group 5. Groups 1 and 2 exhibited severe DNA fragmentation, which was observed as DNA laddering. Nanocurcumin significantly reduced the expression of the P53 and UCP2 genes in groups 3 and 4, with very low or undetectable DNA laddering in both groups. Vaccination with nanoaluminum adjuvants can cause genotoxic effects, which can be mediated by the inflammatory response and oxidative stress, and nanocurcumin can protect against these toxic effects through the modulation of oxidative stress regulators and gene expression.

In silico analysis of the wild-type and mutant-type of BRCA2 gene

BackgroundThe aim of this study was to conduct an in silico analysis of a novel compound heterozygous variant in breast cancer susceptibility gene 2 (BRCA2) to clarify its structure–function relationship and elucidate the molecular mechanisms underlying triple-negative breast cancer (TNBC).MethodsA tumor biopsy sample was obtained from a 42-year-old Chinese woman during surgery, and a maxBRCA™ test was conducted using the patient’s whole blood. We obtained an experimentally determined 3D structure (1mje.pdb) of the BRCA2 protein from the Protein Data Bank (PDB) as a relatively reliable reference. Subsequently, the wild-type and mutant structures were predicted using SWISS-MODEL and AlphaFold, and the accuracy of these predictions was assessed through the SAVES online server. Furthermore, we utilized a high ambiguity-driven protein–protein docking (HADDOCK) algorithm and protein–ligand interaction profiler (PLIP) to predict the pathogenicity of the mutations and elucidate pathogenic mechanisms that potentially underlies TNBC.ResultsHistological examination revealed that the tumor biopsy sample exhibited classical pathological characteristics of TNBC. Furthermore, the maxBRCA™ test revealed two compound heterozygous BRCA2 gene mutations (c.7670 C > T.pA2557V and c.8356G > A.pA2786T). Through performing in silico structural analyses and constructing of 3D models of the mutants, we established that the mutant amino acids valine and threonine were located in the helical domain and oligonucleotide binding 1 (OB1), regions that interact with DSS1.ConclusionOur analysis revealed that substituting valine and threonine in the helical domain region alters the structure and function of BRCA2 proteins. This mutation potentially affects the binding of proteins and DNA fragments and disrupts interactions between the helical domain region and OB1 with DSS1, potentially leading to the development of TNBC. Our findings suggest that the identified compound heterozygous mutation contributes to the clinical presentation of TNBC, providing new insights into the pathogenesis of TNBC and the influence of compound heterozygous mutations in BRCA2.

Experimental biological studies of novel tetradentate hydrazone Cu(II) complexes for potential applications in medicinal chemistry

A series of mononuclear Cu(II) complexes were synthesized using a potential tetradentate hydrazone ligand obtained from the reaction between phenylhydrazine groups and 2,5-thiophenedicarboxaldehyde. The structures of ligands and complexes were elucidated through various spectroscopic techniques, confirming their composition. All complexes were found to adopt four-coordinated geometries, indicating the formation of stable structures. Spectroscopic analysis revealed that the hydrazone ligand coordinated with the Cu(II) metal ions as a dibasic tetradentate ligand by utilizing the phenolic oxygen and azomethine nitrogen atoms. The binding affinity of the complexes with calf thymus DNA (CT-DNA) was investigated using absorption and viscosity measurements, demonstrating their interaction through the intercalation mode. The binding studies showed that the Cu(II) complexes exhibited varying degrees of binding affinity, with Cu(L4) demonstrating the highest affinity, followed by Cu(L1), Cu(L2), and Cu(L3). Moreover, the DNA fragmentation properties of the Cu(II) complexes were evaluated, suggesting their potential utilization in pharmaceutical applications. The obtained results highlight the significance of these novel complexes in the field of medicinal chemistry.

Involvement of circular RNAs in the control of porcine ovarian cell functions: Upregulation by ciR-00596 and downregulation by ciR-00646

The current understanding of the role of circular RNAs (circRNAs) in regulating ovarian functions is inadequate. To assess the impact of ciR-00596 and ciR-00646 on the regulation of basic porcine ovarian granulosa cell functions, we conducted upregulation (utilizing overexpressing vectors) and downregulation (utilizing shRNA vectors) of these circRNAs. The relative expression of both circRNAs, cell viability and proliferation (accumulation of PCNA, cyclin B1, and XTT-positive cells), cytoplasmic (accumulation of bax and caspase-3) and nuclear (DNA fragmentation) apoptosis, and the release of progesterone, testosterone, estradiol, IGF-I, and oxytocin were evaluated. Transfection of cells with the ciR-00596 overexpression vector resulted in increases in cell viability and proliferation and the release of progesterone and IGF-I, while it decreased the cytoplasmic and nuclear apoptosis, testosterone, estradiol, and oxytocin output. CiR-00596 inhibition had the opposite effects. The overexpression of ciR-00646 decreased cell viability and proliferation, and the release of progesterone, IGF-I, and oxytocin, while increasing cytoplasmic and nuclear apoptosis and the output of testosterone and estradiol. Our findings are the first to show the stimulatory action of ciR-00596 and the inhibitory effect of ciR-00646 on ovarian cell functions, including the cell cycle, apoptosis, and secretory activity.

Cryopreservation of spermatozoa from black-and-gold howler monkeys (Alouatta caraya) using egg yolk-based or soy lecithin-based extenders.

There are more than 200 species and subspecies of Neotropical Primates of which more than 40% are listed as threatened by the IUCN Red List of Threatened Species. Both in situ and ex situ conservation programs can benefit from the use of assisted reproductive technologies. The objective of this study was to evaluate, for the first time, cryopreservation techniques for Alouatta caraya semen. Semen samples were collected from five adult males, analyzed, and frozen in either Test-egg yolk or Test-soy lecithin-based extenders containing either 3 or 4% glycerol. Frozen-thawed samples were analyzed at 10, 40, and 80min post-thaw. Egg yolk-based extenders were overall better than soy lecithin-based extenders. There was no significant difference between 3 and 4% glycerol in any of the parameters analyzed, however, 4% glycerol in egg yolk-based extender produced more favorable results for total motility, intact plasma membrane, lipidperoxidation, and DNA fragmentation index. This study brought novel information on semen characteristics and cryopreservation aspects for A. caraya, which can help shape future experiments to improve the outcome of frozen-thawed sperm for this and other species of Neotropical primates.

Exploring Gut Microbiome Composition and Circulating Microbial DNA Fragments in Patients with Stage II/III Colorectal Cancer: A Comprehensive Analysis.

Colorectal cancer (CRC) significantly contributes to cancer-related mortality, necessitating the exploration of prognostic factors beyond TNM staging. This study investigates the composition of the gut microbiome and microbial DNA fragments in stage II/III CRC. A cohort of 142 patients with stage II/III CRC and 91 healthy controls underwent comprehensive microbiome analysis. Fecal samples were collected for 16S rRNA sequencing, and blood samples were tested for the presence of microbial DNA fragments. De novo clustering analysis categorized individuals based on their microbial profiles. Alpha and beta diversity metrics were calculated, and taxonomic profiling was conducted. Patients with CRC exhibited distinct microbial composition compared to controls. Beta diversity analysis confirmed CRC-specific microbial profiles. Taxonomic profiling revealed unique taxonomies in the patient cohort. De novo clustering separated individuals into distinct groups, with specific microbial DNA fragment detection associated with certain patient clusters. The gut microbiota can differentiate patients with CRC from healthy individuals. Detecting microbial DNA fragments in the bloodstream may be linked to CRC prognosis. These findings suggest that the gut microbiome could serve as a prognostic factor in stage II/III CRC. Identifying specific microbial markers associated with CRC prognosis has potential clinical implications, including personalized treatment strategies and reduced healthcare costs. Further research is needed to validate these findings and uncover underlying mechanisms.

Effects of long and short ejaculatory abstinence on sperm parameters: a meta-analysis of randomized-controlled trials

PurposeThis study aimed to investigate the effects of ejaculatory abstinence on sperm parameters.MethodsThis analysis was registered in PROSPERO (CRD42023472124). We performed a search on PubMed using the following text terms: ((“sperm parameters” OR “sperm analysis” [Mesh]) AND (“sperm DNA fragmentation” OR “DNA fragmentation” [Mesh]) AND (“sexual abstinence” [Mesh] OR “abstinence”)) and an advanced search in Scopus using the terms (“sperm parameters” OR “sperm parameters” OR “DNA fragmentation”) AND (“abstinence”). The sperm parameters that were investigated were sperm volume, total sperm motility, progressive sperm motility, sperm concentration, sperm morphology, and sperm DNA fragmentation (SDF). A two-day cut-off as a “short” or “long” abstinence period has been defined.ResultsThirteen studies published between 2013 and 2022 were included in this meta-analysis. A total of 2,315 patients, ranging from 6 to 836 from each cohort, were enrolled in the study. We showed that longer abstinence time was associated with greater sperm concentration (mean difference [MD]: 8.19; p <0.01), sperm volume (MD: 0.96; p <0.01), and higher SDF (MD: 3.46; p <0.01), but lower progressive sperm motility (MD: −1.83; p <0.01). Otherwise, no statistically significant difference was observed in patients with longer vs. shorter abstinence times regarding total sperm motility (MD: −1.83; p = 0.06). Meta-regression analysis showed that days of abstinence were positively and linearly related to sperm concentration (slope: 3.74; p <0.01) and SDF (slope: 0.65; p = 0.044).ConclusionsAccording to our data, short ejaculatory abstinence is associated with better sperm quality. Indeed, a higher percentage of progressive sperm motility and lower levels of SDF have been reported in a short abstinence cohort. In contrast, the long abstinence group reported a higher sperm concentration.Systematic review registrationhttps://www.crd.york.ac.uk/PROSPERO/, identifier CRD42023472124.

Assessing testicular morphofunctionality under Roundup WG® herbicide exposure in zebrafish.

Glyphosate-based herbicides, like Roundup WG® (RWG) used for a range of crops, such as corn, soybean, coffee, sugarcane, rice, apple, and citrus, can reach aquatic ecosystems and impact non-target organisms like fish. Thus, the fish were exposed to three RWG concentrations plus one negative control, which represents the concentration allowed for inland Brazilian waters and concentrations found in surface water worldwide (0.0, 0.065, 0.65, and 6.5mg a.i./L) for 7 and 15days. Morphological analysis revealed significant alterations in the testicular structure, particularly in Sertoli cell extensions and cytoplasmic bridges between germ cells. Subcellular compartments also displayed alterations, including dilated mitochondria and the loss of electron density and autophagic vesicles. Gene transcript levels related to autophagy and steroidogenic regulation were upregulated in exposed fish. Germ cell quality was also affected, increasing ROS (reactive oxygen species) production and DNA fragmentation. The study highlighted the RWG reproductive toxicity, providing valuable insights into understanding the morphofunctional alterations in somatic and germ cells of Danio rerio. In conclusion, the environmental relevant concentrations used in this study were toxic to male somatic and germ cells, which raises a concern about the concentrations considered safe for human and animal use.

Enhanced Therapeutic Efficacy Against Melanoma through Exosomal Delivery of Hesperidin.

Melanoma, characterized as the most aggressive and metastatic form of skin cancer, currently has limited treatment options, predominantly chemotherapy and radiation therapy. However, the drawbacks associated with parenterally administered chemotherapy underscore the urgent need for alternative compounds to combat melanoma effectively. Hesperidin (HES), a flavonoid present in various citrus fruits, exhibits promising anticancer activity. Nevertheless, the clinical utility of HES is hindered by challenges such as poor water solubility, a short half-life, and low oral bioavailability. In response to these limitations, we introduced a novel approach by formulating HES-loaded exosomes (Exo-HES). Isolation of exosomes was achieved through the ultracentrifugation method, and HES was efficiently loaded using the sonication method. The resulting formulations displayed a desirable particle size (∼106 nm) and exhibited a spherical morphology, as confirmed by scanning electron and atomic force microscopy. In vitro studies conducted on B16F10 cell lines demonstrated higher cytotoxicity of Exo-HES compared to free HES, supported by enhanced cellular uptake validated through coumarin-6-loaded exosomes. This superior cytotoxicity was further evidenced by DNA fragmentation, increased generation of free radicals (ROS), loss of mitochondrial membrane potential, and effective inhibition of colony formation. The antimetastatic properties of Exo-HES were confirmed through wound healing and transwell migration assays. Oral pharmacokinetics studies revealed a remarkable increase of approximately 2.5 times in oral bioavailability and half-life of HES when loaded into exosomes. Subsequent in vivo experiments utilizing a B16F10-induced melanoma model in Swiss mice established that Exo-HES exhibited superior anticancer activity compared to HES after oral administration. Importantly, no biochemical, hematological, or histological toxicities were observed in tumor-bearing mice treated with Exo-HES. These findings suggest that exosomes loaded with HES represent a promising nanocarrier strategy to enhance the therapeutic effectiveness of hesperidin in melanoma treatment.