Ultrasound-responsive microbubbles and nanodroplets: A pathway to targeted drug delivery.

Ultrasound-responsive agents have shown great potential as targeted drug delivery agents, effectively augmenting cell permeability and facilitating drug absorption. This review focuses on two specific agents, microbubbles and nanodroplets, and provides a sequential overview of their drug delivery process. Particular emphasis is given to the mechanical response of the agents under ultrasound, and the subsequent physical and biological effects on the cells. Finally, the state-of-the-art in their pre-clinical and clinical implementation are discussed. Throughout the review, major challenges that need to be overcome in order to accelerate their clinical translation are highlighted.

Predicting nanocarriers' efficacy in 3D models with Brillouin microscopy.

Thanks to their unique nanoscale properties, nanomedicines can overcome some of the shortcomings of conventional therapies. For better predictive screening, it is important to assess their performance in three-dimensional (3D) multicellular tumour spheroids (MCTS) that can recapitulate the physiological barriers found in real tumours. Today, the evaluation of drug delivery nanosystems in MCTS is mainly explored by means of microscopy techniques that are invasive and require fluorescent labels which modify the composition and fate of the carriers. In recent years, a new quantitative microscopy technique based on Brillouin light scattering (BLS) has been proposed that uses the interaction of laser light with picosecond timescale density fluctuations in the sample. Because it is label-free, all-optical and non-destructive, BLS has gained interest in the pharmaceutical and biomedical fields. In this work, we implemented a fast BLS spectrometer and used the Brillouin frequency shift at the center of the MCTS as a quantitative readout for drug efficacy. We first investigated the ability of this setup to quantify drug efficacy in MCTS grown in classical multiwell plates and concluded that the low number of samples available in the multiwells limits the statistical significance of the results. To improve the throughput, we then combined the microscope with agarose microwells designed to fabricate a large number of MCTS and test 50 MCTS in less than a minute. Using this platform, we assessed the efficacy of polymeric nanoparticles (NPs) loaded with a platinum derivative anticancer drug (dichloro(1,2-diaminocyclohexane)platinum(II)) in reducing the growth of colorectal cancer cells (HCT-116) in MCTS. We observe a time- and dose-dependent decrease in the frequency shift, revealing the progressive loss of mechanical integrity in the MCTS. These results demonstrate that BLS probing of MCTS grown in agarose microwells is a promising tool for high-throughput screening of nanocarriers in 3D models.

Metal pollution in freshwater fish: A key indicator of contamination and carcinogenic risk to public health.

Metals are micropollutants that cannot be degraded by microorganisms and are infiltrated into various environmental media, including both freshwater and marine water. Metals from polluted water are absorbed by many aquatic species, especially fish. Fish is a staple food in the diets of many regions in the world; hence, both the type and concentration of metals accumulated and transferred from contaminated water sources to fish must be determined and assessed. In this study, the heavy metal concentration was determined and assessed in fish collected from freshwater sources via published literature and Estimated Daily Intake (EDI), Target hazard quotient (THQ), and Carcinogenic Risk (CR) analyses, aiming to examine the metal pollution in freshwater fish. The fish was used as a bioindicator, and Geographic information system (GIS) was sued to map the polluted regions. The results confirmed that Pb was detected in fish sampled at 28 locations, Cr at 24 locations, Cu and Zn at 30 locations, with values Pb detected ranging from 0.0016 mg kg-1 to 44.3 mg kg-1, Cr detected ranging from 0.07 mg kg-1 to 27 mg kg-1, Cu detected ranging from 0.031 mg kg-1 to 35.54 mg kg-1, and Zn detected ranging from 0.242 mg kg-1 to 103.2 mg kg-1. The strongest positive associations were discovered between Cu-Zn (r = 0.74, p < 0.05) and Cr-Zn (r = 0.57, p < 0.05). Spatial distribution maps depicting the consumption of fish as food and its corresponding Pb and Cr intake revealed a higher incidence of both carcinogenic and non-carcinogenic health concerns attributed to Pb and Cr in the region with populations consuming the fish.

Nanoemulsions Embedded in Alginate Beads as Bioadhesive Nanocomposites for Intestinal Delivery of the Anti-Inflammatory Drug Tofacitinib.

Oral administration of nanoparticles (NPs) is a promising strategy to overcome solubility and stability issues of many active compounds. However, this route faces major obstacles related to the hostile gastrointestinal (GI) environment, which impairs the efficacy of orally administered nanomedicines. Here, we propose nanocomposites as a promising approach to increase the retention time of NPs in the intestinal tract by using bio- and mucoadhesive matrixes able to protect the cargo until it reaches the targeted area. A microfluidic-based approach has been applied for the production of tailored nanoemulsions (NEs) of about 110 nm, used for the encapsulation of small hydrophobic drugs such as the anti-inflammatory JAK-inhibitor tofacitinib. These NEs proved to be efficiently internalized into a mucus-secreting human intestinal monolayer of Caco-2/HT29-MTX cells and to deliver tofacitinib to subepithelial human THP-1 macrophage-like cells, reducing their inflammatory response. NEs were then successfully encapsulated into alginate hydrogel microbeads of around 300 µm, which were characterized by rheological experiments and dried to create a long-term stable system for pharmaceutical applications. Finally, ex vivo experiments on excised segments of rats' intestine proved the bioadhesive ability of NEs embedded in alginate hydrogels compared to free NEs, showing the advantage that this hybrid system can offer for the treatment of intestinal pathologies.

Epigenetics regulation of prostate cancer: Biomarker and therapeutic potential.

Prostate cancer (CaP) is the second leading cause of cancer death and displays a broad range of clinical behavior from relatively indolent to aggressive metastatic disease. The etiology of most cases of CaP is not understood completely, which makes it imperative to search for the molecular basis of CaP and markers for early diagnosis. Epigenetic modifications, including changes in DNA methylation patterns, histone modifications, miRNAs, and lncRNAs are key drivers of prostate tumorigenesis. These epigenetic defects might be due to deregulated expression of the epigenetic machinery, affecting the expression of several important genes like GSTP1, RASSF1, CDKN2, RARRES1, IGFBP3, RARB, TMPRSS2-ERG, ITGB4, AOX1, HHEX, WT1, HSPE, PLAU, FOXA1, ASC, GPX3, EZH2, LSD1, etc. In this review, we highlighted the most important epigenetic gene alterations and their variations as a diagnostic marker and target for therapeutic intervention of CaP in the future. Characterization of epigenetic changes involved in CaP is obscure and adequate validation studies are still required to corroborate the present results that would be the impending future of transforming basic research settings into clinical practice.

Fatty Acids in Waste Tissues: The Nutraceutical Value of Gonads and Livers from the Moroccan Hypophthalmichthys molitrix and Cyprinus carpio Fishes.

Fishes are an important component of human nutrition, mainly acting as source of essential fatty acids in the prevention of cardiovascular disorders. The increase in their consumption has led to a growth of fishes waste; therefore, the disposal and recycling of waste has become a key issue to address, in accordance with circular economy principles. The Moroccan Hypophthalmichthys molitrix and Cyprinus carpio fishes, living in freshwater and marine environments, were collected at mature and immature stages. The fatty acid (FA) profiles of liver and ovary tissues were investigated by GC-MS and compared with edible fillet tissues. The gonadosomatic index, the hypocholesterolemic/hypercholesterolemic ratio, and the atherogenicity and thrombogenicity indexes were measured. Polyunsaturated fatty acids were found to be abundant in the mature ovary and fillet of both species, with a polyunsaturated fatty acids/saturated fatty acids ratio ranging from 0.40 to 1.06 and a monounsaturated fatty acids/polyunsaturated fatty acids ratio between 0.64 and 1.84. Saturated fatty acids were found to be highly abundant in the liver and gonads of both species (range 30-54%), as well as monounsaturated fatty acids (range 35-58%). The results suggested that the exploitation of fish wastes, such as the liver and ovary, may represent a sustainable strategy for the achievement of high value-added molecules with nutraceutical potential.

Long Non Coding RNA in Triple Negative Breast Cancer: A Promising Biomarker in Tumorigenesis.

Globally, Triple-negative breast cancer (TNBC) is an unsurpassed variant of breast cancer (BC) with a very high fatality rate, and disease burden. Nevertheless, the deficit of diagnostic markers and focused treatment are major hurdles for potent therapeutics. They are also the reason for bad outcomes and causes of a worse prognosis and a high rate of flare up in patients with TNBC diagnosis. Long non-coding RNAs (lncRNA) are a new class of molecules that have recently gained interest in healthcare management due to their potential as biomarkers for human diseases especially cancers. The growing interest in lncRNA in clinical practice has created an unmet need for developing assays to test lncRNA quickly and accurately for early diagnostics. These lncRNA modulate multiple stages of tumor development, including growth, proliferation, invasion, angiogenesis, and metastases, by controlling several genes and changing metabolic networks. Highly invasive phenotype and chemo resistance are prominent characteristics of TNBC subtypes that require accurate diagnostic and prognostic instruments involving lncRNA. This review focusses on the evolving purpose and coalition of lncRNAs in TNBC and accentuates their capable effects in diagnosis and treatment of cancer. Moreover, the extensive literature analysis of our review creates an opportunity in the translational application concerning the TNBC lncRNAs described until now. The depiction of lncRNAs enrolled in TNBC is comprehensive, and sufficient substantiation studies are the need of the hour to authenticate the current outcomes and create imminent upcoming of elemental research setting into clinical practice.

Hazard and health risk assessment of exposure to pharmaceutical active compounds via toxicological evaluation by zebrafish.

The uncontrolled or continuous release of effluents from wastewater treatment plants leads to the omnipresence of pharmaceutical active compounds (PhACs) in the aquatic media. Today, this is a confirmed problem becoming a main subject of twin public and scientific concerns. However, still little information is available about the long-term impacts of these PhACs on aquatic organisms. In this review, efforts were made to reveal correlation between the occurrence in the environment, ecotoxicological and health risks of different PhACs via toxicological evaluation by zebrafish (Danio rerio). This animal model served as a bioindicator for any health impacts after the exposure to these contaminants and to better understand the responses in relation to human diseases. This review paper focused on the calculation of Risk Quotients (RQs) of 34 PhACs based on environmental and ecotoxicological data available in the literature and prediction from the ECOSAR V2.2 software. To the best of the authors' knowledge, this is the first report on the risk assessment of PhACs by the two different methods as mentioned above. RQs showed greater difference in potential environmental risks of the PhACs. These differences in risk values underline the importance of environmental and experimental factors in exposure conditions and the interpretation of RQ values. While the results showed high risk to Danio rerio of the majority of PhACs, risk qualification of the others varied between moderate to insignifiant. Further research is needed to assess pharmaceutical hazards when present in wastewater before discharge and monitor the effectiveness of treatment processes. The recent new advances in the morphological assessment of toxicant-exposed zebrafish larvae for the determination of test compounds effects on the developmental endpoints were also discussed. This review emphasizes the need for strict regulations on the release of PhACs into environmental media in order to minimize their toxicity to aquatic organisms.

Anthropic impact on soil heavy metal contamination in riparian ecosystems of northern Algeria.

The aim of this research was to highlight the impact of urbanization and agriculture on soil quality, mainly by focusing on heavy metal accumulation (Cd, Cu, Fe, Mn, Ni, Pb and Zn) in the fragile riparian ecosystems of the Medjerda river (Souk-Ahras, Algeria). This study was performed in three cultivated soils (0-10, 10-20, 20-60 cm depth) along an increasing gradient of anthropogenic pressure in non-urban (NU), peri-urban (PU) and urban areas (U). Geo-accumulation index (Igeo), Enrichment factor (EF), Pollution load index (PLI) and Potential ecological risk index (RI) were calculated, as well as the potential non-carcinogenic risk for humans (HI). Additionally, to corroborate the role of Bufo spinosus D. as biosentinel, a skin biopsy was performed to quantify the concentration of heavy metals in both terrestrial and aquatic ecosystems. The results showed that when compared to NU, U and PU areas were richer in heavy metals. In particular, Igeo-Cd displayed strongly contaminated soil in U (>3), EF showed high enrichment of heavy metals (>2) for all the soils except for Ni, PLI presented no contamination for all (<1) while RI was significantly higher in U (>300), denoting a strong impact of heavy metals on soil quality. However, HI was below 1 for all the studied areas, although the highest values were related to U and PU. The skin biopsy showed the highest values for Cu, Fe and Pb in PU (0.328, 0.713 and 0.524 mg kg-1, respectively) similarly to trends observed in the soil of that area. This study shed light on the rising pollution of heavy metals due to urbanization and agricultural input in these fragile ecosystems where Bufo spinosus D. plays the role of potential bio-indicator for environmental pollution.

Time- and dose-dependent biological effects of a sub-chronic exposure to realistic doses of salicylic acid in the gills of mussel Mytilus galloprovincialis.

Among nonsteroidal anti-inflammatory drugs (NSAIDs) commonly found in seawater and wastewater, salicylic acid (SA) represents one of the most persistent and hazardous compounds for aquatic organisms. This study was therefore designed to elucidate the biological effects of SA in mussel Mytilus galloprovincialis. During a sub-chronic exposure (12 days), mussels were exposed to five realistic concentrations of SA (C1: 0.05 µg/L; C2: 0.5 µg/L; C3: 5 µg/L; C4: 50 µg/L; C5: 100 µg/L) and gills, selected as the target organ, were collected at different time points (T3: 3 days; T5: 5 days; T12: 12 days). Exposure to SA induced no histological alterations in mussel gills, despite a relevant hemocyte infiltration was observed throughout the exposure as a defensive response to SA. Temporal modulation of glutathione S-transferase (GST), catalase (CAT), and superoxide dismutase (SOD) activities suggested the occurrence of antioxidant and detoxifying responses against SA exposure, while lipid peroxidation (LPO), except for a partial increase at T3, was prevented. Inhibition of the cholinergic system was also reported by reduced acetylcholinesterase (AChE) activity, mainly at T12. Overall, findings from this study contribute to enlarge the current knowledge on the cytotoxicity of SA, on non-target aquatic organisms, and might for the enhancement of new ecopharmacovigilance programs and optimization of the efficacy of wastewater treatment plants for mitigation of pharmaceutical pollution in coastal areas.

Effect of Oxidative Stress on Reproduction and Development.

There is a growing amount of literature on the effects of oxidative stress resulting from the imbalance between prooxidants and antioxidants [...].

Comparative Fatty Acid Profiling of Edible Fishes in Kuala Terengganu, Malaysia.

The aim of this study was to compare the relative nutritional benefit of edible Malaysian fishes from the coast of Terengganu in Malaysia, as well as to perform a taxonomical characterization and metal assessment. Discrimination between species was carried out by a morphological and molecular approach by evaluating the total concentrations of metals by ICP-MS analyses and the fatty acids (FA) composition using the GC-MS approach on the fish fillet tissues. The taxonomical studies detected fishes of 11 families and 13 species. The heavy metal assessment showed that all detected elements did not exceed the regulatory limit stated by Malaysian Food Regulations. The proportion of saturated fatty acids (SFA) ranged from 33 to 58.34%, followed by the polyunsaturated fatty acids (PUFA) values from 24 to 51.8%, and the lowest proportion was of monounsaturated fatty acids (MUFA), ranging from 12.7 to 35.9%. The ω-3/ω-6 PUFA and PUFA/SFA ratios were determined in the range 1.1 to 7.4 and 0.35 to 1.6, respectively. The C20:5 ω-3 and C22:6 ω-3 acids were detected at levels comparable to those found in the corresponding species from similar tropical marine ecosystems. The high FA values can be useful biochemical tools for comparing the relative nutritional benefits of these biodiverse and non-toxic edible Malaysian fishes.

Chemistry of Tropical Eucheumatoids: Potential for Food and Feed Applications.

The use of seaweeds as additives in animal nutrition may be a valid option to traditional feed as they represent a rich source of minerals, carbohydrates and antioxidants. The aim of this study was to analyze the chemical composition and in vitro antioxidant capacity of two tropical eucheumatoids, Kappaphycus alvarezii and Kappaphycus striatus, in Malaysian wild offshore waters. The chemical analysis was performed via inductively coupled plasma-optical emission spectroscopy for evaluating the concentration of toxic (Cd, Pb, Hg, As) and essential elements (Mn, Fe, Cu, Ni, Zn, Se); NMR spectroscopy was used for carrageenans investigation. Furthermore, the soluble and fat-soluble antioxidant capacities were determined by FRAP, DPPH and ABTS assays. The chemical analysis revealed a higher content of trace elements in K. alvarezii as compared to K. striatus, and both exhibited a high mineral content. No significant differences in metal concentrations were found between the two species. Both samples showed a mixture of prevailing κ- and t-carrageenans. Finally, the levels of soluble and fat-soluble antioxidants in K. alvarezii were significantly higher than in K. striatus. Our findings suggest that K. alvarezii could be used as a potential feed additive because of its favorable chemical and nutritional features.

Biological Effects Assessment of Antibiofouling EDCs: Gaeta Harbor (South Italy) Benthic Communities' Analysis by Biodiversity Indices and Quantitative gpx4 Expression.

The most representative organisms of the Harbor of Gaeta Gulf in South Italy were analyzed for biofouling by visual census and confirmed later by molecular approach on an artificial Conatex panel dipped 3 m into a eutrophic area during the Covid-19 pandemic. Mitochondrial Cytochrome C oxidase subunit 1(COI) gene was sequenced from 20 different marine species (flora: 2 families, 2 orders; fauna: 16 families, 11 orders) to test whether the morphology-based assignment of the most common biofouling member was supported by DNA-based species identification. Twelve months of submersion resulted in generation of sufficient data to obtain a facies climax represented mainly by the bivalve mollusk, Mytilus galloprovincialis. Specific diversity and variations of the biofouling biomasses were analyzed using two different anti-biofouling paints: an endocrine disrupting chemical (EDC)-containing metal biocide, and a biocide-free paint. Also, their effects on detoxification and reproductive health of M. galloprovincialis were evaluated using glutathione S-transferase enzymatic activity and RTqPCR expression of the fertility antioxidant gene glutathione peroxidase 4 (gpx4). The obtained data provide useful indications on which future investigations may be focused and may become a potential management tool for a harbor biofouling database to assist local administrations in EDCs protection of autochthonous benthic communities and their fertility using innovative antifouling paints.

Antioxidative Defense and Fertility Rate in the Assessment of Reprotoxicity Risk Posed by Global Warming.

The objective of this review is to briefly summarize the recent progress in studies done on the assessment of reprotoxicity risk posed by global warming for the foundation of strategic tool in ecosystem-based adaptation. The selected animal data analysis that was used in this paper focuses on antioxidative markers and fertility rate estimated over the period 2000-2019. We followed a phylogenetic methodology in order to report data on a panel of selected organisms that show dangerous effects. The oxidative damage studies related to temperature fluctuation occurring in biosentinels of different invertebrate and vertebrate classes show a consistently maintained physiological defense. Furthermore, the results from homeothermic and poikilothermic species in our study highlight the influence of temperature rise on reprotoxicity.

The Ageing Process Affects the Antioxidant Defences and the Poly (ADPribosyl)ation Activity in Cistus Incanus L. Leaves.

The ageing process in living organisms is characterised by the accumulation of several deleterious changes occurring in cells and tissues. The increase of reactive oxygen species with the advancement of age is responsible for the oxidative damage to proteins, lipids and DNA, enhancing the risk of diseases. The antioxidant response and the activation of the poly(ADP-ribosyl)ation process represent the first defences activated by organisms at all life stages to counteract damage to cell structures and genomic material. The regulation of poly(ADP ribosyl)ation with age is little known in plants, especially in combination with antioxidant defences modulation. In this study, the relationships between poly (ADP-ribose) polymerase (PARP) activity and enzymatic and non-enzymatic antioxidant pool have been studied together with the photosynthetic apparatus efficiency in the Mediterranean species Cistus incanus L., examining leaves at different developmental stages: young, mature and senescent. The photosynthetic performance was evaluated by chlorophyll a fluorescence measurement, the total soluble and fat-soluble antioxidant capacity, as well as the activities of enzymes superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and glutathione-S-transferase (GST), were determined by spectrophotometer, PARP activity was assessed by radioactive labelling. The highest photochemical activity was observed in young leaves, together with the highest GST activity. With the progress of the ageing process, the non-enzymatic antioxidant pool (namely ascorbic acid, α-tocopherol) declined, reaching the lowest value in senescent leaves, whereas PARP activity rose significantly. The overall results indicate that the decline of photosynthetic apparatus efficiency during senescence is due to the reduction of specific defences against oxidative damages, which increase the damages to DNA, as demonstrated by PARP activity rise.

Seasonal and pharmaceutical-induced changes in selenoprotein glutathione peroxidase 4 activity in the reproductive dynamics of the soil biosentinel Podarcis sicula (Chordata: Reptilia).

There is rising concern for the interaction of environmental contaminants with brain transcriptome and the potential effect on reproductive processes. The present study sought to determine selenoprotein glutathione peroxidase 4 (gpx4) transcriptional activity in the brain and testis of the soil biosentinel, Podarcis sicula, through the main phases of the reproductive cycle and whether pharmaceuticals exert an endocrine disruption. Based on gpx4 cloned amminoacids sequence (GenBank AEX09236.1.), we used a bioinformatic approach to assess the structural role. Specifically, we detected seasonally the reactive oxygen species (ROS) level using electron spin resonance spectroscopy and gpx4 transcriptional activity using quantitative real-time polymerase chain reaction. In addition, the impact of pharmaceuticals was assessed after 21-days of treatment with ICI 182,780 and human chorionic gonadotropin administration in mating and winter stasis, respectively. Bioinformatic data shows the gpx4 proteic activity and a phylogenetic profile. ROS contents in lizard brain are significantly less than in testis and display higher levels after treatments. Brain gpx4 expression gives statistically significant seasonal differences, opposite trends in testis and altered expression in both tissues, with evidence of testis morphological and DNA disruption. Taken together, these results provide direct evidence that gpx4 in P. sicula plays a seasonal regulatory role and may be a reliable biomarker for reproductive health toxicity screening.

Pharmacokinetics of rectal levetiracetam as add-on treatment in dogs affected by cluster seizures or status epilepticus.

BACKGROUND: Levetiracetam can be used for seizure control alone or in combination with other antiepileptic medications. A previous study achieved the minimum targeted serum drug concentration after rectal administration of levetiracetam in healthy dogs. The purpose of the present study was to determine the pharmacokinetics of rectal LEV in dogs presented for cluster seizures or status epilepticus and potentially in treatment with other anti-epileptic drugs. Furthermore, preliminary information on response to this treatment as add-on to the standard treatment protocol is reported. RESULTS: Eight client-owned dogs were enrolled. Plasma levetiracetam concentrations (measured at 0, 30, 60, 90, 120, 180, 240, 360, 720, and 1440 min after drug administration) reached the minimum target concentration (5 µg/ml) at 30 min in all but one patient. At T1 (30 min) the mean concentration was 28.2 ± 15.5 µg/ml. Plasma concentrations remained above the targeted minimum concentration in all patients until 240 min and in 7/8 until 360 min. Six out of eight patients experienced no seizures in the 24-h period after hospitalization and were classified as "responders". CONCLUSIONS: Minimum plasma levetiracetam concentration can be reached after rectal administration of 40 mg/kg in dogs affected by cluster seizures and status epilepticus and concurrently receiving other antiepileptic drugs. These preliminary results may encourage the evaluation of rectal levetiracetam as an additional treatment option for cluster seizures and status epilepticus in a larger number of dogs.

Protamine-like proteins' analysis as an emerging biotechnique for cadmium impact assessment on male mollusk Mytilus galloprovincialis (Lamarck 1819).

Here we report the industrial pollution effects due to cadmium on the reproductive health of Mytilus galloprovincialis. Mussels were removed from the biofouling of a Conatex panel after one year exposition at a polluted site near a disposal metallurgical factory. A high cadmium bioaccumulation was observed in the testis of mussels housed at the polluted site, with respect to a control site, as determined by inductively coupled plasma-mass spectrometry, along with a 10 fold increase in metallothionein 20 kDa gene (mt20) expression levels determined by qPCR. Furthermore, mussels transferred into laboratory tanks from the reference site, and exposed to 1.5, 5 and 10 µM CdCl2, revealed a 1.7, 3.2 and 4.5 fold expression increase in the testis mt20, respectively, and a positive correlation with cadmium bioaccumulation was found. To evaluate a potential detrimental risk of such alterations on spermatozoa, we carried out electrophoretic analyses on their protamine-like proteins. As determined by AU-PAGE, after 1.5 µM CdCl2 exposure, protamine-like proteins also display major alterations with respect to those obtained after 5 and 10 µM CdCl2 exposure. All protamine-like proteins isolated from the polluted biofouling were in an aggregated form and displayed the same reduced DNA binding affinity of the protamine-like proteins obtained after 1.5 µM CdCl2 as demonstrated EMSA with sperm genomic DNA. Our results contribute to the studies concerning cadmium induced testis alterations and highlight protamine-like proteins' analysis as an emerging biotechnique for cadmium impact assessment on Mytilus galloprovincialis, for the sensitivity of the in vivo and in vitro changes of protamine-like proteins' state and their DNA binding affinity.

Baseline levels of metabolites in different tissues of mussel Mytilus galloprovincialis (Bivalvia: Mytilidae).

The Mediterranean mussel Mytilus galloprovincialis (Lamarck 1819) is a popular shellfish commonly included in human diet and is routinely used as bioindicator in environmental monitoring programmes worldwide. Recently, metabolomics has emerged as a powerful tool both in food research and ecotoxicology for monitoring mussels' freshness and assessing the effects of environmental changes. However, there is still a paucity of data on complete metabolic baseline of mussel tissues. To mitigate this knowledge gap, similarities and differences in metabolite profile of digestive gland (DG), gills (G), and posterior adductor muscle (PAM) of aquaculture-farmed M. galloprovincialis were herein investigated by a proton nuclear magnetic resonance (1H NMR)-based metabolomic approach and discussed considering their physiological role. A total of 44 metabolites were identified in mussel tissues and grouped in amino acids, energy metabolites, osmolytes, neurotransmitters, nucleotides, alkaloids, and miscellaneous metabolites. A PCA showed that mussel tissues clustered separately from each other, suggesting a clear differentiation in their metabolic profiles. A Venn diagram revealed that mussel DG, G and PAM shared 27 (61.36%) common metabolites, though with different concentrations. Osmolytes were found to dominate the metabolome of all tissues. The DG exhibited higher level of glutathione and carbohydrates. The G showed greater level of osmolytes and the exclusive presence of neurotransmitters, namely acetylcholine and serotonin. In PAM higher levels of energetics-related metabolites were found. Overall, findings from this study are helpful for a better understanding of mussel tissue-specific physiological functions as well as for future NMR-based metabolomic investigations of marine mussel health and safety.