Valentina Notarstefano

Microplastics are particles smaller than five millimeters deriving from the degradation of plastic objects present in the environment. Microplastics can move from the environment to living organisms, including mammals. In this study, six human placentas, collected from consenting women with physiological pregnancies, were analyzed by Raman Microspectroscopy to evaluate the presence of microplastics. In total, 12 microplastic fragments (ranging from 5 to 10 μm in size), with spheric or irregular shape were found in 4 placentas (5 in the fetal side, 4 in the maternal side and 3 in the chorioamniotic membranes); all microplastics particles were characterized in terms of morphology and chemical composition. All of them were pigmented; three were identified as stained polypropylene a thermoplastic polymer, while for the other nine it was possible to identify only the pigments, which were all used for man-made coatings, paints, adhesives, plasters, finger paints, polymers and cosmetics and personal care products.

The widespread use of plastics determines the inevitable human exposure to its by-products, including microplastics (MPs), which enter the human organism mainly by ingestion, inhalation, and dermal contact. Once internalised, MPs may pass across cell membranes and translocate to different body sites, triggering specific cellular mechanisms. Hence, the potential health impairment caused by the internalisation and accumulation of MPs is of prime concern, as confirmed by numerous studies reporting evident toxic effects in various animal models, marine organisms, and human cell lines. In this pilot single-centre observational prospective study, human breastmilk samples collected from N. 34 women were analysed by Raman Microspectroscopy, and, for the first time, MP contamination was found in 26 out of 34 samples. The detected microparticles were classified according to their shape, colour, dimensions, and chemical composition. The most abundant MPs were composed of polyethylene, polyvinyl chloride, and polypropylene, with sizes ranging from 2 to 12 µm. MP data were statistically analysed in relation to specific patients' data (age, use of personal care products containing plastic compounds, and consumption of fish/shellfish, beverages, and food in plastic packaging), but no significant relationship was found, suggesting that the ubiquitous MP presence makes human exposure inevitable.

The ubiquitous presence of microplastics (MPs) and their health effects is a recent scientific topic. However, the investigation of MPs in human/biological matrices has several limitations due to analytical methods and sample treatment protocols. In this study, the presence of MPs in the urine samples of six volunteers from different cities in the south of Italy (three men and three women) was investigated by Raman microspectroscopy. The analysis pinpointed four pigmented microplastic fragments (4-15 μm size), with irregular shapes, which were characterized in terms of morphology and chemical composition. Polyethylene vinyl acetate (PVA), polyvinyl chloride (PVC), polypropylene (PP), and polyethylene (PE) MPs were found in four samples (PVA and PVC in one female sample and PP and PE in three male samples). This preliminary study suggests that MPs could pass through the gastrointestinal tract and are eliminated through biological processes.

Microplastics (MPs) are defined as plastic particles smaller than 5 mm. They have been found almost everywhere they have been searched for and recent discoveries have also demonstrated their presence in human placenta, blood, meconium, and breastmilk, but their location and toxicity to humans have not been reported to date. The aim of this study was twofold: 1. To locate MPs within the intra/extracellular compartment in human placenta. 2. To understand whether their presence and location are associated with possible structural changes of cell organelles. Using variable pressure scanning electron microscopy and transmission electron microscopy, MPs have been localized in ten human placentas. In this study, we demonstrated for the first time the presence and localization in the cellular compartment of fragments compatible with MPs in the human placenta and we hypothesized a possible correlation between their presence and important ultrastructural alterations of some intracytoplasmic organelles (mitochondria and endoplasmic reticulum). These alterations have never been reported in normal healthy term pregnancies until today. They could be the result of a prolonged attempt to remove and destroy the plastic particles inside the placental tissue. The presence of virtually indestructible particles in term human placenta could contribute to the activation of pathological traits, such as oxidative stress, apoptosis, and inflammation, characteristic of metabolic disorders underlying obesity, diabetes, and metabolic syndrome and partially accounting for the recent epidemic of non-communicable diseases.

The presence of microplastics (MPs) in human fluids and organs is a great concern, since, as highlighted by recent studies on animal models, they could cause alterations of several physiological functions, including reproduction. In this study, semen samples collected from men living in a polluted area of the Campania Region (Southern Italy), were analyzed to assess the presence of MPs. N. 16 pigmented microplastic fragments (ranging from 2 to 6 μm in size) with spheric or irregular shapes were found in six out of ten samples. All the detected MPs were characterized in terms of morphology (size, colour, and shape) and chemical composition by Raman Microspectroscopy. Chemical composition showed the presence of polypropylene (PP), polyethylene (PE), polyethylene terephthalate (PET), polystyrene (PS), polyvinylchloride (PVC), polycarbonate (PC), polyoxymethylene (POM) and acrylic, suggesting ingestion and/or inhalation as a route of exposure to environmental MPs. In this work, we propose for the first time a mechanism by which MPs pass into the semen most likely through the epididymis and seminal vesicles, which are the most susceptible to inflammation.

Abstract Black Soldier Fly (BSF) meal is considered as an alternative, emerging and sustainable ingredient for aquafeed production. However, results on fish physiological responses are still fragmentary and often controversial, while no studies are available on fish behavior in response to these new diets. The present work represents the first comprehensive multidisciplinary study aimed to investigate zebrafish physiological and behavioural responses to BSF-based diets. Five experimental diets characterized by increasing inclusion levels (0, 25, 50, 75 and 100% respect to fish meal) of full fat BSF prepupae meal were tested during a 2-months feeding trial. Prepupae were cultured on coffee silverskin growth substrate enriched with a 10% Schizochytrium sp. to improve insects’ fatty acids profile. The responses of zebrafish were assayed through biometric, histological, gas chromatographic, microbiological, spectroscopic, molecular and behavioural analyses. Results evidenced that BSF-based diets affected fish fatty acid composition, while behavioural tests did not show differences among groups. Specifically, a 50% BSF inclusion level diet represented the best compromise between ingredient sustainability and proper fish growth and welfare. Fish fed with higher BSF inclusions (75 and 100%) showed hepatic steatosis, microbiota modification, higher lipid content, fatty acid modification and higher expression of immune response markers.

A major challenge for development of sustainable aquafeeds is its dependence on fish meal and fish oil. Replacement with more sustainable, nutritious and safe ingredients is now a priority. Over the last years, among several alternatives proposed, insects have received great attention as possible candidates. In particular, the black soldier fly ( Hermetia illucens ; BSF) represents a concrete example of how the circular economy concept can be applied to fish culture, providing a valuable biomass rich in fat and protein valorising organic by-products. In the last decade, several studies have been published about the use of different BSF dietary inclusion levels for various fish species including experimental models. Varying and encouraging results have been obtained in this research field using a plethora of laboratory methodological approaches that can be applied and coupled to obtain a comprehensive view of the BSF-based diets effects on fish physiology, health, and quality. The present review aims to explore some of the most promising laboratory approaches like histology, infrared spectroscopy, gut microbiome sequencing, molecular biology, fish fillets’ physico-chemical and sensory properties, essential for a better understanding of fish welfare and fillet quality, when BSF is used as aquafeed ingredient. In particular, great importance has been given to European finfish species and experimental models.

Reliable point-of-care (POC) rapid tests are crucial to detect infection and contain the spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The emergence of several variants of concern (VOC) can reduce binding affinity to diagnostic antibodies, limiting the efficacy of the currently adopted tests, while showing unaltered or increased affinity for the host receptor, angiotensin converting enzyme 2 (ACE2). We present a graphene field-effect transistor (gFET) biosensor design, which exploits the Spike-ACE2 interaction, the crucial step for SARS-CoV-2 infection. Extensive computational analyses show that a chimeric ACE2-Fragment crystallizable (ACE2-Fc) construct mimics the native receptor dimeric conformation. ACE2-Fc functionalized gFET allows in vitro detection of the trimeric Spike protein, outperforming functionalization with a diagnostic antibody or with the soluble ACE2 portion, resulting in a sensitivity of 20 pg/mL. Our miniaturized POC biosensor successfully detects B.1.610 (pre-VOC), Alpha, Beta, Gamma, Delta, Omicron (i.e., BA.1, BA.2, BA.4, BA.5, BA.2.75 and BQ.1) variants in isolated viruses and patient's clinical nasopharyngeal swabs. The biosensor reached a Limit Of Detection (LOD) of 65 cps/mL in swab specimens of Omicron BA.5. Our approach paves the way for a new and reusable class of highly sensitive, rapid and variant-robust SARS-CoV-2 detection systems.

The Mediterranean Sea is particularly endangered by microplastics (MPs), polymers, and additives. These contaminants can be ingested by fishes and, hence, translocate into tissues. We aimed to quantify MPs, polyethylene terephthalate (PET), polycarbonate (PC), bisphenol A (BPA), and p-phthalic acid (PTA) in the edible muscles of swordfish (Xiphias gladius) and bluefin tuna (Thunnus thynnus) caught in the Mediterranean Sea. The MPs were extracted from muscles and characterized by stereomicroscopy and Raman microspectroscopy, while the polymers (PET and PC) and additives (BPA and PTA) were identified by LC-MS/MS. The number of MPs ranged from 140 to 270 no. kg−1 in swordfish and from 160 to 270 no. kg−1 in tuna. The most frequent MP polymer was polypropylene in swordfish (33%) and in tuna (34.7%), while the most abundant pigments were PB115, PB116, PBr101/102. A similar level of plastic contamination was revealed in these two fish species with differences in shapes, colors, pigments and polymers of MPs.

Many man-made chemical compounds are recognized as endocrine disruptors and once released into the environment are likely to spread and bioaccumulate in wild species. Due to their lipophilic nature, these substances pass through the cell membrane or bind to specific receptors activating physiological responses that in the long run can cause reproductive impairment, physiological disorders, including the occurrence of metabolic syndromes. One significant source of contamination is represented by the consumption of polluted food. As a consequence, different environmental pollutants, with similar or different modes of action, can accumulate in organisms and biomagnify along the food web, finally targeting humans. The aim of this study was to analyze, under controlled conditions, the effects induced by the consumption of contaminated diets, focusing on the effects exerted at hepatic level. Juvenile seabream were fed for 21days a diet enriched with different combinations of pollutants, nonylphenol (NP), tert-octylphenol (t-OP) and bisphenol A (BPA). The different diets containing 5mg/kg bw of each contaminant, were formulated as follows: NP+tOP, BPA+NP, BPA+tOP and NP+BPA+tOP (NBO). EDCs, at the doses administered, showed low biomagnification factor (BMF), suggesting that these pollutants hardly accumulate in muscles. The results obtained at hepatic level pinpointed the steatotic effect of all the administered diets, associated to a modulation of the expression of genes involved in lipid metabolism (ppars, fas, lpl, and hsl). Results were compared to those obtained in previous studies in which fish were fed single pollutants evidencing that the administration of mixture of contaminants exerts a milder lipogenic effect, highlighting the contrasting/antagonistic interaction establishing among chemicals. Noteworthy was the setup of a new chromatographic method to detect the presence of the selected chemical in fish muscle and the application of Fourier Transform Infrared (FT-IR) analysis to evaluate pollutant-induced changes in the liver macromolecular building.

Endocrine disrupting chemicals (EDCs) are known to disrupt normal metabolism and can influence the incidence of obesity in animals and humans. EDCs can exert adverse effects at low concentrations, often in a non-monotonic dose-related fashion. Among EDCs, Bisphenol A (BPA) is extensively used in the production of polycarbonate plastic, and is among the most abundant contaminants in the world. Diethylene glycol dibenzoate (DGB), an approved alternative to phthalates in the production of plastic and latex products, however, is less abundant and its effects are almost completely unknown. The aim of this study is to provide information on the hepatic effects of BPA and DGB on lipid metabolism, and investigate possible links between these contaminants and the increased incidence of obesity. In the present study, we exposed zebrafish to three different BPA doses (5; 10; 20 μg/L) and five different doses of DGB (0.01; 0.1; 1; 10; 100 μg/L) for a period of 21 days, and investigated transcript levels for genes involved in lipid metabolism as well as measuring liver content of phosphates, lipids and proteins. The results demonstrate disruptive effects of BPA and DGB on lipid metabolism in a non-monotonic dose-related fashion. The lowest dose of BPA increased the storage of triglycerides and promoted fatty acid synthesis, while the highest concentration promoted de novo lipogenesis and cholesterologenesis. Exposure to DGB was also found to affect lipid metabolism leading to increased lipid production and mobilization in a non-monotonic dose-related fashion. Analysis of BPA and DGB by FT-IR revealed that exposure to both compounds lead to changes in the biochemical composition of liver. The findings provide a support for the hypothesis that BPA and DGB may be among the environmental contaminants with obesogenic property.

Abstract Fourier‐transform infrared hyperspectral imaging (FTIR‐HSI) provides hyperspectral images containing both morphological and chemical information. It is widely applied in the biomedical field to detect tumor lesions, even at the early stage, by identifying specific spectral biomarkers. Pancreatic neoplasms present different prognoses and are not always easily classified by conventional analyses. In this study, tissue samples with diagnosis of pancreatic ductal adenocarcinoma and pancreatic neuroendocrine tumor were analyzed by FTIR‐HSI and the spectral data compared with those from healthy and dysplastic samples. Multivariate/univariate approaches were complemented to hyperspectral images, and definite spectral markers of the different lesions identified. The malignant lesions were recognizable both from healthy/dysplastic pancreatic tissues (high values of phospholipids and triglycerides with shorter, more branched and less unsaturated alkyl chains) and between each other (different amounts of total lipids, phosphates and carbohydrates). These findings highlight different metabolic pathways characterizing the different samples, well detectable by FTIR‐HSI.

Abstract Loggerhead sea turtle ( C. caretta ) is the official European bioindicator of marine litter in the Mediterranean Sea. In 2019, 8 sea turtles, out of 28 specimens loggerhead on the Adriatic coast of Molise, were subjected to necropsy. The intestinal contents were collected and the microplastics until 0.45 μm were extracted. Qualitative and quantitative assessments were performed by stereomicroscope observation and spectroscopic analyses (attenuated total reflection-Fourier transform infrared spectroscopy, ATR-FTIR and Raman microspectroscopy, RMS). Moreover, the analytical quantification of polyethylene terephthalate (PET), polycarbonate (PC), para phthalic acid (PTA) and bisphenol A (BPA) in fat and liver tissues was performed by LC-MS/MS. Microparticles ranging from 0.45 μm to 1 mm were found in all turtles, for a total of 623, while plastic litter greater than 1 mm were found only in 4 specimens (ranging from 0.03 to 0.11 g). Nineteen different polymers and 10 pigments, including polyester (100% of animals), high-density polyethylene (50%) and polypropylene (50%) were identified. BPA, PTA and PET were detected in fat and liver tissues of all animals, while PC was found only in 50%. A major prevalence was registered in the abdominal fat tissue, although only PC compounds were significantly higher in abdominal tissue ( p < 0.05), except for free PTA with liver tissue being the most contaminated ( p < 0.05). Microplastics and additives surely impact the health status of turtles that showed gastrointestinal impairment and an important level of contamination in tissues. Graphical abstract

Di-isononyl phthalate (DiNP) is a high molecular weight phthalate commonly used as a plasticizer. It was introduced as a replacement for bis (2-ethylhexyl) phthalate (DEHP) which is used in the production of plasticized polyvinyl chloride (PVC). The purpose of this study was to investigate for the first time the effect of DiNP on female reproductive physiology in Danio rerio. Fish were exposed to five different doses of DiNP plus control (0 μg/L; 0.42 μg/L; 4.2 μg/L; 42 μg/L; 420 μg/L; 4200 μg/L) for a period of 21 days. We evaluated fish fecundity, oocyte growth, autophagic and apoptotic processes, as well as changes in morphological and biochemical composition of oocytes, using, qPCR analysis, histology and Fourier transform infrared imaging. The results demonstrate a non-monotonic dose response to DiNP. Greater differences were observed at the lowest (0.42 μg/L) and higher concentrations (420 μg/L; 4200 μg/L) of DiNP. The findings provide evidence that exposure to DiNP adversely affect oocytes growth and maturation, leading to abnormal gonadal development and reproduction in zebrafish.

Endometriosis is a chronic gynaecological disease characterised by the presence of endometrial cells in extra-uterine regions. One of the main factors impacting on the fertility of women affected by endometriosis is the poor oocyte quality. Granulosa Cells (GCs) regulate oocyte development and maintain the appropriate microenvironment for the acquisition of its competence; hence, the dysregulation of these functions in GCs can lead to severe cellular damages also in oocytes. In this study, luteinized GCs samples were separately collected from both ovaries of women affected by Unilateral Ovarian Endometriosis and analysed by infrared and Raman microspectroscopy. The spectral data were compared with those of GCs from women with diagnosis of tubal, idiopathic or male infertility (taken as control group). The coupling of these two spectroscopic techniques sheds new light on the alteration induced by this pathology on GCs metabolism and biochemical composition. In fact, the study revealed similar biochemical modifications in GCs from both ovaries of women affected by unilateral ovarian endometriosis, such as the alteration of the protein pattern, the induction of oxidative stress mechanisms, and the deregulation of lipid and carbohydrate metabolisms. These evidences suggest that unilateral endometriosis impairs the overall ovarian functions, causing alterations not only in the ovary with endometriotic lesions but also in the contralateral "healthy" one.

Tooth whitening is one of the most requested dental treatments, but it still presents some side effects. Indeed, the bleaching agent can generate patients' discomfort and dental hard tissue damages, not achieving an efficient and long-lasting treatment with optimum whitening effect. To overcome these limitations, the bleaching agents containing nano-hydroxyapatite can represent a reliable solution to avoid these detrimental effects.In this study, human third molars were treated with commercial bleaching agents, containing nano-hydroxyapatite (nHA) and 6% (at-home treatment), 12% and 18% (in-office treatments) of hydrogen peroxide (HP), named respectively G1, G2 and G3. The results were evaluated descriptively and analytically using Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy (ATR-FTIR), Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray spectroscopy (EDS), comparing the treated groups with a commercial gel containing 10% carbamide peroxide (CONV) and with a non-treated control group (CTRL).ATR-FTIR analysis revealed a similar composition in carbonates and phosphates for CTRL, G1 and G2 groups, which showed no significant differences in their spectral profiles; an increased amount of organic matter was found in G3, while CONV displayed an intermediate behavior. SEM analysis did not highlight significant changes in the enamel microstructure of G1 and CONV when compared to CTRL; the pattern observed in G2 presented a slight increase of enamel irregularities, while G3 displayed a partial removal of the aprismatic layer and microporosities. No evident effects due to nHA were observed in the structure of the hydroxyapatite component of G1, G2 and CONV, if compared to CTRL, while G3 showed a slight loss of crystallinity. In all groups, EDS identified slight changes in the concentration of chemical elements O and Ca, while the Ca/P ratio was similar when compared to CTRL.The obtained results suggest that the application of the tested commercial bleaching agents, with a concentration of HP up to 12%, does not alter the morphological and chemical composition of the enamel surface and maintains its crystallinity.

Abstract Over the last years, the potential use of Black Soldier Fly meal (BSF) as a new and sustainable aquafeed ingredient has been largely explored in several fish species. However, only fragmentary information is available about the use of BSF meal-based diets in sturgeon nutrition. In consideration of a circular economy concept and a more sustainable aquaculture development, the present research represents the first comprehensive multidisciplinary study on the physiological effects of a BSF diet during sturgeon culture in an aquaponic system. Siberian sturgeon ( Acipenser baerii ) juveniles were fed over a 60-days feeding trial on a control diet (Hi0) and a diet containing 50% of full-fat BSF meal respect to fish meal (Hi50). Physiological responses of fish were investigated using several analytical approaches, such as gas chromatography-mass spectrometry, histology, Fourier Transformed Infrared Spectroscopy (FTIR), microbiome sequencing and Real-time PCR. While aquaponic systems performed optimally during the trial, Hi50 group fish showed lower diet acceptance that resulted in growth and survival reduction, a decrease in hepatic lipids and glycogen content (FTIR), a higher hepatic hsp70.1 gene expression and a worsening in gut histological morphometric parameters. The low feed acceptance showed by Hi50 group sturgeon highlighted the necessity to improve the palatability of BSF-based diet designed for sturgeon culture.

Teeth are characterized by a specific chemical composition and microstructure, which are related to their nature, permanent and deciduous, and to the sides, lingual and vestibular. Deeper knowledge in this topic could be useful in clinical practice to develop new strategies in restorative dentistry and in the choice of materials with the best performances. In this study, Raman MicroSpectroscopy (RMS), Scanning Electron Microscopy (SEM), Energy Dispersive X-ray Spectrometry (EDS), and Vickers MicroHardness (VMH) were exploited to: (1) identify the microstructure and the chemical/elemental composition of permanent and deciduous human teeth, also characterizing their lingual and vestibular sides, and (2) validate a new multidisciplinary analytical approach, for obtaining multiple information on calcified tissues. All applied techniques evidenced differences between permanent and deciduous teeth both in the lingual and vestibular sides. In particular, scanning electron micrographs identified areas with an irregular appearance in the vestibular and lingual sides, which presented also different VMH values. Moreover, RMS and EDS displayed a different chemical/elemental composition in outer and inner enamel and dentin, in terms of Mineral/Matrix, Crystallinity, Carbonates/phosphates, and concentrations by weight (%) of calcium, phosphorous, carbon, magnesium, and sodium. A good linear correlation was found between RMS spectral profiles and EDS and VMH measurements, suggesting that RMS may be considered a useful and non-destructive diagnostic tool for obtaining multiple information on calcified tissues.

We herein report the synthesis and multi-technique characterization of [Ru2Cl((2-phenylindol-3-yl)glyoxyl-l-leucine-l-phenylalanine)4], a novel diruthenium(ii,iii) complex obtained by reacting [Ru2(μ-O2CCH3)4Cl] with a dual indolylglyoxylyl dipeptide anticancer agent. We soon realised that the compound is very stable under several different conditions including aqueous buffers or organic solvents. It is also completely unreactive toward proteins. The high stability is also suggested by cellular experiments in a glioblastoma cell line. Indeed, while the parent ligand exerts high cytotoxic effects in the low μM range, the complex is completely non-cytotoxic against the same line, most probably because of the lack of ligand release. To investigate the reasons for such high stability, we carried out DFT calculations that are fully consistent with the experimental findings. The results highlight that the stability of [Ru2Cl((2-phenylindol-3-yl)glyoxyl-l-leucine-l-phenylalanine)4] relies on the nature of the ligand, including its steric hindrance that prevents the reaction of any nucleophilic group with the Ru2 core. Ligand displacement is the key step to allow reactivity with the biological targets of metal-based prodrugs. Accordingly, we discuss the implications of some important aspects that should be considered when active molecules are chosen as ligands for the synthesis of paddle-wheel-like complexes with medicinal applications.

In the present study, the cytotoxic effects of 5-azacytidine on primary Oral Squamous Cell Carcinoma cells (OSCCs) from human biopsies, and on Cancer Stem Cells (CSCs) from the same samples, were investigated by an in vitro Fourier Transform InfraRed Microscospectroscopy (FTIRM) approach coupled with multivariate analysis. OSCC is an aggressive tumoral lesion of the epithelium, accounting for ~90% of all oral cancers. It is usually diagnosed in advanced stages, and this causes a poor prognosis with low success rates of surgical, as well as radiation and chemotherapy treatments. OSCC is frequently characterised by recurrence after chemotherapy and by the development of a refractoriness to some employed drugs, which is probably ascribable to the presence of CSCs niches, responsible for cancer growth, chemoresistance and metastasis. The spectral information from FTIRM was correlated with the outcomes of cytotoxicity tests and image-based cytometry, and specific spectral signatures attributable to 5-azacytidine treatment were identified, allowing us to hypothesise the demethylation of DNA and, hence, an increase in the transcriptional activity, together with a conformational transition of DNA, and a triggering of cell death by an apoptosis mechanism. Moreover, a different mechanism of action between OSSC and CSC cells was highlighted, probably due to possible differences between OSCCs and CSCs response.

Oral squamous cell carcinoma represents the most aggressive and frequent form of head and neck cancer. Due to drug resistance, the 5-year survival rate of patients with advanced disease is less than 50%. In order to identify molecular targets for effective oral cancer treatment, we focused on paraoxonase-2 enzyme. Indeed, based on data previously obtained from preliminary immunohistochemistry and Western blot analyses performed on tissue specimens, the enzyme was found to be upregulated in tumor compared with normal oral mucosa. Therefore, paraoxonase-2 gene silencing was achieved in HSC-3 and HOC621 oral cancer cell lines, and the effect on cell proliferation, viability, apoptosis induction and sensitivity to cisplatin and 5-fluorouracil treatment was evaluated. Fourier Transform InfraRed Microspectroscopy analyzed alterations of cellular macromolecules upon treatment. Enzyme level and cell proliferation were also determined in cisplatin-resistant clones obtained from HOC621 cell line, as well as in parental cells. Reported data showed that paraoxonase-2 knockdown led to a reduction of cell proliferation and viability, as well as to an enhancement of sensitivity to cisplatin, together with the activation of apoptosis pathway. Spectroscopical data demonstrated that, under treatment with cisplatin, oxidative damage exerted on lipids and proteins was markedly more evident in cells down-regulating paraoxonase-2 compared to controls. Interestingly, enzyme expression, as well as cell proliferation were significantly higher in cisplatin-resistant compared with control HOC621 cells. Taken together these results seem to candidate the enzyme as a promising target for molecular treatment of this neoplasm.

Abstract Bone homeostasis is the equilibrium between organic and inorganic components of the extracellular matrix (ECM) and cells. Alteration of this balance has consequences on bone mass and architecture, resulting in conditions such as osteoporosis (OP). Given ECM protein mutual regulation and their effects on bone structure and mineralization, further insight into their expression is crucial to understanding bone biology under normal and pathological conditions. This study focused on Type I Collagen, which is mainly responsible for structural properties and mineralization of bone, and selected proteins implicated in matrix composition, mineral deposition, and cell‐matrix interaction such as Decorin, Osteocalcin, Osteopontin, Bone Sialoprotein 2, Osteonectin and Transforming Growth Factor beta. We developed a novel multidisciplinary approach in order to assess bone matrix in healthy and OP conditions more comprehensively by exploiting the Fourier Transform Infrared Imaging (FTIRI) technique combined with histomorphometry, Sirius Red staining, immunohistochemistry, and Western Blotting. This innovatory procedure allowed for the analysis of superimposed tissue sections and revealed that the alterations in OP bone tissue architecture were associated with warped Type I Collagen structure and deposition but not with changes in the total protein amount. The detected changes in the expression and/or cooperative or antagonist role of Decorin, Osteocalcin, Osteopontin, and Bone Sialoprotein‐2 indicate the deep impact of these NCPs on collagen features of OP bone. Overall, our strategy may represent a starting point for designing targeted clinical strategies aimed at bone mass preservation and sustain the FTIRI translational capability as upcoming support for traditional diagnostic methods.

Clear orthodontic aligners have recently seen increasing popularity. The thermoplastic materials present several advantages, even if it is known that all plastic products can be subjected to environmental and mechanical degradation, leading to the release of microplastics (MPs). Their ingestion could cause oxidative stress and inflammatory lesions. This study aims to evaluate the potential detachment of MPs by clear aligners due to mechanical friction simulated with a 7-day protocol in artificial saliva. The study was performed on orthodontic clear aligners from different manufacturers: Alleo (AL); FlexiLigner (FL); F22 Aligner (F22); Invisalign® (INV); Lineo (LIN); Arc Angel (ARC), and Ortobel Aligner (OR). For each group, two aligners were immersed in artificial saliva for 7 days and stirred for 5 h/day, simulating the physiological teeth mechanical friction. After 7 days, the artificial saliva was filtered; then, filters were analyzed by Raman Microspectroscopy (RMS) and Scanning Electron Microscopy (SEM), respectively to chemically identify the polymeric matrix and to measure the number and size of the detected MPs. RMS spectra revealed that AL, FL, LIN, ARC, and OR aligners were composed by polyethylene terephthalate, while F22 and INV ones by polyurethane. SEM analysis showed that the highest number of MPs was found in ARC and the lowest in INV (p < 0.05). As regards MPs' size, no statistically significant difference was found among groups, with most MPs ranging from 5 to 20 μm. Noteworthy, a highly significant correlation (p < 0.0001) was highlighted between the distribution of MPs size and the different typologies of aligners. This in vitro study highlighted for the first time the detachment of MPs from clear aligners due to mechanical friction. This evidence may represent a great concern in the clinical practice since it could impact human general health.

A nanodelivery system based on palladium nanoparticles (PdNP) and cisplatin (CisPt) was developed by physisorption of the drug onto the PdNP synthesized via a green redox process, using d-glucose and polyvinylpyrrolidone (PVP) as reducing and stabilizing/capping agents, respectively. UV-vis analysis and H2-evolution measurements were carried out to prove the nanoparticles' capability to act as bimodal theranostic nanomedicine, i.e., having both plasmonic and photocatalytic properties. XPS, XRD, and TEM allowed light to be shed on the chemical composition and morphology of the PdNP. The analysis of the UV-visible spectra evidenced plasmonic peak changes for the hybrid nanoparticle-drug assembly (Pd@CisPt), which pointed to a significant interaction of CisPt with the NP surface. The drug loading was quantitatively estimated by ICP-OES measurements, while DLS and AFM confirmed the strong association of the drug with the nanoparticle surface. The test of SOD-like activity in a cell-free environment proved the maintenance of the antioxidant capability of PdNP also in the Pd@CisPt systems. Finally, Pd@CisPt tested in prostate cancer cells (PC-3 line) unveiled the antitumoral action of the developed nanomedicine, related to reactive oxygen species (ROS) generation, with a condition of protein misfolding/unfolding and DNA damage, as evidenced by cytotoxicity and MitoSOX assays, as well as Raman microspectroscopy, respectively. Cell imaging by confocal microscopy evidenced cellular uptake of the nanoparticles, as well as dynamic processes of copper ion accumulation at the level of subcellular compartments. Finally, cell migration studies upon treatment with Pd@CisPt evidenced a tunable response between the inhibitory effect of CisPt and the enhanced rate of cell migration for the metal NP alone, which pointed out the promising potential of the developed theranostic nanomedicine in tissue regeneration.

Oral Squamous Cells Carcinoma (OSCC) is characterised by the risk of recurrence and the onset of a refractoriness response to chemotherapy drugs.

Collagen is one of the main components of the extracellular matrix (ECM), involved, among all, in the maintenance of the structural support of tissues. In fibrotic diseases, collagen is overexpressed, and its production determines the formation of a significantly stiffer ECM. The cross-linking of high-resolution analytical tools, able to investigate both the tridimensional organization and the secondary structure of collagen in fibrotic diseases, could be useful to identify defined markers correlating the status of this protein with specific pathological conditions. To this purpose, an innovative multidisciplinary approach based on Phase-Contrast MicroComputed Tomography, Transmission Electron Microscopy, and Fourier Transform Infrared Imaging Spectroscopy was exploited on leiomyoma samples and adjacent myometrium to characterize microstructural collagen features. Uterine leiomyoma is a common gynecological disorder affecting women in fertile age. It is characterized by a massive collagen production due to the repairing processes occurring at myometrium level, and, hence, it represents a valuable model to investigate collagen self-organization in a pathological condition. Moreover, to evaluate the sensitivity of this multidisciplinary approach, the effects of eicosapentaenoic (EPA) and docosahexaenoic (DHA) omega-3 fatty acids in collagen reduction were also investigated.

The pleiotropic effects of glucocorticoids in metabolic, developmental, immune and stress response processes have been extensively investigated; conversely, their roles in reproduction are still less documented. It is well known that stress or long-lasting therapies can cause a strong increase in these hormones, negatively affecting reproduction. Moreover, the need of glucocorticoid (GC) homeostatic levels is highlighted by the reduced fertility reported in the zebrafish glucocorticoid receptor mutant (nr3c1ia30/ia30) line (hereafter named gr−/−). Starting from such evidence, in this study, we have investigated the role of glucocorticoid receptor (Gr) in the reproduction of female zebrafish. Key signals orchestrating the reproductive process at the brain, liver, and ovarian levels were analyzed using a multidisciplinary approach. An impairment of the kiss-GnRH system was observed at the central level in (gr−/−) mutants as compared to wild-type (wt) females while, in the liver, vitellogenin (vtg) mRNA transcription was not affected. Changes were instead observed in the ovary, particularly in maturing and fully grown follicles (classes III and IV), as documented by the mRNA levels of signals involved in oocyte maturation and ovulation. Follicles isolated from gr−/− females displayed a decreased level of signals involved in the acquisition of competence and maturation, causing a reduction in ovulation with respect to wt females. Fourier transform infrared imaging (FTIRI) analysis of gr−/− follicle cytoplasm showed major changes in macromolecule abundance and distribution with a clear alteration of oocyte composition. Finally, differences in the molecular structure of the zona radiata layer of gr−/− follicles are likely to contribute to the reduced fertilization rate observed in mutants.

Preeclampsia is a human pregnancy-specific disease characterized by abnormal placentation that usually presents with maternal hypertension and proteinuria. The main hallmark of preeclampsia, impaired trophoblast migration, and the subsequent disruption of uterine arteries remodeling lead to several molecular alterations in the placental compartments with those occurring in the chorionic villi being of the utmost importance. Given the essential role of the endocannabinoid system during preimplantation and trophoblast migration, we have combined the histological and hyperspectral imaging analyses to shed light on the involvement of two cannabinoid receptors in the macromolecular alterations related to preeclampsia. The results obtained by immunohistochemistry showed a significant increase in the protein levels of cannabinoid receptor 1 (CB1) in the preeclamptic chorionic villi. However, no changes were reported regarding transient receptor potential vanilloid 1 (TRPV-1) levels either in the bulk placental samples or chorionic villi when comparing control and preeclamptic patients. Histological analysis and Fourier-transform infrared spectroscopy (FTIRI) showed an increase in collagen deposition together with higher levels of lipid peroxidation and phosphorylated compounds in the pathological villi. Since CB1 enhancement has been described as promoting fibrosis and oxidative stress in several tissues, we proposed that the higher receptor abundance in preeclampsia could be triggering similar molecular effects in preeclamptic term placentas.

During their life, women are likely to develop uterine diseases, which often compromise their fertile and perimenopausal age. Besides benign lesions like leiomyomas, several malignant neoplasms can occur, such as the uterine leiomyosarcoma, which represents the most frequent malignancy among the rarest uterine cancers. It presents several variants similar to both benign and malignant neoplasms, and sometimes it shares symptoms with the benign counterpart. In this scenario, for a correct diagnosis and a successful prognosis, it is mandatory to detect new reliable markers which strengthen histopathological outcomes and let define a more appropriate and less harmful therapy. Based on this concerning evidence, in the present study, Fourier Transform Infrared Microspectroscopy has been exploited at a cellular level on uterine leiomyoma and leiomyosarcoma cell lines to (1) identify specific spectral biomarkers able to distinguish between benign and malignant lesions, and (2) evaluate the efficacy of eicosapentaenoic and docosahexaenoic acids (respectively EPA and DHA), already successfully tested. Results evidenced reliable differences in the spectral signature of benign and malignant cells, mainly in terms of lipids and nucleic acids composition. Moreover, even if EPA and DHA seemed to exert different effects on the tested cell lines, no cytotoxic and/or anti-apoptotic actions were observed after omega-3 based treatments.

This review presents several alternatives to replace antibiotic therapy and make the European aquaculture industry more friendly and environmentally sustainable. The first part of this review highlights the growing importance of the aquaculture industry worldwide for its ability to supply low-cost proteins and lipids. The second part discusses different strategies for these replacements, from recombinant vaccines to diets with low environmental impact and rich in bioactive molecules that can benefit other species. Specifically, the beneficial effects of bioactive compounds present within insect meals are discussed. In addition, particular focus is placed on the importance of adopting sustainable protocols for fish farming, including supplements such as probiotics, prebiotics, and synbiotics capable of modulating the gut microbiota as the second brain. Those feed additives can stimulate European farmed species’ immunological systems, growth, and welfare.

One of the main factors for the osseointegration of dental implants is the development of an adequate soft tissue barrier, mainly composed by collagen, which protects the implant from bacterial development. The structural features of the peri-implant collagen are influenced by the implant components and, in particular, by the type of the surface. In the clinical practice, healing abutments are characterized by smooth surfaces, named machined. Recently, a new laser technique, Synthegra, has been developed to obtain a topography-controlled surface with micrometric regular pores that seems reducing the risk of peri-implantitis. Based on this background, this study aims investigating the structural organization and spatial distribution of collagen surrounding healing abutments characterized by laser-treated and machined surfaces.Gingiva portions surrounding custom-made healing abutments (HA), characterized by alternated laser-treated and machined surfaces, were collected and analyzed by combining Fourier Transform InfraRed Imaging (FTIRI) spectroscopy, a non-invasive and high-resolution bidimensional analytical technique, with histological and multivariate analyses.Masson's trichrome staining, specific for collagen, highlighted a massive presence of collagen in all the analyzed samples, evidencing a surface-related spatial distribution. The nature of collagen, investigated by the FTIRI spectroscopy, appeared more abundant close to the laser-treated surface, with a perpendicular disposition of the bundles respect to the HA; conversely, a parallel distribution was observed around the machined surface. A different secondary structure was also found, with a higher amount of triple helices and a lower quantity of random coils in collagen close to the laser treated surfaces.FTIRI spectroscopy demonstrates that the use of a laser treated transmucosal surface can improve the morphological organization of the peri-implant collagen, which presents a distribution more similar to that of natural teeth.This trial is registered with ClinicalTrials.gov Identifier: (Registration Number: NCT05754970). Registered 06/03/2023, retrospectively registered, https://clinicaltrials.gov/show/NCT05754970 .

Currently, various analytical techniques, including scanning electron microscopy, X-Ray diffraction, microcomputed tomography, and energy dispersive X-ray spectroscopy, are available to study the structural or elemental features of hard dental tissues. In contrast to these approaches, Raman Microspectroscopy (RMS) has the great advantage of simultaneously providing, at the same time and on the same sample, a morpho-chemical correlation between the microscopic information from the visual analysis of the sample and its chemical and macromolecular composition. Hence, RMS represents an innovative and non-invasive technique to study both inorganic and organic teeth components in vitro. The aim of this narrative review is to shed new light on the applicative potential of Raman Microspectroscopy in the dental field. Specific Raman markers representative of sound and pathological hard dental tissues will be discussed, and the future diagnostic application of this technique will be outlined. The objective and detailed information provided by this technique in terms of the structure and chemical/macromolecular components of sound and pathological hard dental tissues could be useful for improving knowledge of several dental pathologies. Scientific articles regarding RMS studies of human hard dental tissues were retrieved from the principal databases by following specific inclusion and exclusion criteria.

The marine microalgae Ostreopsis cf. ovata are a well-known producer of palytoxin (PlTXs) analogues, i.e. ovatoxins (OVTXs) among others, which arouse concern for animal and human health. Both in field and laboratory studies, presence of OVTXs, detected in species directly feeding on O. cf. ovata, was frequently correlated with impairment on organisms' physiology, development and behaviour, while similar knowledge is still lacking for animals feeding on contaminated preys. In this study, transfer and toxicity of OVTXs were evaluated in an exposure experiment, in which gilthead seabream Sparus aurata was fed with bivalve mussel Mytilus galloprovincialis, contaminated by a toxic strain of O. cf. ovata. Mussels exposed to O. cf. ovata for 21 days accumulated meanly 188 ± 13 μg/kg OVTXs in the whole tissues. Seabreams fed with OVTX-contaminated mussels started to reject the food after 6 days of contaminated diet. Although no detectable levels of OVTXs were measured in muscle, liver, gills and gastro-intestinal tracts, the OVTX-enriched diet induced alterations of lipid metabolism in seabreams livers, displaying a decreased content of total lipid and fatty acid, together with overexpression of fatty acid biosynthetic genes, downregulation of β-oxidation genes and modulation of several genes related to lipid transport and regulation. Results from this study would suggest the hypothesis that OVTXs produced by O. cf. ovata may not be subject to bioaccumulation in fish fed on contaminated preys, being however responsible of significant biological effects, with important implications for human consumption of seafood products.

Abstract Human pharmaceuticals represent a major challenge in natural environment. A better knowledge on their mechanisms of action and adverse effects on cellular pathways is fundamental to predict long-term consequences for marine wildlife. The FTIRI Imaging (FTIRI) spectroscopy represents a vibrational technique allowing to map specific areas of non-homogeneous biological samples, providing a unique biochemical and ultrastructural fingerprint of the tissue. In this study, FTIRI technique has been applied, for the first time, to characterize (i) the chemical building blocks of digestive glands of Mytilus galloprovincialis , (ii) alterations and (iii) resilience of macromolecular composition, after a 14-days exposure to 0.5 µg/L of carbamazepine (CBZ), valsartan (VAL) and their mixture, followed by a 14-days recovery period. Spectral features of mussels digestive glands provided insights on composition and topographical distribution of main groups of biological macromolecules, such as proteins, lipids, and glycosylated compounds. Pharmaceuticals caused an increase in the total amount of protein and a significant decrease of lipids levels. Changes in macromolecular features reflected the modulation of specific molecular and biochemical pathways thus supporting our knowledge on mechanisms of action of such emerging pollutants. Overall, the applied approach could represent an added value within integrated strategies for the effects-based evaluation of environmental contaminants.

Resveratrol (RES) is a stilbenoid polyphenol with interesting antitumor activity compromised by its poor solubility and bioavailability; thus, new approaches are necessary to improve its therapeutic effectiveness. In the present study, bovine serum albumin coated layered double hydroxide (LDH-BSA) was employed to encapsulate RES in order to overcome the above-mentioned usage limits. To evaluate the feasibility of neutral RES complexation with cationic LDH, we carried out molecular dynamics simulation in order to predict its structure and stability. In the supramolecular complex formed with LDH, RES disposes itself in the interlamellar region of LDH where it is stabilized by intermolecular interactions. The physico-chemical characteristics of the resulting nanocomplexes were studied by X-ray powder diffraction, transmission electron microscopy, and attenuated total reflection Fourier transform infrared spectroscopy. The encapsulation efficiency and drug release studies were also performed. The combined experimental and computational approach were highly effective in giving insight into the interaction mode of the neutral RES with the charged LDH. Finally, the nanohybrid's anticancer ability was evaluated in human lung cancer cell line (A549) resulting in higher activity with respect to bare RES. Overall, the results showed that the nanocomposites are suitable for biomedical applications as delivery agents of RES.

Different Follicle Stimulating Hormone (FSH) formulation and Luteinizing Hormone (LH) are used in Assisted Reproductive Technology (ART) to induce follicles development and oocytes maturation, but it is still under debate which protocol is to be preferred. In the present study, the different effects on cumulus cells (CCs) of three controlled ovarian stimulation (COS) protocols, based on urinary FSH, recombinant FSH, or human Menopausal Gonadotropin (hMG) administration, were assessed. CCs were obtained from 42 normal-responders women undergoing COS, randomly divided into three groups according to the used gonadotropin formulation. Differences were found in the expression of genes belonging to the endocannabinoid system (the receptors CNR1, CNR2 and TRPV1, and the enzymes involved in the metabolisms of anandamide, NAPE-PLD and FAAH, and 2-acylglycerol, DAGL and MAGL); consistently, changes in lipid (PPARα, and FASN) and carbohydrate (GLUT1 and GLUT9) metabolisms, in CCs’ macromolecules composition (highlighted by Fourier Transform Infrared Microspectroscopy, FTIRM), and in the number of retrieved oocytes were found. For the first time, statistically significant evidence on the differences related to each COS protocol on the endocannabinoid system, metabolism and macromolecular composition of CCs was found, representing a proof of concept to be further confirmed in a larger cohort of patients.

Multicellular spheroids are the new frontier for studying how the tumour micro-environment interferes with drug uptake and response, since they can reproduce a three-dimensional cellular organisation mimicking the behaviour of in vivo solid tissues. In this study, we exploited Focal Plane Array - Fourier Transform Infrared Imaging spectroscopy to characterize the biochemical features, in terms of distribution and composition of the meaningful macromolecules (lipids, proteins, sugars and nucleic acids), of malignant pleural mesothelioma spheroid sections, and, as further extent, to investigate the penetrating effects of cisplatin within the spheroid mass. The hyperspectral imaging analysis evidenced, in untreated spheroids, the occurrence of a replicative outer region and a hypoxic inner one, as suggested by the band area ratios related to lipid alkyl chains (2925/2960) and glycogen (1020/1650), which showed the highest values in the inner region. Moreover, the HCA spectroscopic images showed, after cisplatin treatment, an increase of the band area ratio related to lipid carbonyl ester moiety (1740/2925), suggesting the occurrence of lipid peroxidation; furthermore, the band area ratio related to nucleic acids (1240/1220) revealed a DNA fragmentation along all regions of spheroids that may be related to apoptotic mechanisms, whereas a reduction of the band area ratios related to glycogen and carbohydrates (1020/1650 and 1054/1650, respectively) appeared consistent with an inhibition of cell division. The few spectral differences between the outer and the inner regions of cisplatin-treated spheroids pointed out the diffuse penetrating effect of the drug.

Isofuranodiene (IFD) is a sesquiterpene occurring in several plant species, which proved to have multiple anticancer activities. IFD has a lipophilic nature and, hence, a very low water solubility and a poor bioavailability; moreover, it is not stable, undergoing the "Cope rearrangement" to the less active curzerene. The use of appropriate delivery systems can thus be considered as a valid tool to enhance IFD bioavailability, solubility, stability and at the same time also to improve its intracellular uptake and pharmacological activity. Within this frame, monoolein (GMO) nanoparticles loaded with IFD were prepared and their enhanced anticancer activity, compared to pristine IFD, was assessed. In this study, for the first time, an in vitro Fourier Transform Infrared and Raman Microspectroscopy approaches were exploited to evaluate the effects of IFD, alone and loaded in GMO nanoparticles, on MDA-MB 231 breast cancer cell line. The anti-cancer effects of IFD were evidenced by both the spectroscopic techniques and discriminated from the GMO-induced changes in the culture environment; moreover, a synergistic effect of IFD and GMO administration can be envisaged by the experimental results.

Gestational diabetes mellitus (GDM) and small-for-gestational-age (SGA) are two metabolic-related diseases that could affect women during pregnancy. Considering that the chorionic villi (CVs) are crucial structures for the feto-maternal exchange, the alterations in their conformation have been linked to an imbalanced metabolic environment of placenta. In this study, a multidisciplinary approach has been carried out to describe the changes occurring in the placental CVs of GDM and SGA patients. The results revealed higher levels of superoxide dismutase 1 (SOD-1) and catalase (CAT), especially in the GDM placentae, which could be correlated with the hyperglycemic environment characteristic of this pathology. Furthermore, spectroscopy and histologic analyses revealed that both pathologies modify the placental lipid composition altering its structure. However, SGA induces lipid peroxidation and reduces collagen deposition within the CVs. Since the endocannabinoid system (ECS) is involved in placentation and different metabolic activities, the cannabinoid receptor 1 (CB1) and transient receptor potential cation channel subfamily V member 1 (TRPV-1) were analyzed. No changes have been observed either at general or specific levels in the CVs comparing control and pathological samples, suggesting the non-involvement of the cannabinoid system in these two pathologies.

Does the molecular and metabolic profile of human mural granulosa cells (GCs) correlate with oocyte fate?A close relation between the metabolic profile of mural GCs and the fate of the corresponding oocyte was revealed by the analysis of selected biomarkers defined by GC Fourier transform infrared microspectroscopy (FTIRM) analysis.In ART, oocyte selection is mainly based on the subjective observation of its morphological features; despite recent efforts, the success rate of this practice is still unsatisfactory. FTIRM is a well-established vibrational technique recently applied to evaluate oocytes quality in several experimental models, including human.GCs retrieved from single-follicle aspirates were obtained with informed consent from 55 women undergoing controlled ovarian stimulation for IVF treatment. GCs were analysed by FTIRM to retrospectively correlate their spectral features with the fate of the companion oocytes. The study has been conducted between March 2016 and September 2017.Patients were selected according to the following inclusion criteria: age <40 years; non-smokers; no ovarian infertility diagnosis (only tubal, idiopathic and male infertility); regular ovulatory menstrual cycles (25-30 days) with FSH < 10 IU/I on Day 3 of the menstrual cycle; sperm sample with a total motility count after treatment ≥300.000; number of retrieved oocytes ≥8. Based on the clinical outcome of the corresponding oocyte, GCs were retrospectively classified into the following experimental groups: clinical pregnancy (CP), fertilization failure (FF), embryo development failure (EDF) and implantation failure (IF). All samples were analysed by the FTIRM technique. The spectral biomarker signature of different oocyte fates was derived by several feature selection procedures ('Leave-one-out' method on factorial discriminant analysis (FDA), variable characterization method and logistic regression method with the multinomial Logit model). ANOVA, permutational multivariate ANOVA, FDA and canonical analysis of principal co-ordinates statistical tools were also applied to validate the identified spectral biomarkers.In total, 284 GCs samples were retrieved and retrospectively classified as FF: (N = 92), EDF (N = 113), IF (N = 56) and CP (N = 23). From the spectral profiles of GCs belonging to CP, FF, EDF and IF experimental groups, 17 spectral biomarkers, were identified by several feature selection procedures (P < 0.0001). These biomarkers were then validated by applying multivariate tools, to evaluate their ability to segregate GCs samples into the four experimental groups. FDA showed a clear separation along the F1-axis (62.75% of discrimination) between GCs from oocytes able (CP, IF groups) or not (FF, EDF groups) to develop into embryos; the F2-axis (24.14% of discrimination) segregated the embryos that gave pregnancy (CP) from those that failed implantation (IF). The confusion matrix (total percentage of correctness = 80.25%) obtained from this analysis pinpointed that GCs from oocytes unable to develop into embryos (FF, EDF) were better characterized than those from oocytes able to give viable embryos (CP, IF). ANOVA (P < 0.05) analysis pinpointed that: each experimental group showed specific macromolecular traits, ascribable to different biological and metabolic characteristics of GCs; these metabolic features were likely associated with different oocytes fates, but not to patient characteristics, since from the same patient we obtained GCs with different metabolic profiles.The study is based on a small sample size but provides proof of concept that the GCs' metabolic profile is associated with the companion oocyte fate. The generated model should be further tested on a larger cohort of patients, classified in a similar manner, to assess the potential predictive value of this approach. Ultimately, validity of the proposed approach should be tested in a RCT.For the first time, the FTIRM analysis of human GCs has demonstrated an approach to better understand the molecular crosstalk between follicular cells and oocytes and has identified potential spectral biomarkers for improving human IVF success rate.The study was funded by GFI 2014 grant. The authors declare that there is no conflict of interest.

The present study was conducted to examine, for the first time in Nothobranchius furzeri, the effects of mating in different aged breeders with particular emphasis on reproductive fitness and the effects of parental aging on offspring gametogenesis. In N. furzeri, the increase of maternal and paternal age is often a predictable indicator of the upcoming deterioration on their natural habitat (i.e., ephemeral puddles) during African dry season. We previously revealed that elderly parents respond to their physiological decline state by decelerating offspring's development and growth rate. In the present study, we focused on the effects of different age parents at the onset of female offspring's sexual maturity since interaction between growth and reproduction traits generally occurs in vertebrates and could represent a life strategy. For this purpose, four different age breeder groups were set up. The age-specific breeder differences in reproductive performance were examined by analyzing the reproductive age-related fitness, the gametogenesis process in F1 females by histology, the offspring oocytes quality analyzing the gene expression of age-related molecular markers, like sirt1 and foxo3a, and the biochemical composition of vitellogenic oocytes using a spectroscopic approach. Results suggest that both maternal and paternal age affect reproductive performance and could influence the onset of sexual maturity in female offspring. In conclusion, these findings highlighted the effects of parental aging on life history traits in the short-lived model N. furzeri. Our results suggested that the advanced sexual maturity in offspring from young parents could be related to an adaptive response to the temporary habitat conditions.

Black Soldier Fly (BSF) meal is considered an alternative, emerging, and sustainable ingredient for aquafeed formulation. However, results on fish physiological responses are still fragmentary and often controversial, and no data are available on the effect of insect meal-based diets on fish reproduction. On this regard, zebrafish, with its relatively short life cycle, represents an ideal experimental model to explore this topic. In this study, female zebrafish were fed for 12 months on a control diet based on fish meal (FM) and fish oil and two experimental diets with full-fat BSF (Hermetia illucens) prepupae meal inclusion, to replace 25% and 50% of FM (BSF25 and BSF50). All diets were isonitrogenous, isolipidic, and isoenergetic. The effects of these two experimental diets on female's reproduction were investigated through a multidisciplinary approach, including the evaluation of growth, gonadosomatic index, spawned/fertilized eggs and hatching rate, adult female carcass and fertilized egg fatty acid composition, histological analysis of the ovary, spectroscopic macromolecular composition of class IV oocytes, and expression of genes involved in fish lipid metabolism in the liver. Results showed that while fish were perfectly able to cope with a 25% insect meal dietary inclusion, a 50% inclusion level caused the overexpression of genes involved in lipid metabolism, a general reduction in the number of spawned eggs, and differences in the frequency rate of previtellogenic oocytes, class III, IV, oocytes and postovulatory follicles and atretic oocytes, in the macromolecular composition of class IV oocytes, and in the fatty acid composition of the fertilized eggs, respect to control and 25% group.

Clear and removable tooth aligners for orthodontics treatments have become an increasingly popular alternative to fixed appliances. Even if protocols suggest removing aligners before eating or drinking, most patients retain them when they drink beverages. Alterations in the material during the daily use could determine a reduction in the application forces, affecting the desired orthodontic movement; the knowledge of how this material reacts when subjected to different aging processes is mandatory to establish the predictability of the orthodontic treatment. According to this, the aim of the present study was to assess a new objective approach, coupling spectroscopic and chemometric tools, to evaluate the changes occurring in Invisalign® aligners, the most widely used brand, exposed in vitro to coffee, tea, Coca Cola® and UV radiation for 24 and 48 h. In particular, ATR-FTIR spectroscopy was utilized to characterize, at the molecular level, the chemical and color modifications in the surfaces of the appliances; the obtained data were submitted to PCA and one-way ANOVA and Tukey’s multiple comparison test. Moreover, a colorimetry analysis was carried out to evaluate any changes in color and transparency. Coffee and tea samples displayed the major color changes between the tested groups. The differences highlighted in the spectral features of coffee, tea and UV-treated samples were mainly ascribable to color and transparency changes, because the chemical properties remained unaltered.

The corpus callosum (CC) is the largest interhemispheric commissure of the mammalian brain, and it includes axons, cortical neurons, and glial cells. It is mainly composed of myelin, a lipidic sheath which is produced by glial cell membranes; myelin is wrapped up around axons and plays a fundamental role in the fast conduction of neuronal electrical signals. The human CC is divided into various anatomical regions, with different axonal composition, including, from front to back, genu, body or trunk, isthmus, and splenium. Corpus callosum undergoes some alterations not only in the presence of specific physiological and pathological conditions, but also because of aging. For the first time, in the present study a hyperspectral imaging analysis of human corpus callosum was performed. The study, carried out on CC autopsy samples collected from human adult males of different ages, was focused mainly on the genu and splenium regions. By combining Fourier-transform infrared imaging and histological analyses with multivariate and univariate ones, the macromolecular composition of these regions was defined, and age-related alterations in the lipid and protein components were identified.

Lyotropic Liquid Crystalline (LLC) nanoparticles represent an emerging class of smart, biocompatible, and biodegradable systems for the delivery of drugs. Among these, structures with complex 3D architectures such as cubosomes are of particular interest. These are non- lamellar assemblies having hydrophobic and hydrophilic portions able to carry drugs of different nature. They can further be modulated including suitable additives to control the release of the active payload, and to promote an active targeting. Starting from monoolein (GMO) cubic phase, different concentrations of mannose-based esters were added, and the eventual structural modifications were monitored to ascertain the effects of the presence of glycolipids. Moreover, the structural properties of these nanosystems loaded with Dexamethasone (DEX), a very well-known anti-inflammatory steroid, were also studied. Experiments were carried out by synchrotron Small Angle X-ray Scattering (SAXS), Raman Microspectroscopy (RMS) and Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) measurements. The drug delivery potential (i.e. entrapment efficiency and release properties) of the obtained nanoparticles was evaluated. Finally, in vitro cytocompatibility and anti-inflammatory activity studies of the prepared formulations were carried. Inclusion of mannose-based surfactants up to 10 mol% influenced the structural parameters of Im3m cubic phase and swollen cubic phases were obtained with the different glycolipids with lattice parameters significantly higher than GMO. A complete cytocompatibility and an increased DEX activity were observed, thus suggesting the possibility to use GMO/glycolipids nanoparticles to formulate innovative drug delivery systems.

Once they reach the aquatic environment, microplastics (MPs) are accidentally ingested by aquatic biota, thus entering the food chain with possible negative effects. The present study investigated, for the first time, MP presence in cuttlefish (Sepia officinalis) eggs and their association with embryonic development. Cuttlefish eggs were sampled from four different sites along the Marche region (Senigallia, Ancona, Numana, and San Benedetto del Tronto). Embryo and hatchling biometric parameters were evaluated and the internal structural integrity was examined through histological analysis. MPs were detected and characterized in embryos and yolk samples. MPs were identified in all sites (size < 5 µm), however, their presence has not been associated with an impairment of either embryo or hatchling internal structures. Noteworthy, the highest number of MPs (in both yolk and embryo samples) were found in Numana (37% of the total amount), where the lowest hatchling size was observed. On the other hand, the highest embryo mantle length was associated with the lowest number of MPs detected (9%) in Ancona. Overall, only MP fragments and sphere types (74 and 26%, respectively) were observed, and the most frequent polymers were Polyvinyl chloride (52%), Polypropylene, and Cellulose acetate (15% both). Further studies are needed to assess the possible MP effects on the yolk quality and assimilation.

This in vitro study aimed assessing the remineralization potential of three commercial fluoride-based toothpastes in permanent teeth with natural white spot lesions (WSLs). A multidisciplinary approach based on Raman microspectroscopy (RMS), Scanning electron microscopy (SEM), Energy-dispersive x-ray spectroscopy (EDS), and Vickers microhardness (VMH) was exploited.N = 12 human molars with natural WSLs in the proximal-vestibular zone were selected and divided into 4 groups (n = 3) according to the different treatments: HAF (hydroxyapatite with fluoride ions); SMF (sodium monofluorophosphate with arginine); SF (sodium fluoride with enzymes), and CTRL (untreated group). All toothpastes tested contained 1450 ppm of fluoride. Teeth samples were submitted to the following protocol: a 7-day pH cycling treatment, with two daily exposures (2 min each time) to the commercial toothpastes described above. The surface micromorphology (SEM), the chemical/elemental composition (RMS and EDS), and the Vickers microhardness (VMH) were evaluated. Statistical analysis was performed.A remarkable remineralization of WSLs in SEM images was observed in all treated groups compared to CTRL. In particular, HAF and SF displayed higher values of VMH, phosphates amount (I960), crystallinity (FWHM960), and lower ones of C/P (I1070/I960) with respect to CTRL. Intermediate values were found in SMF, higher than CTRL but lower with respect to HAF and SF. As regards the Ca/P ratio, statistically significant differences (p < 0.05) were found between SF and the other groups.All the tested dentifrices have shown to remineralize the WSLs. SF and HAF have comparable capability in hardness recovery and crystallinity; however, SF shows the best remineralizing potential according to both micromorphological and chemical analyses. Clinical relevance The daily use of toothpastes containing hydroxyapatite partially replaced with fluoride, sodium monofluorophosphate with arginine and sodium fluoride toothpaste associated with enzymes represents a preventive, therapeutic, effective, and non-invasive tool for remineralize WSLs.

The aim of this work was to provide, for the first time, a protocol for isolation and characterization of stem cells from porcine amniotic membrane in view of their potential uses in regenerative medicine. From three samples of allanto-amnion recovered at delivery, the amniotic membrane was stripped from overlying allantois and digested with trypsin and collagenase to isolate epithelial (amniotic epithelial cells [AECs]) and mesenchymal cells, respectively. Proliferation, differentiation, and characterization studies by molecular biology and flow cytometry were performed. Histological examination revealed very few mesenchymal cells in the stromal layer, and a cellular yield of AECs of 10 × 106/gram of digested tissue was achieved. AECs readily attached to plastic culture dishes displaying typical cuboidal morphology and, although their proliferative capacity decreased to the fifth passage, AECs showed a mean doubling time of 24.77 ± 6 h and a mean frequency of one fibroblast colony-forming unit (CFU-F) for every 116.75 plated cells. AECs expressed mesenchymal stem cell (MSC) mRNA markers (CD29, CD166, CD90, CD73, CD117) and pluripotent markers (Nanog and Oct 4), whereas they were negative for CD34 and MHCII. Mesodermic, ectodermic, and endodermic differentiation was confirmed by staining and expression of specific markers. We conclude that porcine amniotic membrane can provide an attractive source of stem cells that may be a useful tool for biomedical research.

Management of dredged sediments results in an environmental and social cost. Based on their level of contamination, they can be intended for beach nourishment or for alternative uses. Sediment quality is established considering their specific chemical contamination level and setting up bioassays to evaluate their toxic effects on living organisms. The integration of these different Line of Evidence (LOE) generates toxicity indexes, the Hazard Chemical Quotient (HQc), and the Hazard Ecotoxicological Quotient (HQe), which are further elaborated using the SediQualSoft software, finally providing evidence on the levels of sediment contamination. In this study, four different dredged sediments were analysed. Except for one, which was sampled in a reference area, the others were assigned to the same class of toxicity, despite they presented different levels of chemical and ecotoxicological toxicity. As a novelty, this study introduces transcriptomics as a new LOE, to provide a new tool to better categorize sediment toxicity. C. gigas embryos were exposed to sediment elutriates, sampled at 5 and 18 h post-fertilization (hpf), and the expression of a set of genes involved in immune and stress response (hsp70, gpx, sod, dehf1, galectin, lysozyme, tg) was analysed by Real-time PCR. Molecular results suggested that the 18 hpf stage represents a sensitive window of exposure during development and can be suggested as a critical time point for ecotoxicity studies. Finally, by multivariate statistical analysis, integrating the well-established LOEs with molecular data, it was demonstrated that transcriptomics could be a useful and novel LOE with the ability to provide greater accuracy in the assessment of sediment toxicity.

The agricultural sector is required to produce food at the same pace as population growth, while accounting for pollution and costs. For this reason, conservative agricultural practices have been employed worldwide. Attenuated Total Reflectance–Fourier Transform Infrared (ATR-FTIR) spectroscopy has the ability to provide a snapshot of the macromolecular composition of a sample in a timely and cost-effective way and it has been widely applied in the field of agriculture to assess food quality. The aim of this study was to exploit ATR-FTIR spectroscopy to assess the impact of different soil tillage methods (conventional tillage, CT; minimum tillage, MT, and no tillage, NT) and nitrogen fertilization levels (0, 90 and 180 kg N ha−1) on the macromolecular composition of leaves and caryopses of durum wheat (Triticum turgidum subsp. durum). The analysis of the spectral data revealed that the quality of durum wheat, in terms of protein content, grown on soil with no tillage was not reduced. Indeed, with regards to caryopses, the different tillage methods influenced only the lipid and hemicellulose content, whereas the macromolecular composition of leaves was sensitive to tillage methods mostly during the early stage of growth. Moreover, no relevant effects were found in leaves and caryopses when different fertilizer concentrations were used. These results provide important knowledge supporting the adoption of both no-tillage soil treatments and reduced fertilization dosage for the development of durum wheat management strategies and support the use of spectroscopy for conservative agriculture practices.

Summary paragraph Microplastics are particles smaller than five millimetres obtained from the degradation of plastic objects abandoned in the environment. Microplastics can move from the environment to living organisms and, in fact, they have been found in fishes and mammals. Six human placentas, prospectively collected from consenting women with uneventful pregnancies, were analyzed by Raman Microspectroscopy to evaluate the presence of microparticles. Detected microparticles were characterized in terms of morphology and chemical composition. 12 microparticles, ranging from 5 to 10 μm in size, were found in 4 out of 6 placentas: 5 in the foetal side, 4 in the maternal side and 3 in the chorioamniotic membranes. All the analyzed microparticles were pigmented: three of them were identified as stained polypropylene, while for the other nine it was possible to identify only the pigments, which are all used for man-made coatings, paints and dyes. Here we show, for the first time, the presence of microparticles and microplastics in human placenta. This sheds new light on the impact of plastic on human health. Microparticles and microplastics in the placenta, together with the endocrine disruptors transported by them, could have long-term effects on human health.

ATR-FTIR (Attenuated Total Reflectance Fourier Transform InfraRed) spectroscopy, combined with chemometric, represents a rapid and reliable approach to obtain information about the macromolecular composition of food and plant materials. With a single measurement, the chemical fingerprint of the analyzed sample is rapidly obtained. Hence, this technique was used for investigating 13 differently processed tea leaves (green, black and white) all grown and processed in European tea gardens, and their vacuum-dried tea brews, prepared using both hot and cold water, to observe how the components differ from tea leaf to the in-cup infusion. Spectra were collected in the 1800–600 cm−1 region and were submitted to Principal Component Analysis (PCA). The comparison of the spectral profiles of leaves and hot and cold infusions of tea from the same country, emphasizes how they differ in relation to the different spectral regions. Differences were also noted among the different countries. Furthermore, the changes observed (e.g., at ~1340 cm−1) due to catechin content, confirm the antioxidant properties of these teas. Overall, this experimental approach could be relevant for rapid analysis of various tea types and could pave the way for the industrial discrimination of teas and of their health properties without the need of time-consuming, lab chemical assays.

Bones offer a great amount of information on ancient populations regarding both their lifestyle habits and the influence of the living area. Bones are composed by an inorganic component, i.e., carbonated hydroxyapatite (Ca10[(PO4)6−x(CO3)x](OH)2), and an organic matrix (mainly proteins and collagen). After death, bones are subjected to diagenetic processes, with changes in structure, morphology, and chemical composition. All these modifications strictly depend on several factors, including the nearby environment, the climate, and the burial modality. Hence, a precise knowledge of the diagenetic processes affecting bones after death is mandatory. In this study, archeological human bones from the Garamantian necropolis of Fewet (Libyan Sahara) were analyzed by ATR–FTIR spectroscopy to elucidate the role of the burial location and modality, as well as the highly arid environment in the diagenesis rate. Several spectral parameters related to structural and chemical features of the organic and mineral components (i.e., AmideI/PO4, C/P, MM, FWHM603, and IRSF indexes) were statistically analyzed. Spectral data were compared with those from modern ruminants from the same site to evaluate a possible time-dependent correlation between the chemical composition and the diagenetic processes. A mild diagenesis was found in all human bones, even though it had a variable degree depending on the burial location.

The potential toxicity of microplastics is a growing concern for the scientific community. The loggerhead sea turtle (Caretta caretta) is particularly vulnerable to plastic and microplastic accidental ingestion due to its long-life cycle, late reproductive maturity age and seasonal migrations. The transfer of microplastics from the female to the eggs is a possibility that should be investigated since embryonic development is already threatened by several environmental factors.The present study aimed to investigate the presence of microplastics in the yolk and the liver of loggerhead sea turtle embryos and successively evaluated the possible effects of microplastics occurrence in the yolk and the liver on the embryonic health status considering as a biomarker the number of melanomacrophages in the hepatic tissue. Raman Microspectroscopy was performed to identify the microplastics after alkaline digestion (10 % KOH) of yolk and liver samples. Histological analysis was applied to liver anatomical investigation. Microplastics were found in the yolk and liver of loggerhead sea turtle late-stage embryos for the first time. All microplastics had dimensions lower than 5 µm and were characterized by a high variety of polymers and colors suggesting their diverse origins. Despite the eggs showing a higher number of microplastics in yolk samples than the liver, a positive correlation was observed only between the number of melanomacrophages in the liver and microplastics in the same tissue. This result suggested that microplastics could exert their effect once they reached the tissues while in the yolk, they did not impair the physiological function of the embryo. Future studies should investigate the presence of microplastics in other embryonic tissues and the possible relation with other stress biomarkers.

Raman MicroSpectroscopy (RMS) is a powerful label-free tool to probe the effects of drugs at a cellular/subcellular level.

The potential toxicity of microplastics is a growing concern for the scientific community. The loggerhead sea turtle (Caretta caretta) is particularly inclined to accidently ingest plastic and microplastic due to its long-life cycle features. The possible transfer of microplastics from the female to the eggs should be investigated. The present study investigated the presence of microplastics in yolk and liver samples evaluating the number of melanomacrophages in the hepatic tissue as a possible biomarker of microplastics impact on the embryonic health status. The biometric parameters and liver histological analysis of 27 and 48 embryos (from two different nests respectively) at the 30 stage of development were analyzed. Raman Microspectroscopy was performed to identify the microplastics after alkaline digestion (10% KOH) of yolk and portion of liver from 5 embryos at the 30 developmental stage per nest. Microplastics were found in yolk and liver of loggerhead sea turtles at late embryonic stage for the first time. All microplastics were smaller than 5 μm and were made of polymers and colors suggesting their diverse origins. A total number of 21 microplastics, with dimensions lower than 5 μm, were found between the two nests (11 and 10 microplastics respectively). Only two shape categories were identified: spheres and fragments. The most frequent polymers observed were polyethylene, polyvinyl chloride and acrylonitrile butadiene styrene (31.5%, 21.1% and 15.8% respectively). Despite the eggs showing a higher number of microplastics in yolk samples than liver (15 and 6 microplastics in yolk and liver respectively), a positive correlation was observed only between the number of melanomacrophages (r = 0.863 p < 0.001) and microplastics in the liver. This result may suggest that microplastics could exert some effects on the hepatic tissues. Future studies should investigate this aspect and the possible relation between microplastics and other stress biomarkers.

The marine microalgae Ostreopsis cf. ovata is a well-known producer of palytoxin (PlTXs) analogues, i.e. ovatoxins (OVTXs) among others, which arouse concern for animal and human health. The presence and effects of OVTXs are frequently reported in marine species directly feeding on O. cf. ovata, while similar knowledge is still lacking for animals feeding on contaminated preys. In this study, transfer and toxicity of OVTXs along the food chain were evaluated in a trophic-transfer experiment, using the bivalve mussel Mytilus galloprovincialis, initially fed on a toxic strain of O. cf. ovata, and subsequently given as food to the gilthead seabream Sparus aurata, a carnivorous fish naturally feeding on mussels. Mussel fed on O. cf. ovata for 21 days accumulated meanly 188 µg/kg OVTXs in the whole tissues. Seabreams fed with OVTX-contaminated mussels started to reject the food after 6 days of contaminated diet. Despite no detectable levels of OVTXs were measured in their muscle, liver, gills and gastro-intestinal tracts, the OVTX-enriched diet induced alterations of lipid metabolism in seabreams livers, displaying a decreased content of total lipid and fatty acid, together with overexpression of fatty acid biosynthetic genes, downregulation of β-oxidation genes and modulation of several genes related to lipid transport and regulation. Results from this study would suggest the hypothesis that OVTXs produced by O. cf. ovata may not be subject to bioaccumulation being however responsible of significant biological effects, with important implications for human consumption of seafood products.

The presence of microplastics (MPs) in human fluids and organs is a great concern, since, as highlighted by recent studies on animal models, they could cause alterations of several physiological functions, including reproduction. In this study, N. 10 semen samples collected from men living in a polluted area of the Campania Region (Southern Italy), were analyzed by Raman Microspectroscopy to evaluate the presence of MPs and to characterize them in terms of morphology (size, color, and shape) and chemical composition. In total, N. 16 pigmented microplastic fragments (ranging from 2 to 6 μm in size) with spheric or irregular shapes were found in six of ten samples. Chemical composition showed the presence of polypropylene (PP), polyethylene (PE), polyethylene terephthalate (PET), polystyrene (PS), polyvinylchloride (PVC), polycarbonate (PC), polyoxymethylene (POM) and acrylic, suggesting ingestion and/or inhalation as a route of exposure to environmental MPs. In this work, we propose for the first time a mechanism by which MPs pass into the semen most probably through the epididymis and the seminal vesicles, which are the most susceptible to inflammation. Given the global decline in seminal quality in recent decades, action is therefore needed to stop the exponential increase in plastic waste as it impacts the reproductive process.

Abstract Fast, reliable and point-of-care systems to detect the SARS-CoV-2 infection are crucial to contain viral spreading and to adopt timely clinical treatments. Many of the rapid detection tests currently in use are based on antibodies that bind viral proteins 1 . However, newly appearing virus variants accumulate mutations in their RNA sequence and produce proteins, such as Spike, that may show reduced binding affinity to these diagnostic antibodies, resulting in less reliable tests and in the need for continuous update of the sensing systems 2 . Here we propose a graphene field-effect transistor (gFET) biosensor which exploits the key interaction between the Spike protein and the human ACE2 receptor. This interaction is one of the determinants of host infections and indeed recently evolved Spike variants were shown to increase affinity for ACE2 receptor 3 . Through extensive computational analyses we show that a chimeric ACE2-Fc construct mimics the ACE2 dimer, normally present on host cells membranes, better than its soluble truncated form. We demonstrate that ACE2-Fc functionalized gFET is effective for in vitro detection of Spike and outperforms the same chip functionalized with either a diagnostic antibody or the soluble ACE2. Our sensor is implemented in a portable, wireless, point-of-care device and successfully detected both alpha and gamma virus variants in patient’s clinical samples. As incomplete immunization, due to vaccine roll-out, may offer new selective grounds for antibody-escaping virus variants 4 , our biosensor opens to a class of highly sensitive, rapid and variant-robust SARS-CoV-2 detection systems.

ObjectiveThe role of the endocannabinoid system (ECS) in the pathogenesis of endometriosis is still under investigation. In this study, the changes on ECS related to apoptosis and autophagy processes have been analized in human granulosa cells (GCs) from women affected by endometriomaDesignThis prospective non-randomized study has been carried out from January to September 2014 on women undergoing a controlled ovarian hyperstimulation for an IVF treatment. In particular, GCs were collected from both ovaries of 10 women with a diagnosis of unilateral ovarian endometrioma at the time of oocytes retrieval. 9 women with male, idiopathic or tubal-factor infertility diagnosis were selected for the control group. The three experimental groups matched for female age (36.2±4.1 vs 35.4±2.6)Materials and MethodsGCs obtained from follicles aspirates were isolated from red blood cells and follicular fluid by density gradient centrifugation. Q-PCRs were performed with the SYBR green method in an iQ5 iCycler thermal cycler using βactin and GAPDH as reference genes. Data are presented as mean ± S.D. Two-Way ANOVA followed by Tukey test as Multiple comparisons test, was used for comparison among experimental groups. All statistical analyses were performed using the statistical software package Prism5 (Graphpad Software, Inc. USA) with significance accepted at P<0.05ResultsResults are showed in table. A meaningful increase of Endocannabinoids receptors (CNR1, CNR2, GPR55 and TRPV1) gene expression was found in GCs both from ovaries with endometrioma and from contralateral “healthy” ones. In addition, an increase of autophagyc (BECN1 and LC3-II) and apoptotic (Caspase3) signals, and a decrease of the antiapoptotic signal (Survivin) have been detected.ConclusionsTabled 1GENE NAMEControlCONTRALATERAL OVARYOVARY WITH ENDOMETRIOMASignificanceCNR11.18±0.18a2.00±0.88b3.53±0.18ba vs b=P<0.05CNR21.07±0.07a3.30±1.31b4.32±0.83ba vs b=P<0.05GPR552.94±1.02a27.56±3.014b33.10±1.11ba vs b=P<0.001TRPV11.96±0.01a10.13±0.12b14.98±1.65ca vs b=P<0.05SURVIVIN27.54±2.27a2.54±0.56b1.09±0.15ba vs b=P<0.0001CASPASE32.29±1.13a54.47±4.36b63.30±2.29ca vs b=P<0.05BECLIN11.20±0.02a1.52±0.01a3.01±0.21ba vs b=P<0.05LC3-II1.96±0.01a3.41±0.11b4.27±.59ca vs b=P<0.05 Open table in a new tab ObjectiveThe role of the endocannabinoid system (ECS) in the pathogenesis of endometriosis is still under investigation. In this study, the changes on ECS related to apoptosis and autophagy processes have been analized in human granulosa cells (GCs) from women affected by endometrioma The role of the endocannabinoid system (ECS) in the pathogenesis of endometriosis is still under investigation. In this study, the changes on ECS related to apoptosis and autophagy processes have been analized in human granulosa cells (GCs) from women affected by endometrioma DesignThis prospective non-randomized study has been carried out from January to September 2014 on women undergoing a controlled ovarian hyperstimulation for an IVF treatment. In particular, GCs were collected from both ovaries of 10 women with a diagnosis of unilateral ovarian endometrioma at the time of oocytes retrieval. 9 women with male, idiopathic or tubal-factor infertility diagnosis were selected for the control group. The three experimental groups matched for female age (36.2±4.1 vs 35.4±2.6) This prospective non-randomized study has been carried out from January to September 2014 on women undergoing a controlled ovarian hyperstimulation for an IVF treatment. In particular, GCs were collected from both ovaries of 10 women with a diagnosis of unilateral ovarian endometrioma at the time of oocytes retrieval. 9 women with male, idiopathic or tubal-factor infertility diagnosis were selected for the control group. The three experimental groups matched for female age (36.2±4.1 vs 35.4±2.6) Materials and MethodsGCs obtained from follicles aspirates were isolated from red blood cells and follicular fluid by density gradient centrifugation. Q-PCRs were performed with the SYBR green method in an iQ5 iCycler thermal cycler using βactin and GAPDH as reference genes. Data are presented as mean ± S.D. Two-Way ANOVA followed by Tukey test as Multiple comparisons test, was used for comparison among experimental groups. All statistical analyses were performed using the statistical software package Prism5 (Graphpad Software, Inc. USA) with significance accepted at P<0.05 GCs obtained from follicles aspirates were isolated from red blood cells and follicular fluid by density gradient centrifugation. Q-PCRs were performed with the SYBR green method in an iQ5 iCycler thermal cycler using βactin and GAPDH as reference genes. Data are presented as mean ± S.D. Two-Way ANOVA followed by Tukey test as Multiple comparisons test, was used for comparison among experimental groups. All statistical analyses were performed using the statistical software package Prism5 (Graphpad Software, Inc. USA) with significance accepted at P<0.05 ResultsResults are showed in table. A meaningful increase of Endocannabinoids receptors (CNR1, CNR2, GPR55 and TRPV1) gene expression was found in GCs both from ovaries with endometrioma and from contralateral “healthy” ones. In addition, an increase of autophagyc (BECN1 and LC3-II) and apoptotic (Caspase3) signals, and a decrease of the antiapoptotic signal (Survivin) have been detected. Results are showed in table. A meaningful increase of Endocannabinoids receptors (CNR1, CNR2, GPR55 and TRPV1) gene expression was found in GCs both from ovaries with endometrioma and from contralateral “healthy” ones. In addition, an increase of autophagyc (BECN1 and LC3-II) and apoptotic (Caspase3) signals, and a decrease of the antiapoptotic signal (Survivin) have been detected. ConclusionsTabled 1GENE NAMEControlCONTRALATERAL OVARYOVARY WITH ENDOMETRIOMASignificanceCNR11.18±0.18a2.00±0.88b3.53±0.18ba vs b=P<0.05CNR21.07±0.07a3.30±1.31b4.32±0.83ba vs b=P<0.05GPR552.94±1.02a27.56±3.014b33.10±1.11ba vs b=P<0.001TRPV11.96±0.01a10.13±0.12b14.98±1.65ca vs b=P<0.05SURVIVIN27.54±2.27a2.54±0.56b1.09±0.15ba vs b=P<0.0001CASPASE32.29±1.13a54.47±4.36b63.30±2.29ca vs b=P<0.05BECLIN11.20±0.02a1.52±0.01a3.01±0.21ba vs b=P<0.05LC3-II1.96±0.01a3.41±0.11b4.27±.59ca vs b=P<0.05 Open table in a new tab

The use of pig models for preclinical testing is well established, and the availability of stem cells from this species would open the way to preclinical studies for application of cell therapy. According to the developmental stage from which they are obtained, stem cells are classified as being embryonic, fetal, or adult. Embryonic stem cells have unlimited self-renewing capacity and multilineage differentiation potential, but their clinical application seems to be hindered by the high tumorigenic rate after transplantation. Mesenchymal stem cells (MSC) derived from adult tissues are considered to be more limited in their potential and the risk of the immunological rejection of the transplanted stem cells by the recipient is an important limiting factor. The MSC derived from extra-fetal tissues could overcome many of these restrictions. Indeed, in veterinary medicine, MSC isolated from equine term placenta were the ideal candidates for tendon disease treatment, specifically for their plasticity and their reduced immunogenicity compared to bone marrow-derived cells. Extra-fetal derived MSC in porcine have been isolated from the umbilical cord matrix and amniotic fluid. The aim of this work was to provide, for the first time, an isolation protocol and the characterisation of stem cells from porcine amniotic membrane, which could hold potential uses in regenerative medicine. The amnion is a thin, avascular membrane made of an epithelial layer and an outer layer of connective tissue. From 3 samples of allanto-amnion retrieved at delivery, each amniotic membrane was stripped from the overlying allantois and, for isolation of the epithelial cells, it was digested with trypsin. After removal of epithelial cells, the stromal layer was digested with collagenase to obtain amniotic mesenchymal cells. The cellular yield from term amnion resulted only in epithelial cells (AEC) at a concentration of 10 × 106 for 1 g of digested tissue while no MSC were obtained. Histology, indeed, revealed very few cells in the stromal layer. The AEC readily attached to plastic culture dishes. Culture was established in DMEM-HG medium, supplemented with 10% serum and 10 ng mL–1 of EGF where the cells proliferated robustly. The AEC displayed typical cuboidal morphology. These cells showed a mean of 31 ± 0.24 cell population doublings after 31 days. The mean frequency of colony-forming unit fibroblasts was 1 for each of the 75 plated cells. The AEC expressed MSC mRNA markers (CD29, CD166, CD90, CD73, CD117) and pluripotent markers (Nanog and Oct4), while were negative for CD34 and MHC-II. Osteogenic, adipogenic, and neurogenic differentiations were confirmed by von Kossa, Red Oil, and Nissle stains, respectively, and by expression of specific markers (osteocalcin and osteopontin for osteogenic differentiation, adiponectin and leptin for adipogenic differentiation, and glial fibrillary acid protein and nestin for neurogenic differentiation). We conclude that porcine amnion contain unique and primitive cells whose potential is as yet undefined. Ease of collection and propagation of AEC make this tissue an attractive candidate as a resource for stem cell biotechnology and biomedical research.

Up to date, endometriosis’ possible impact on follicle cells metabolism has never been highlighted. In this light, the aim of this study was to evaluate, by FT-IR microspectroscopy, metabolic changes on granulosa cells (GCs) isolated from endometriosis affected ovaries and contralateral healthy ones This prospective non-randomized study has been conducted from January to September 2014 on women undergoing a controlled ovarian hyperstimulation for an IVF treatment. In particular, GCs were collected from both ovaries of 10 women with a diagnosis of unilateral ovarian endometrioma at the time of oocytes retrieval. 9 women with male, idiopathic or tubal-factor infertility diagnosis were selected for the control group. The three experimental groups matched for female age (36.2±4.1 vs 35.4±2.6) GCs obtained from follicles aspirates were isolated from red blood cells and follicular fluid by density gradient centrifugation. FTIR analysis was performed by using a Bruker Vertex 70 Interferometer with a Hyperion 3000 Vis-IR microscope. QPCR analysis Supported FTIR results. . Data are presented as mean ± S.D. Two-Way ANOVA followed by Tukey test as Multiple comparisons test, was used for comparison among experimental groups. All statistical analyses were performed using the statistical software package Prism5 (Graphpad Software, Inc. USA) with significance accepted at P<0.05 Results are showed in table This preliminary study represents the first approach to evaluate the macromolecular and biochemical changes on GCs from both ovaries in patients with unilateral endometrioma. These findings pinpoint that this pathology causes relevant changes in the metabolism and amount of proteins, lipids, carbohydrates and nucleic acids, and increases peroxidative processes. It is impressive that the same biochemical alterations have been found both in ovaries with endometrioma and in the contralateral “healthy” ones. All these results could be useful in developing new strategies to prevent the detrimental effects of endometriosis on fertility.Tabled 1ControlCONTRALATERAL OVARYOVARY WITH ENDOMETRIOMASIGNIFICANCELIPIDS/CELL (%)7.89±1.18a11.55±0.27b11.80±0.39ba vs b=P<0.0001PHOSPHOLIPIDS/LIPIDS (%)5.92±1.79a12.50±0.24b12.02±0.55ba vs b=P<0.0001LIPIDS PEROXIDATION(%)4.21±0.10a7.47±0.73b7.68±0.19ba vs b=P<0.0001UNFOLDED PROTEIN (%)22.14±1.32a30.03±4.02b28.84±1.34ca vs b=P<0.05CARBOHYDRATES/PROTEIN(%)16.06±2.37a10.94±0.13b10.39±0.92ba vs b=P<0.01RNA/DNA(%)10.83±3.62a6.16±1.22b6.14±1.05ba vs b=P<0.05PPARα1.13±0.22a7.00±0.88b8.70±0.97ba vs b=P<0.01PPARγ1.20±0.13a3.30±1.31b4.17±0.83ba vs b=P<0.05FAN1.13±0.41a4.13±0.12b6.23±1.29ba vs b=P<0.01GLUT110.33±0.78a2.19±0.86b1.13±0.39ca vs b=P<0.01GLUT99.08±0.44a1.51±0.80b1.19±0.47ba vs b=P<0.01 Open table in a new tab

The oocyte and the surrounding cumulus cells (CCs) are deeply linked by a complex bidirectional cross-talk. In this light, the molecular analysis of the CCs is nowadays considered to be precious in providing information on oocyte quality. It is now clear that miRNAs play a key role in several ovarian functions, such as folliculogenesis, steroidogenesis, and ovulation. Thus, in this study, specific miRNAs, together with their target genes, were selected and investigated in CCs to assess the response of patients with normal (NR) and low (LR) ovarian reserve to two different controlled ovarian stimulation (COS) protocols, based on rFSH and hMG. Moreover, a Fourier transform infrared microspectroscopy (FTIRM) analysis was performed to evaluate DNA conformational changes in CCs and to relate them with the two COS protocols. The results evidenced a modulation of the expression of miRNAs and related target genes involved in CCs' proliferation, in vasculogenesis, angiogenesis, genomic integrity, and oocyte quality, with different effects according to the ovarian reserve of patients. Moreover, the COS protocols determined differences in DNA conformation and the methylation state. In particular, the results clearly showed that treatment with rFSH is the most appropriate in NR patients with normal ovarian reserve, while treatment with hMG appears to be the most suitable in LR patients with low ovarian reserve.

In the field of reproductive technology, the biochemical composition of female gametes has been successfully investigated with the use of vibrational spectroscopy. Currently, in assistive reproductive technology (ART), there are no shared criteria for the choice of oocyte, and automatic classification methods for the best quality oocytes have not yet been applied. In this paper, considering the lack of criteria in Assisted Reproductive Technology (ART), we use Machine Learning (ML) techniques to predict oocyte quality for a successful pregnancy. To improve the chances of successful implantation and minimize any complications during the pregnancy, Fourier transform infrared microspectroscopy (FTIRM) analysis has been applied on granulosa cells (GCs) collected along with the oocytes during oocyte aspiration, as it is routinely done in ART, and specific spectral biomarkers were selected by multivariate statistical analysis. A proprietary biological reference dataset (BRD) was successfully collected to predict the best oocyte for a successful pregnancy. Personal health information are stored, maintained and backed up using a cloud computing service. Using a user-friendly interface, the user will evaluate whether or not the selected oocyte will have a positive result. This interface includes a dashboard for retrospective analysis, reporting, real-time processing, and statistical analysis. The experimental results are promising and confirm the efficiency of the method in terms of classification metrics: precision, recall, and F1-score (F1) measures.