p57Kip2 Phosphorylation Modulates Its Localization, Stability, and Interactions.

p57Kip2 is a member of the cyclin-dependent kinase (CDK) Interacting Protein/Kinase Inhibitory Protein (CIP/Kip) family that also includes p21Cip1/WAF1 and p27Kip1. Different from its siblings, few data are available about the p57Kip2 protein, especially in humans. Structurally, p57Kip2 is an intrinsically unstructured protein, a characteristic that confers functional flexibility with multiple transient interactions influencing the metabolism and roles of the protein. Being an IUP, its localization, stability, and binding to functional partners might be strongly modulated by post-translational modifications, especially phosphorylation. In this work, we investigated by two-dimensional analysis the phosphorylation pattern of p57Kip2 in different cellular models, revealing how the human protein appears to be extensively phosphorylated, compared to p21Cip1/WAF1 and p27Kip1. We further observed clear differences in the phosphoisoforms distributed in the cytosolic and nuclear compartments in asynchronous and synchronized cells. Particularly, the unmodified form is detectable only in the nucleus, while the more acidic forms are present in the cytoplasm. Most importantly, we found that the phosphorylation state of p57Kip2 influences the binding with some p57Kip2 partners, such as CDKs, LIMK1 and CRM1. Thus, it is necessary to completely identify the phosphorylated residues of the protein to fully unravel the roles of this CIP/Kip protein, which are still partially identified.

When the Embryo Meets the Endometrium: Identifying the Features Required for Successful Embryo Implantation.

Evaluation of the optimal number of embryos, their quality, and the precise timing for transfer are critical determinants in reproductive success, although still remaining one of the main challenges in assisted reproduction technologies (ART). Indeed, the success of in vitro fertilization (IVF) treatments relies on a multitude of events and factors involving both the endometrium and the embryo. Despite concerted efforts on both fronts, the overall success rates of IVF techniques continue to range between 25% and 30%. The role of the endometrium in implantation has been recently recognized, leading to the hypothesis that both the "soil" and the "seed" play a central role in a successful pregnancy. In this respect, identification of the molecular signature of endometrial receptivity together with the selection of the best embryo for transfer become crucial in ART. Currently, efforts have been made to develop accurate, predictive, and personalized tests to identify the window of implantation and the best quality embryo. However, the value of these tests is still debated, as conflicting results are reported in the literature. The purpose of this review is to summarize and critically report the available criteria to optimize the success of embryo transfer and to better understand current limitations and potential areas for improvement.

An improved in vitro model simulating the feto-maternal interface to study developmental effects of potentially toxic compounds: The example of titanium dioxide nanoparticles.

The study of developmental effect of xenobiotics in humans is limited and often relies on epidemiological data. Whether and to which extent potentially toxic compounds may cross the placental barrier, and whether adverse effects on embryo development are the consequence of direct or indirect placental-mediated action is debated. The availability of in vitro models simulating the feto-maternal interface could contribute to elucidate this issue. Here, we report the development of a novel in vitro model using murine blastocyst derived trophoblast stem cells (TSC) to mimic the placental barrier and mouse embryoid bodies (EBs) to represent the embryonic tissues. We demonstrate that this model can be used for translocation studies, as well as embryotoxicity assessment of titanium dioxide nanoparticles (TiO2NPs). By evaluating trans-epithelial electrical resistance, translocation of fluorescein isothiocyanate-dextran beads and expression of junctional complex proteins, we show that TSCs cultured on transwell inserts under differentiating condition form syncytia. We also show that TiO2NPs administered in the upper transwell compartment are able to reach the lower compartment and interfere with EB differentiation when no TSC are cultured on the insert. On the contrary, when TSC are present, NPs translocate to a lesser extent and do not affect EB development. These results indicate that the proposed in vitro model is suitable to study the correlation between translocation and toxicity of TiO2NPs and suggest a direct effect of the particles on EB development. We propose that this model could be exploited to study developmental effect of other xenobiotics.

A practical approach to the management of thyroid dysfunction during pregnancy.

Pregnancy has an important impact on the thyroid gland and its function. Thyroid activity changes as a consequence of the novel physiological state of pregnancy and requires a complex hormonal and metabolic adaptation, which is possible only in the presence of a perfectly functioning thyroid gland. In fact, thyroid function is crucial for the success of the implantation and the progression of pregnancy. Abnormal thyroid function is very common among childbearing age women, explaining the high incidence of thyroid diseases that occur during pregnancy. Aim of this work is to analyze the adaptive events that characterize the thyroid function during pregnancy, exploring their hormonal, metabolic and molecular mechanisms. Moreover, the interpretation of the laboratory data necessary to monitor the thyroid functioning during normal pregnancy or in the presence of thyroid abnormalities will be discussed.

The process of ovarian aging: it is not just about oocytes and granulosa cells.

Ovarian age is classically considered the main cause of female reproductive infertility. In women, the process proceeds as an ongoing decline in the primordial follicle stockpile and it is associated with reduced fertility in the mid-thirties, irregular menstruation from the mid-forties, cessation of fertility, and, eventually, menopause in the early fifties. Reproductive aging is historically associated with changes in oocyte quantity and quality. However, besides the oocyte, other cellular as well as environmental factors have been the focus of more recent investigations suggesting that ovarian decay is a complex and multifaceted process. Among these factors, we will consider mitochondria and oxidative stress as related to nutrition, changes in extracellular matrix molecules, and the associated ovarian stromal compartment where immune cells of both the native and adaptive systems seem to play an important role. Understanding such processes is crucial to design treatment strategies to  slow down ovarian aging and consequently prolong reproductive lifespan and, more to this, alleviaingt side effects of menopause on the musculoskeletal, cardiovascular, and nervous systems.

Silica encapsulation of ZnO nanoparticles reduces their toxicity for cumulus cell-oocyte-complex expansion.

BACKGROUND: Metal oxide nanoparticles (NPs) are increasingly used in many industrial and biomedical applications, hence their impact on occupational and public health has become a concern. In recent years, interest on the effect that exposure to NPs may exert on human reproduction has grown, however data are still scant. In the present work, we investigated whether different metal oxide NPs interfere with mouse cumulus cell-oocyte complex (COC) expansion. METHODS: Mouse COCs from pre-ovulatory follicles were cultured in vitro in the presence of various concentrations of two types of TiO2 NPs (JRC NM-103 and NM-104) and four types of ZnO NPs (JRC NM-110, NM-111, and in-house prepared uncoated and SiO2-coated NPs) and the organization of a muco-elastic extracellular matrix by cumulus cells during the process named cumulus expansion was investigated. RESULTS: We show that COC expansion was not affected by the presence of both types of TiO2 NPs at all tested doses, while ZnO NM-110 and NM-111 induced strong toxicity and inhibited COCs expansion at relatively low concentration. Medium conditioned by these NPs showed lower toxicity, suggesting that, beside ion release, inhibition of COC expansion also depends on NPs per se. To further elucidate this, we compared COC expansion in the presence of uncoated or SiO2-coated NPs. Differently from the uncoated NPs, SiO2-coated NPs underwent slower dissolution, were not internalized by the cells, and showed an overall lower toxicity. Gene expression analysis demonstrated that ZnO NPs, but not SiO2-coated ZnO NPs, affected the expression of genes fundamental for COC expansion. Dosimetry analysis revealed that the delivered-to-cell mass fractions for both NPs was very low. CONCLUSIONS: Altogether, these results suggest that chemical composition, dissolution, and cell internalization are all responsible for the adverse effects of the tested NPs and support the importance of a tailored, safer-by-design production of NPs to reduce toxicity.

Molecular Signaling Regulating Endometrium-Blastocyst Crosstalk.

Implantation of the embryo into the uterine endometrium is one of the most finely-regulated processes that leads to the establishment of a successful pregnancy. A plethora of factors are released in a time-specific fashion to synchronize the differentiation program of both the embryo and the endometrium. Indeed, blastocyst implantation in the uterus occurs in a limited time frame called the "window of implantation" (WOI), during which the maternal endometrium undergoes dramatic changes, collectively called "decidualization". Decidualization is guided not just by maternal factors (e.g., estrogen, progesterone, thyroid hormone), but also by molecules secreted by the embryo, such as chorionic gonadotropin (CG) and interleukin-1ß (IL-1 ß), just to cite few. Once reached the uterine cavity, the embryo orients correctly toward the uterine epithelium, interacts with specialized structures, called pinopodes, and begins the process of adhesion and invasion. All these events are guided by factors secreted by both the endometrium and the embryo, such as leukemia inhibitory factor (LIF), integrins and their ligands, adhesion molecules, Notch family members, and metalloproteinases and their inhibitors. The aim of this review is to give an overview of the factors and mechanisms regulating implantation, with a focus on those involved in the complex crosstalk between the blastocyst and the endometrium.

Relevance to investigate different stages of pregnancy to highlight toxic effects of nanoparticles: The example of silica.

Amorphous silica nanoparticles (SiO2NPs) have been recognized as safe nanomaterial, hence their use in biomedical applications has been explored. Data, however, suggest potential toxicity of SiO2 NPs in pregnant individuals. However, no studies relating nanoparticle biokinetic/toxicity to the different gestational stages are currently available. In this respect, we have investigated the possible embryotoxic effects of three-size and two-surface functionalization SiO2NPs in mice. After intravenous administration of different concentrations at different stages of pregnancy, clinical and histopathological evaluations, performed close to parturition, did not show signs of maternal toxicity, nor effects on placental/fetal development, except for amino-functionalized 25 nm NPs. Biodistribution was studied by ICP-AES 24 h after administration, and demonstrates that all particles distributed to placenta and conceptuses/fetuses, although size, surface charge and gestational stage influenced biodistribution. Our data suggest the need of comprehensive toxicological studies, covering the entire gestation to reliably assess the safety of nanoparticle exposure during pregnancy.

The unrecognized occupational relevance of the interaction between engineered nanomaterials and the gastro-intestinal tract: a consensus paper from a multidisciplinary working group.

BACKGROUND: There is a fundamental gap of knowledge on the health effects caused by the interaction of engineered nanomaterials (ENM) with the gastro-intestinal tract (GIT). This is partly due to the incomplete knowledge of the complex physical and chemical transformations that ENM undergo in the GIT, and partly to the widespread belief that GIT health effects of ENM are much less relevant than pulmonary effects. However, recent experimental findings, considering the role of new players in gut physiology (e.g. the microbiota), shed light on several outcomes of the interaction ENM/GIT. Along with this new information, there is growing direct and indirect evidence that not only ingested ENM, but also inhaled ENM may impact on the GIT. This fact, which may have relevant implications in occupational setting, has never been taken into consideration. This review paper summarizes the opinions and findings of a multidisciplinary team of experts, focusing on two main aspects of the issue: 1) ENM interactions within the GIT and their possible consequences, and 2) relevance of gastro-intestinal effects of inhaled ENMs. Under point 1, we analyzed how luminal gut-constituents, including mucus, may influence the adherence of ENM to cell surfaces in a size-dependent manner, and how intestinal permeability may be affected by different physico-chemical characteristics of ENM. Cytotoxic, oxidative, genotoxic and inflammatory effects on different GIT cells, as well as effects on microbiota, are also discussed. Concerning point 2, recent studies highlight the relevance of gastro-intestinal handling of inhaled ENM, showing significant excretion with feces of inhaled ENM and supporting the hypothesis that GIT should be considered an important target of extrapulmonary effects of inhaled ENM. CONCLUSIONS: In spite of recent insights on the relevance of the GIT as a target for toxic effects of nanoparticles, there is still a major gap in knowledge regarding the impact of the direct versus indirect oral exposure. This fact probably applies also to larger particles and dictates careful consideration in workers, who carry the highest risk of exposure to particulate matter.

New frontiers in nanotoxicology: Gut microbiota/microbiome-mediated effects of engineered nanomaterials.

It has been recently recognized that the gut microbiota, the community of organisms living within the gastrointestinal tract is an integral part of the human body, and that its genoma (the microbiome) interacts with the genes expressed by the cells of the host organism. Several important physiological functions require the cooperation of microbiota/microbiome, whose alterations play an important role in several human diseases. On this basis, it is probable that microbiota/microbiome may in part be involved in many biological effects of engineered nanomaterials (ENMs). There are still few reports on the possible toxicological effects of ENMs on microbiota/microbiome, and on their possible clinical consequences. Available data suggest that several ENMs, including carbon nanotubes (CNTs), titanium dioxide, cerium dioxide, zinc oxide, nanosilica and nanosilver may affect the microbiota and that clinical disorders such as colitis, obesity and immunological dysfunctions might follow. On the other hand, other ENMs such as iron nanoparticles may show advantages over traditional iron-based supplemental treatment because they do not interfere with the microbiota/microbiome, and some ENM-based therapeutic interventions might be employed for treating intestinal infections, while sparing the microbiota. The final section of the review is focused on the possible future developments of the research in this field: new in vitro and in vivo models, possible biomarkers and new pathophysiological pathways are proposed and discussed, as well as the possibility that metabolic changes following ENMs/microbiota interactions might be exploited as a fingerprint of ENM exposure. The potential toxicological relevance of physico-chemical modifications of ENMs induced by the microbiota is also highlighted.

Multi-walled carbon nanotubes directly induce epithelial-mesenchymal transition in human bronchial epithelial cells via the TGF-ß-mediated Akt/GSK-3ß/SNAIL-1 signalling pathway.

BACKGROUND: Multi-walled carbon nanotubes (MWCNT) are currently under intense toxicological investigation due to concern on their potential health effects. Current in vitro and in vivo data indicate that MWCNT exposure is strongly associated with lung toxicity (inflammation, fibrosis, granuloma, cancer and airway injury) and their effects might be comparable to asbestos-induced carcinogenesis. Although fibrosis is a multi-origin disease, epithelial-mesenchymal transition (EMT) is recently recognized as an important pathway in cell transformation. It is known that MWCNT exposure induces EMT through the activation of the TGF-ß/Smad signalling pathway thus promoting pulmonary fibrosis, but the molecular mechanisms involved are not fully understood. In the present work we propose a new mechanism involving a TGF-ß-mediated signalling pathway. METHODS: Human bronchial epithelial cells were incubated with two different MWCNT samples at various concentrations for up to 96 h and several markers of EMT were investigated. Quantitative real time PCR, western blot, immunofluorescent staining and gelatin zymographies were performed to detect the marker protein alterations. ELISA was performed to evaluate TGF-ß production. Experiments with neutralizing anti-TGF-ß antibody, specific inhibitors of GSK-3ß and Akt and siRNA were carried out in order to confirm their involvement in MWCNT-induced EMT. In vivo experiments of pharyngeal aspiration in C57BL/6 mice were also performed. Data were analyzed by a one-way ANOVA with Tukey's post-hoc test. RESULTS: Fully characterized MWCNT (mean length < 5 µm) are able to induce EMT in an in vitro human model (BEAS-2B cells) after long-term incubation at sub-cytotoxic concentrations. MWCNT stimulate TGF-ß secretion, Akt activation and GSK-3ß inhibition, which induces nuclear accumulation of SNAIL-1 and its transcriptional activity, thus contributing to switch on the EMT program. Moreover, a significant increment of nuclear ß-catenin - due to E-cadherin repression and following translocation to nucleus - likely reinforces signalling for EMT promotion. In vivo results supported the occurrence of pulmonary fibrosis following MWCNT exposure. CONCLUSIONS: We demonstrate a new molecular mechanism of MWCNT-mediated EMT, which is Smad-independent and involves TGF-ß and its intracellular effectors Akt/GSK-3ß that activate the SNAIL-1 signalling pathway. This finding suggests potential novel targets in the development of therapeutic and preventive approaches.

Twenty years of in vitro nanotoxicology: how AI could make the difference.

More than two decades ago, the advent of Nanotechnology has marked the onset of a new and critical field in science and technology, highlighting the importance of multidisciplinary approaches to assess and model the potential human hazard of newly developed advanced materials in the nanoscale, the nanomaterials (NMs). Nanotechnology is, by definition, a multidisciplinary field, that integrates knowledge and techniques from physics, chemistry, biology, materials science, and engineering to manipulate matter at the nanoscale, defined as anything comprised between 1 and 100 nm. The emergence of nanotechnology has undoubtedly led to significant innovations in many fields, from medical diagnostics and targeted drug delivery systems to advanced materials and energy solutions. However, the unique properties of nanomaterials, such as the increased surface to volume ratio, which provides increased reactivity and hence the ability to penetrate biological barriers, have been also considered as potential risk factors for unforeseen toxicological effects, stimulating the scientific community to investigate to which extent this new field of applications could pose a risk to human health and the environment.

Maternal exposure to zinc oxide nanoparticles causes cochlear dysfunction in the offspring.

Introduction: Zinc oxide nanoparticles (ZnO NPs) have been engineered and are largely used in material science and industry. This large and increasing use justifies a careful study about the toxicity of this material for human subjects. The concerns regard also the reproductive toxicity and the fetotoxicity. Materials and methods: The effect of the exposure to ZnO NPs on the cochlear function was studied in a group of pregnant CD1 mice and in their offspring. This study is part of a larger toxicological study about the toxicity of ZnO NPs during pregnancy. Four groups were analyzed and compared, exposed and non-exposed dams and their offspring. The cochlear function was quantitatively assessed by means of Distortion Product Otoacoustic Emissions (DPOAEs). Results and discussion: A large statistically significant difference was found between the non-exposed dams offspring and the exposed dams offspring (p = 1.6 · 10-3), whose DPOAE levels were significantly lower than those of non-exposed dams offspring and comparable to those of the adults. The DPOAE levels of the exposed and non-exposed dams were very low and not significantly different. This occurrence is related to the fact that these mice encounter a rapid aging process. Conclusion: Our findings show that maternal exposure to ZnO NPs does not reflect in overt toxicity on fetal development nor impair offspring birth, however it may damage the nervous tissue of the inner ear in the offspring. Other studies should confirm this result and identify the mechanisms through which ZnO NPs may affect ear development.

Chronic endometritis and recurrent reproductive failure: a systematic review and meta-analysis.

Background: The endometrium holds a crucial role in reproduction by supporting blastocyst adhesion, cytotrophoblast invasion and fetal development. Among the various uterine disorders, endometritis, particularly chronic endometritis (CE), has gained attention due to its association with adverse reproductive outcomes (recurrent pregnancy loss (RPL), recurrent implantation failure (RIF), and infertility). The association between CE and adverse reproductive outcomes stresses the necessity for comprehensive diagnostic and therapeutic strategies to optimize fertility outcomes and support individuals in their journey towards parenthood. Aim: To explore the relationship between CE and reproductive disorders. Methods: Following PRISMA guidelines, a systematic review and meta-analysis using published data from 1990 to 2024 were carried out. Results: A population of 1,038 women was included. Regarding CE-infertility association, a positive correlation was found, with 19.46% CE rate in infertile women compared to 7.7% in controls (OR: 2.96, 95% CI 1.53-5.72, p 0.001). No significant association was observed between RIF and CE (OR: 1.10, 95% CI 0.26-4.61, p 0.90), CE rates in both groups were relatively comparable, with 6.35% in women with RIF and 5.8% in controls. On the opposite, a strong association between CE and RPL was found, reporting a CE rate of 37.6% in RPL cases compared to 16.4% in controls (OR: 3.59, 95% CI 2.46-5.24, p < 0.00001). Conclusions: CE appears to be associated to infertility and RPL, while no significant association was noted in cases of RIF. Systematic review registration: https://www.crd.york.ac.uk/prospero/#recordDetails PROSPERO, identifier CRD42024541879.

Effect of Pravastatin on Placental Expression of Epidermal Growth Factor-like Domain 7 in Early-Onset Pre-Eclampsia: A New Potential Mechanism of Action.

The primary intervention for pre-eclampsia (PE) remains iatrogenic delivery, which can be very preterm and not optimal for the fetus. Although many efforts have been made to prevent and manage PE, there is still a dearth of drugs to treat its pathophysiological progression. Pravastatin (PRA), a hydrophilic statin, has gained interest for the prevention and treatment of PE. The aim of the present study was to evaluate the ability of PRA to modulate factors involved in placentation, such as Epidermal Growth Factor-Like Domain 7 (EGFL7), in human chorionic villous culture from healthy controls and women with PE. A total of 18 women were enrolled: 10 controls and 8 cases. Chorionic villous explants were maintained in culture for 24 h with or without 10 µM Pravastatin, and the expression of EGFL7 and NOTCH1 pathway members was evaluated by qRT-PCR and Western blot analysis. The rationale of the present study was to establish an ex vivo model to identify potential different responses to PRA treatment of chorionic villous explants in order to clarify the molecular mechanism of PRA in the prevention and treatment of PE and to predict whether there are specific clinical conditions that modulate the response to the drug treatment. Within PE patients, two different groups were identified: the high responders, whose villous cultures exhibit significantly increased expressions of the EGFL7 and Notch pathways after PRA incubation; and the low responders, who are high-risk PE patients in which prophylaxis failed to prevent PE and PRA was not able to modulate EGFL7 expression. In conclusion, we identified EGFL7 as a new factor regulated by PRA, placing interest in early discrimination between low- and high- risk women, in which the well-known pharmacological prophylaxis seems to be ineffective, and to explore new potential prevention strategies.

Interactions of engineered nanoparticles with organs protected by internal biological barriers.

Engineered nanomaterials may exert adverse effects on human health which, in turn, may be linked to their propensity to cross biological barriers in the body. Here, available evidence is discussed, based on in vivo studies for interactions of commercially relevant nanoparticles with critical internal barriers. The internal barriers in focus in this review are the blood-brain barrier, protecting the brain, the blood-testis barrier, protecting the male germ line, and the placenta, protecting the developing fetus. The route of exposure (pulmonary, gastro-intestinal, intravenous, intraperitoneal, dermal), and, hence, the portal of entry of nanoparticles into the body, is of critical importance. Different physico-chemical properties, not only size, may determine the ability of nanoparticles to breach biological barriers; the situation is further compounded by the formation of a so-called corona of biomolecules on the surfaces of nanoparticles, the composition of which may vary depending on the route of exposure and the translocation of nanoparticles from one biological compartment to another. The relevance of nanoparticle interactions with internal biological barriers for their impact on the organs protected by these barriers is also discussed.

Biodistribution and toxicity of pegylated single wall carbon nanotubes in pregnant mice.

BACKGROUND: Single wall carbon nanotubes (SWCNTs) are considered promising nanoparticles for industrial and biomedical applications; however their potential toxicity in several biological systems, including the feto-placental unit, has been demonstrated. Functionalization of SWCNTs with polyethylene glycol chains (PEG-SWCNTs) dramatically reduces their toxicity, and for this reason PEG-SWCNTs are candidates for biomedical applications. However, no data are available on their safety for the developing embryo, in spite of the clinical and social relevance of this topic. The purpose of this study is therefore to investigate the safety of PEG-SWCNTs for their use as biomedical carriers in pregnancy. METHODS: For toxicological studies, amino-functionalized PEG-SWCNT were intravenously injected in CD1 pregnant mice at different doses (range 0.1-30 µg/mouse), in single or multiple administrations. For biodistribution studies, fluorescently labeled PEG-SWCNTs were obtained by acylation of terminal PEG amino groups with near infrared emitting fluorochromes (PEG-SWCNT-750) and injected at the dosage of 10 µg/mouse, at either day 5.5 (when the placenta is still developing) or day 14.5 of gestation (when the maturation of the placenta is complete). RESULTS: We found no adverse effects both on embryos and dams up to the dose of 10 µg/mouse. At the dose of 30 µg/mouse, occasional teratogenic effects, associated with placental damage, were detected both when administered as a single bolus (1 out of 10 dams; 1 malformed embryo) or as multiple doses (2 out of 10 dams; 5 malformed embryos). The difference in the prevalence of dams with malformed embryos between the 30 µg exposed group and controls approached the statistical significance (p = 0.06). Hepatic damage in dams was seen only in the multiple exposure group (4 out of 10; p = 0.04 when compared with the single exposure group or controls). PEG-SWCNT-750 reached the conceptus when administered early in pregnancy. At later stages, PEG-SWCNT-750 were detected in the placenta and the yolk sac, but not in the embryo. CONCLUSIONS: PEG-SWCNTs may cause occasional teratogenic effects in mice beyond a threshold dose. Such effect might depend on their ability to reach the feto-placenta unit. Although not automatically transferable to humans, these data should be considered if exposing women during pregnancy.

Leptin attenuates ischemia-reperfusion injury in the rat liver.

Leptin is an adipocytokine that reduces ischemic damage in several organs including brain and heart. STAT3 activation is a key step for the attainment of leptin effects in various tissues. We evaluated the possible effect of leptin on liver viability and STAT3 activation, in a rat model of ischemia-reperfusion injury. Rat livers, flushed and stored with Belzer solution (4° C for 24 h), were warmly reperfused (3.5 ml/min/g liver for 1 h at 37° C with O(2) ) with Krebs-Ringer bicarbonate. Treatment group underwent an identical protocol with the adjunct of Leptin (10 ng/ml). Liver effluent was harvested to assess LDH and AST output. Liver tissue was used for pSTAT3 expression (western blot and immunostaining), optical microscopy, TUNEL, and Cell Death Detection assays. The pSTAT3 expression was enhanced by administration of leptin. In parallel, LDH and AST output were reduced (P = 0.04 and P = 0.02 for LDH and AST, respectively). Optical microscopy, TUNEL, and Cell Death Detection assay results demonstrated increased viability in livers treated with leptin in comparison with others (Optical microscopy P = 0.02; TUNEL P = 0.01; Cell death Detection assay P = 0.003). In conclusion, cold storage and reperfusion with leptin reduce liver ischemia-reperfusion injury. This effect is associated with an increased expression of pSTAT-3.

Predicting the cytotoxicity of nanomaterials through explainable, extreme gradient boosting.

Nanoparticles (NPs) are a wide class of materials currently used in several industrial and biomedical applications. Due to their small size (1-100 nm), NPs can easily enter the human body, inducing tissue damage. NP toxicity depends on physical and chemical NP properties (e.g., size, charge and surface area) in ways and magnitudes that are still unknown. We assess the average as well as the individual importance of NP atomic descriptors, along with chemical properties and experimental conditions, in determining cytotoxicity endpoints for several nanomaterials. We employ a multicenter cytotoxicity nanomaterial database (12 different materials with first and second dimensions ranging between 2.70 and 81.2 nm and between 4.10 and 4048 nm, respectively). We develop a regressor model based on extreme gradient boosting with hyperparameter optimization. We employ Shapley additive explanations to obtain good cytotoxicity prediction performance. Model performances are quantified as statistically significant Spearman correlations between the true and predicted values, ranging from 0.5 to 0.7. Our results show that i) size in situ and surface areas larger than 200 nm and 50 m2/g, respectively, ii) primary particles smaller than 20 nm; iii) irregular (i.e., not spherical) shapes and iv) positive Z-potentials contribute the most to the prediction of NP cytotoxicity, especially if lactate dehydrogenase (LDH) assays are employed for short experimental times. These results were moderately stable across toxicity endpoints, although some degree of variability emerged across dose quantification methods, confirming the complexity of nano-bio interactions and the need for large, systematic experimental characterization to reach a safer-by-design approach.

Pre-validation of a reporter gene assay for oxidative stress for the rapid screening of nanobiomaterials.

Engineered nanomaterials have been found to induce oxidative stress. Cellular oxidative stress, in turn, can result in the induction of antioxidant and detoxification enzymes which are controlled by the nuclear erythroid 2-related factor 2 (NRF2) transcription factor. Here, we present the results of a pre-validation study which was conducted within the frame of BIORIMA ("biomaterial risk management") an EU-funded research and innovation project. For this we used an NRF2 specific chemically activated luciferase expression reporter gene assay derived from the human U2OS osteosarcoma cell line to screen for the induction of the NRF2 mediated gene expression following exposure to biomedically relevant nanobiomaterials. Specifically, we investigated Fe3O4-PEG-PLGA nanomaterials while Ag and TiO2 "benchmark" nanomaterials from the Joint Research Center were used as reference materials. The viability of the cells was determined by using the Alamar blue assay. We performed an interlaboratory study involving seven different laboratories to assess the applicability of the NRF2 reporter gene assay for the screening of nanobiomaterials. The latter work was preceded by online tutorials to ensure that the procedures were harmonized across the different participating laboratories. Fe3O4-PEG-PLGA nanomaterials were found to induce very limited NRF2 mediated gene expression, whereas exposure to Ag nanomaterials induced NRF2 mediated gene expression. TiO2 nanomaterials did not induce NRF2 mediated gene expression. The variability in the results obtained by the participating laboratories was small with mean intra-laboratory standard deviation of 0.16 and mean inter laboratory standard deviation of 0.28 across all NRF2 reporter gene assay results. We conclude that the NRF2 reporter gene assay is a suitable assay for the screening of nanobiomaterial-induced oxidative stress responses.