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Elisabetta Esposito,1 Elena Pozza,2 Catia Contado,1 Walter Pula,1 Olga Bortolini,3 Daniele Ragno,1 Sofia Toldo,3 Fabio Casciano,4 Agnese Bondi,1 Enrico Zauli,2 Paola Secchiero,4 Giorgio Zauli,3 Elisabetta Melloni4 1Department of Chemical, Pharmaceutical and Agricultural Sciences, University of Ferrara, Ferrara, I-44121, Italy; 2Department of Translational Medicine, University of Ferrara, Ferrara, I-44121, Italy; 3Department of Environmental Sciences and Prevention, University of Ferrara, Ferrara, I-44121, Italy; 4Department of Translational Medicine and LTTA Centre, University of Ferrara, Ferrara, I-44121, ItalyCorrespondence: Elisabetta Esposito; Elisabetta Melloni, Tel +39 0532 455230 ; +39 0532 455936, Email ese@unife.it; elisabetta.melloni@unife.itPurpose: Proliferative vitreoretinal diseases (PVDs) represent a heterogeneous group of pathologies characterized by the presence of retinal proliferative membranes, in whose development retinal pigment epithelium (RPE) is deeply involved. As the only effective treatment for PVDs at present is surgery, we aimed to investigate the potential therapeutic activity of Nutlin-3a, a small non-genotoxic inhibitor of the MDM2/p53 interaction, on ARPE-19 cell line and on human RPE primary cells, as in vitro models of RPE and, more importantly, to formulate and evaluate Nutlin-3a loaded liposomes designed for ophthalmic administration.Methods: Liposomes were produced using an innovative approach by a microfluidic device under selection of different conditions. Liposome size distribution was evaluated by photon correlation spectroscopy and centrifugal field flow fractionation, while the liposome structure was studied by transmission electron microscopy and Fourier-transform infrared spectroscopy. The Nutlin-3a entrapment capacity was evaluated by ultrafiltration and HPLC. Nutlin-3a biological effectiveness as a solution or loaded in liposomes was evaluated by viability, proliferation, apoptosis and migration assays and by morphological analysis.Results: The microfluidic formulative study enabled the selection of liposomes composed of phosphatidylcholine (PC) 5.4 or 8.2 mg/mL and 10% ethanol, characterized by roundish vesicular structures with 150– 250 nm mean diameters. Particularly, liposomes based on the lower PC concentration were characterized by higher stability. Nutlin-3a was effectively encapsulated in liposomes and was able to induce a significant reduction of viability and migration in RPE cell models.Conclusion: Our results lay the basis for a possible use of liposomes for the ocular delivery of Nutlin-3a. Keywords: Nutlin-3a, liposomes, microfluidic, PVDs, CFFF

This study evaluated ethosomes as a novel nanodelivery system for nutlin-3a, a known MDM2 inhibitor and activator of the p53 pathway, to improve nutlin-3a's poor solubility, limiting its bio-distribution and therapeutic efficacy. The potential of nutlin-3a-loaded ethosomes was investigated on two in vitro models of melanoma: the HT144 cell line p53 wild-type and the SK-MEL-28 cell line p53 mutated . Nutlin-3a-loaded ethosomes were characterized for their physicochemical properties and used to treat melanoma cells at different concentrations, considering nutlin-3a solution and empty ethosomes as controls. The biological effects on cells were evaluated 24 and 48 h after treatment by analyzing the cell morphology and viability, cell cycle, and apoptosis rate using flow cytometry and the p53 pathway's activation via Western blotting. The results indicate that ethosomes are delivery systems able to maintain nutlin-3a's functionality and specific biological action, as evidenced by the molecular activation of the p53 pathway and the biological events leading to cell cycle block and apoptosis in p53 wild-type cells. Nutlin-3a-loaded ethosomes induced morphological changes in the HT144 cell line, with evident apoptotic cells and a reduction in the number of viable cells of over 80%. Furthermore, nutlin-3a-loaded ethosomes successfully modulated two p53-regulated proteins involved in survival/apoptosis, with up to a 2.5-fold increase in membrane TRAIL-R2 and up to an 8.2-fold decrease in Notch-1 (Notch intracellular domain, NICD) protein expression. The expression of these molecules is known to be altered or dysfunctional in a large percentage of melanoma tumors. Notably, ethosomes, regardless of their nutlin-3a loading, exhibited the ability to reduce HT144 melanoma cellular migration, as assessed in real time using xCELLigence, likely due to the modification of lipid rafts, suggesting their potential antimetastatic properties. Overall, nutlin-3a delivery using ethosomes appears to be a significantly effective means for upregulating the p53 pathway and downregulating active Notch-1, while also taking advantage of their unexpected ability to reduce cellular migration. The findings of this study could pave the way for the development of specific nutlin-3a-loaded ethosome-based medicinal products for cutaneous use.

Over the last decades, various tissue-clearing techniques have broken the ground for the optical imaging of whole organs and whole-organisms, providing complete representative data sets of three-dimensional biological structures. Along with advancements in this field, the development of fluorescent markers for staining vessels and capillaries has offered insights into the complexity of vascular networks and their impact on disease progression. Here we describe the use of a modified water-soluble chitosan linked to cyanine dyes in combination with ethyl cinnamate-based optical tissue clearing for the 3D visualization of tissue vasculature in depth. The water-soluble fluorescent Chitosans have proven to be an optimal candidate for labeling both vessels and capillaries ex vivo thanks to their increased water solubility, high photostability, and optical properties in the near-infrared window. In addition, the nontoxicity of these markers broadens their applicability to in vitro and in vivo biological applications. Despite the availability of other fluorescent molecules for vascular staining, the present study, for the first time, demonstrates the potential of fluorescent chitosans to depict vessels at the capillary level and opens avenues for advancing the diagnostic field by reducing the complexity and costs of the currently available procedures., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Co-published by Nanjing University and American Chemical Society.)

Microplastics (MPs) are widespread contaminants highly persistent in the environment and present in matrices to which humans are extensively exposed, including food and beverages. MP ingestion occurs in adults and children and is becoming an emerging public health issue. The gastrointestinal system is the most exposed to MP contamination, which can alter its physiology starting from changes in the microbiome. This study investigates by an omic approach the impact of a single intake of a mixture of polyethylene (PE) and polystyrene (PS) MPs on the ecology and metabolic activity of the colon microbiota of healthy volunteers, in an in vitro intestinal model. PE and PS MPs were pooled together in a homogeneous mix, digested with the INFOGEST system, and fermented with MICODE (multi-unit in vitro colon model) at loads that by literature correspond to the possible intake of food-derived MPs of a single meal. Results demonstrated that MPs induced an opportunistic bacteria overgrowth (Enterobacteriaceae, Desulfovibrio spp., Clostridium group I and Atopobium - Collinsella group) and a contextual reduction on abundances of all the beneficial taxa analyzed, with the sole exception of Lactobacillales. This microbiota shift was consistent with the changes recorded in the bacterial metabolic activity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Herein, we report the synthesis of a new hybrid compound based on a 2'-deoxyuridine nucleoside conjugated with a NO photo-donor moiety (dU-t-NO) via CuAAC click chemistry. Hybrid dU-t-NO, as well as two previously reported 2'-deoxyadenosine based hybrids (dAdo-S-NO and dAdo-t-NO), were evaluated for their cytotoxic and cytostatic activities in selected cancer cell lines. dAdo-S-NO and dAdo-t-NO hybrids displayed higher activity with respect to dU-t-NO. All hybrids showed effective release of NO in the micromolar range. The photochemical behavior of the newly reported hybrid, dU-t-NO, was studied in the RKO colon carcinoma cell line, whereas the dAdo-t-NO hybrid was tested in both colon carcinoma RKO and hepatocarcinoma Hep 3B2.1-7 cell lines to evaluate the potential effect of NO released upon irradiation on cell viability. A customized irradiation apparatus for in vitro experiments was also designed.

In this work, the impact of gluten free (GF) breads enriched with spirulina on the ecology of the colon microbiota of non-celiac volunteers was investigated. Simulation of digestion of GF breads was conducted with an in vitro gut model. Microbiomics and metabolomics analyses were done during colon fermentations to study the modulation of the microbiota. From the results, a general increase in Proteobacteria and no reduction of detrimental microbial metabolites were observed in any conditions. Notwithstanding, algae enriched sourdough breads showed potential functionalities, as the improvement of some health-related ecological indicators, like i) microbiota eubiosis; ii) production of bioactive volatile organic fatty acids; iii) production of bioactives terpenes. Our results indicate that a sourdough fermentation and algae enrichment can mitigate the negative effect of GF breads on gut microbiota of non-celiac consumers., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Neurodegenerative diseases are an increasingly common group of diseases that occur late in life with a significant impact on personal, family, and economic life. Among these, Alzheimer's disease (AD) and Parkinson's disease (PD) are the major disorders that lead to mild to severe cognitive and physical impairment and dementia. Interestingly, those diseases may show onset of prodromal symptoms early after middle age. Commonly, the evaluation of these neurodegenerative diseases is based on the detection of biomarkers, where functional and structural magnetic resonance imaging (MRI) have shown a central role in revealing early or prodromal phases, although it can be expensive, time-consuming, and not always available. The aforementioned diseases have a common impact on the visual system due to the pathophysiological mechanisms shared between the eye and the brain. In Parkinson's disease, α-synuclein deposition in the retinal cells, as well as in dopaminergic neurons of the substantia nigra, alters the visual cortex and retinal function, resulting in modifications to the visual field. Similarly, the visual cortex is modified by the neurofibrillary tangles and neuritic amyloid β plaques typically seen in the Alzheimer's disease brain, and this may reflect the accumulation of these biomarkers in the retina during the early stages of the disease, as seen in postmortem retinas of AD patients. In this light, the ophthalmic evaluation of retinal neurodegeneration could become a cost-effective method for the early diagnosis of those diseases, overcoming the limitations of functional and structural imaging of the deep brain. This analysis is commonly used in ophthalmic practice, and interest in it has risen in recent years. This review will discuss the relationship between Alzheimer's disease and Parkinson's disease with retinal degeneration, highlighting how retinal analysis may represent a noninvasive and straightforward method for the early diagnosis of these neurodegenerative diseases.

The recent attention to the risk of potential permanent eye damage triggered by ocular infections has been leading to a deeper investigation of the current antimicrobials. An antimicrobial agent used in ophthalmology should possess the following characteristics: a broad antimicrobial spectrum, prompt action even in the presence of organic matter, and nontoxicity. The objective of this study is to compare the antimicrobial efficacy of widely used ophthalmic antiseptics containing povidone-iodine, chlorhexidine, and liposomes containing ozonated sunflower oil. We determined the minimum inhibitory concentration (MIC) on various microbial strains: Staphylococcus aureus (ATCC 6538), methicillin-resistant Staphylococcus aureus (ATCC 33591), Staphylococcus epidermidis (ATCC 12228), Pseudomonas aeruginosa (ATCC 9027), and Escherichia coli (ATCC 873). Furthermore, we assessed its efficacy in controlling antibiotic resistance, biofilm formation, and bacterial adhesion. All three antiseptic ophthalmic preparations showed significant anti-microbicidal and anti-biofilm activity, with the liposomes containing ozonated sunflower oil with the highest ability to control antibiotic resistance and bacteria adhesion to human corneal cells.

Background: The infection and negative effects of the SARS-CoV-2 (severe acute respiratory syndrome coronavirus) virus are mitigated by vaccines. It is unknown whether vaccination has worked by eliciting robust protective innate immune responses with high affinity., Methods: Twenty healthy volunteers received three doses of Comirnaty (Pfizer Australia Pty Ltd.) and were evaluated 9 months after the second vaccination and 1 month after the booster dose. The exclusion criteria were the presence of adverse effects following the vaccination, a history of smoking, and heterologous immunization. The inclusion criteria were the absence of prior Coronavirus Disease (COVID)-19 history, the absence of adverse effects, and the absence of comorbidities. Specific phenotype and levels of CD107a and granzyme production by blood NK (natural killer) cells were analyzed after exposure to SARS-CoV-2 spike antigen (Wuhan, Alpha B.1.1.7, Delta B.1.617.2, and Omicron B1.1.529 variants), and related with anti-SARS-CoV-2 antibody production., Results: The booster dose caused early NK CD56dim subset activation and memory-like phenotype., Conclusions: We report the relevance of the innate immune response, especially NK cells, to SARS-CoV-2 vaccines to guarantee efficient protection against the infection following a booster dose., Competing Interests: The authors declare no conflict of interest., (© 2023 The Author(s). Published by IMR Press.)

The pivotal role of p53 in the regulation of a vast array of cellular functions has been the subject of extensive research. The biological activity of p53 is not strictly limited to cell cycle arrest but also includes the regulation of homeostasis, DNA repair, apoptosis, and senescence. Thus, mutations in the p53 gene with loss of function represent one of the major mechanisms for cancer development. As expected, due to its key role, p53 is expressed throughout the human body including the eye. Specifically, altered p53 signaling pathways have been implicated in the development of conjunctival and corneal tumors, retinoblastoma, uveal melanoma, and intraocular melanoma. As non-selective cancer chemotherapies as well as ionizing radiation can be associated with either poor efficacy or dose-limiting toxicities in the eye, reconstitution of the p53 signaling pathway currently represents an attractive target for cancer therapy. The present review discusses the role of p53 in the pathogenesis of these ocular tumors and outlines the various pharmacological activators of p53 that are currently under investigation for the treatment of ocular malignancies.

Herein, we investigated the stability and bioaccessibility of phenolics in differently cooked red-skinned onion (RSO) and consequently their impact on the gut microbiota and metabolism of phenolics. In fact, the different processes used to cook vegetables can modify and re-arrange the molecular profiles of bioactive compounds, such as phenolics in phenolic-rich vegetables, such as RSO. Fried and grilled RSO were compared to raw RSO and a blank control and subjected to oro-gastro-intestinal digestion and subsequent colonic fermentation. For upper gut digestion, the INFOGEST protocol was used, and for lower gut fermentation, a short-term batch model, namely, MICODE (multi-unit in vitro colon gut model), was employed. During the process, phenolic compound profile (through high-resolution mass spectrometry) and colon microbiomics (qPCR of 14 core taxa) analyses were performed. According to the results, the degradation driven by the colon microbiota of RSO flavonols resulted in the accumulation of three main metabolites, i.e. , 3-(3'-hydroxyphenyl)propanoic acid, 3-(3'-hydroxyphenyl)acetic acid and 3-(3',4'-dihydroxyphenyl)acetic acid. Also, colonic fermentation of raw onions resulted in a substantial increase in beneficial taxa, which was larger compared to the heat-treated onions, particularly Lactobacillales and beneficial clostridia. Also, a higher level of inhibition of opportunistic bacteria was seen for the raw onion samples, namely, Clostridium perfringens group and Escherichia coli . Thus, our results showed that RSO, and especially the raw one, is an excellent dietary source of flavonols that are strongly metabolized by gut bacteria and can positively modulate the gut microbiota. Although additional in vivo studies are necessary, this work is one of the first to explore how RSO processed with different cooking methods can differently impact the phenolic metabolism and microbiota composition in the large intestine of humans, fine-tuning the antioxidant nature of foods.

Psoriasis is a chronic inflammatory skin disease with an autoimmune component and associated with joint inflammation in up to 30% of cases. To investigate autoreactive T cells, we developed an imiquimod-induced psoriasis-like inflammation model in K5-mOVA.tg C57BL/6 mice expressing ovalbumin (OVA) on the keratinocyte membrane, adoptively transferred with OT-I OVA-specific CD8 + T cells. We evaluated the expansion of OT-I CD8 + T cells and their localization in skin, blood, and spleen. scRNA-seq and TCR sequencing data from patients with psoriatic arthritis were also analyzed. In the imiquimod-treated K5-mOVA.tg mouse model, OT-I T cells were markedly expanded in the skin and blood at early time points. OT-I T cells in the skin showed mainly CXCR3 + effector memory phenotype, whereas in peripheral blood there was an expansion of CCR4 + CXCR3 + OT-I cells. At a later time point, expanded OVA-specific T-cell population was found in the spleen. In patients with psoriatic arthritis, scRNA-seq and TCR sequencing data showed clonal expansion of CCR4 + T CM cells in the circulation and further expansion in the synovial fluid. Importantly, there was a clonotype overlap between CCR4 + T CM in the peripheral blood and CD8 + T-cell effectors in the synovial fluid. This mechanism could play a role in the generation and spreading of autoreactive T cells to the synovioentheseal tissues in psoriasis patients at risk of developing psoriatic arthritis., (© 2023 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

Per- and polyfluoroalkyl substances can be referred to as the most critical group of contaminants of emerging concern. They can accumulate in high concentration in the kidney and are known to potentially affect its function. Nonetheless, there is a lack of knowledge about their morphopathological effect on the glomerular filtration barrier. Since previous research suggests perfluorooctanoic acid (PFOA) induces glomerular protein leakage, the glomerular filtration barrier of 30 carp from the same parental stock (10 unexposed; 10 exposed to 200 ng L -1 of PFOA; and 10 exposed to 2 mg L -1 of PFOA for 56 days) was screened for possible PFOA-induced ultrastructural lesions in order to shed light on the related pathophysiology. PFOA exposure affected the glomerular filtration barrier in carp experimentally exposed to 2 mg L -1 , showing ultrastructural alterations compatible with glomerulonephrosis: podocyte effacement, reduction of filtration slits and filtration slit diaphragms, basement membrane disarrangement, and occurrence of proteinaceous material in the urinary space. The results of the present research confirm the glomerular origin of the PFOA-induced protein leakage and can contribute to the mechanistic comprehension of PFOA's impact on renal function and to the assessment of the exposure effect of environmental pollutants on animals and humans, according to the One Health approach.

The olfactory bulb (OB) is one of two regions of the mammalian brain which undergo continuous neuronal replacement during adulthood. A significant fraction of the cells added in adulthood to the bulbar circuitry is constituted by dopaminergic (DA) neurons. We took advantage of a peculiar property of dopaminergic neurons in transgenic mice expressing eGFP under the tyrosine hydroxylase (TH) promoter: while DA neurons located in the glomerular layer (GL) display full electrophysiological maturation, eGFP+ cells in the mitral layer (ML) show characteristics of immature cells. In addition, they also display a lower fluorescence intensity, possibly reflecting different degrees of maturation. To investigate whether this difference in maturation might be confirmed at the gene expression level, we used a fluorescence-activated cell sorting technique on enzymatically dissociated cells of the OB. The cells were divided into two groups based on their level of fluorescence, possibly corresponding to immature ML cells and fully mature DA neurons from the GL. Semiquantitative real-time PCR was performed to detect the level of expression of genes linked to the degree of maturation of DA neurons. We showed that indeed the cells expressing low eGFP fluorescence are immature neurons. Our method can be further used to explore the differences between these two groups of DA neurons.

Hemp seed bran (HB) is an industrial food byproduct that is generally discarded. Knowledge on the functional capabilities of HB is limited and it is not known the impact of HB on human colon microbiota, where vegetable fibers are metabolized. In this work, we investigated in depth the prebiotic potential of HB and HB protein extract hydrolyzed by alcalase (HBPA) in comparison to fructooligosaccharides (FOS) after human distal colonic fermentation using MICODE (multi-unit in vitro colon gut model). During the 24 h of fermentation, metabolomics (SPME GC/MS) and microbiomics (MiSeq and qPCR) analyses were performed. The results indicated that HBPA on a colonic fermentation had a higher prebiotic index than HB (p < 0.05), and slightly lower to that of FOS (p > 0.05). This feature was described and explained as HBPA colonic fermentation produces beneficial organic fatty acids (e.g. Pentanoic and Hexanoic acids); reduces detrimental phenol derivates (e.g. p-Cresol); produces bioactives VOCs (e.g. Acetophenone or 4-Terpineol); increases beneficial bacteria (e.g. 1.76 fold and 2.07 fold more of Bifidobacterium bifidum and Bacteroides fragilis, respectively) and limits opportunistic bacteria (e.g. 3.04 fold and 2.07 fold less of Bilophila wadsworthia and Desulfovibrio, respectively). Our study evidenced the prebiotic role of HB and HBPA, and within the principles of OneHealth it valorizes a byproduct from the queen plant of sustainable crops as a food supplement., (© 2023. The Author(s).)

Despite curcumin (CUR) inhibiting cell proliferation in vitro by activating apoptotic cell death, its use in pharmacological therapy is hampered by poor solubility, low stability in biological fluids, and rapid removal from the body. Therefore, CUR-derivatives with better biological and chemical-physical characteristics are needed. The bis-ketone moiety of CUR strongly influences its stability in slightly alkaline solutions such as plasma. Here, we considered its replacement with isoxazole, beta-enamine, or oxime groups to obtain more stable derivatives. The evaluation of the chemical-physical characteristics showed that only of the isoxazole derivatives 2 and 22 had better potential than CUR in terms of bioavailability. The UV-visible spectrum analysis showed that derivatives 2 and 22 had better stability than CUR in solutions mimicking the biological fluids. When tested on a panel of cell lines, derivatives 2 and 22 had marked cytotoxicity (IC50 = 0.5 µM) compared with CUR only (IC50 = 17 µM) in the chronic myeloid leukemia (CML)-derived K562 cell line. The derivative 22 was the more selective for CML cells. When administered at the average concentration found for CUR in the blood of patients, derivatives 2 and 22 had potent effects on cell cycle progression and apoptosis initiation, while CUR was ineffective. The apoptotic effect of derivatives 2 and 22 was associated with low necrosis. In addition, derivative 22 was able to reverse drug resistance in K562 cells resistant to imatinib (IM), the reference drug used in CML therapy. The cytotoxicity of derivative 22 on IM-sensitive and resistant cells was associated with upregulation of FOXN3 and CDKN1A expression, G2/M arrest, and triggering of apoptosis. In conclusion, derivative 22 has chemical-physical characteristics and biological effects superior to CUR, which allow us to hypothesize its future use in the therapy of CML and CML forms resistant to IM, either alone or in combination with this drug.

SARS-CoV viruses have been shown to downregulate cellular events that control antiviral defenses. They adopt several strategies to silence p53, key molecule for cell homeostasis and immune control, indicating that p53 has a central role in controlling their proliferation in the host. Specific actions are the stabilization of its inhibitor, MDM2, and the interference with its transcriptional activity. The aim of our work was to evaluate a new approach against SARS-CoV-2 by using MDM2 inhibitors to raise p53 levels and activate p53-dependent pathways, therefore leading to cell cycle inhibition. Experimental setting was performed in the alveolar basal epithelial cell line A549-hACE2, expressing high level of ACE2 receptor, to allow virus entry, as well as p53 wild-type. Cells were treated with several concentrations of Nutlin-3 or RG-7112, two known MDM2 inhibitors, for the instauration of a cell cycle block steady-state condition before and during SARS-CoV-2 infection, and for the evaluation of p53 activation and impact on virus release and related innate immune events. The results indicated an efficient cell cycle block with inhibition of the virion release and a significant inhibition of IL-6, NF-kB and IFN-λ expression. These data suggest that p53 is an efficient target for new therapies against the virus and that MDM2 inhibitors deserve to be further investigated in this field., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lodi, Gentili, Casciano, Romani, Zauli, Secchiero, Zauli, Simioni, Beltrami, Fernandez, Rizzo and Voltan.)

Hyperglycaemia and increased circulating saturated fatty acids are key metabolic features of type 2 diabetes mellitus (T2DM) that contribute to diabetic retinopathy pathogenesis. Contrarily, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) has been shown to improve or prevent T2DM. This study aimed at investigating the effect of TRAIL in an in vitro model of human retinal pigment epithelium: the ARPE-19 cell line, treated with palmitic acid (PA) in the presence of high glucose concentration. PA caused a drop in cellular metabolic activity and cell viability as well as an increase in apoptosis rates, which were paralleled by an upregulation of reactive oxygen species (ROS) generation as well as mitochondrial fragmentation. Despite ARPE-19 cells expressing TRAIL-R2 at the cell surface, TRAIL failed to counteract the cytotoxic effects of PA. However, when TRAIL was used alongside PA and then removed or used alone following PA challenge, it partially attenuated PA-induced lipotoxicity. This effect of TRAIL appeared to rely upon the modulation of inflammation and ROS production. Thus, TRAIL exerted a trophic effect on ARPE-19 cells, which became evident only when the lipotoxic insult was removed. Nevertheless, whether recombinant TRAIL might have a therapeutic potential for the treatment of diabetic retinopathy requires further investigation.

The first weeks of life represent a crucial stage for microbial colonization of the piglets' gastrointestinal tract. Newborns' microbiota is unstable and easily subject to changes under stimuli or insults. Nonetheless, the administration of antibiotics to the sow is still considered as common practice in intensive farming for pathological conditions in the postpartum. Therefore, transfer of antibiotic residues through milk may occurs, affecting the piglets' colon microbiota. In this study, we aimed to extend the knowledge on antibiotic transfer through milk, employing an in vitro dedicated piglet colon model (MICODE-Multi Unit In vitro Colon Model). The authors' focus was set on the shifts of the piglets' microbiota composition microbiomics (16S r-DNA MiSeq and qPCR-quantitative polymerase chain reaction) and on the production of microbial metabolites (SPME GC/MS-solid phase micro-extraction gas chromatography/mass spectrometry) in response to milk with different concentrations of amoxicillin. The results showed an effective influence of amoxicillin in piglets' microbiota and metabolites production; however, without altering the overall biodiversity. The scenario is that of a limitation of pathogens and opportunistic taxa, e.g., Staphylococcaceae and Enterobacteriaceae, but also a limitation of commensal dominant Lactobacillaceae, a reduction in commensal Ruminococcaceae and a depletion in beneficial Bifidobactericeae. Lastly, an incremental growth of resistant species, such as Enterococcaceae or Clostridiaceae, was observed. To the authors' knowledge, this study is the first evaluating the impact of antibiotic residues towards the piglets' colon microbiota in an in vitro model, opening the way to include such approach in a pipeline of experiments where a reduced number of animals for testing is employed. KEY POINTS: • Piglet colon model to study antibiotic transfer through milk. • MICODE resulted a robust and versatile in vitro gut model. • Towards the "3Rs" Principles to replace, reduce and refine the use of animals used for scientific purposes (Directive 2010/63/UE)., (© 2022. The Author(s).)

The retina, the part of the eye, translates the light signal into an electric current that can be sent to the brain as visual information. To achieve this, the retina requires fine-tuned vascularization for its energy supply. Diabetic retinopathy (DR) causes alterations in the eye vascularization that reduce the oxygen supply with consequent retinal neurodegeneration. During DR, the mammalian target of rapamycin (mTOR) pathway seems to coordinate retinal neurodegeneration with multiple anabolic and catabolic processes, such as autophagy, oxidative stress, cell death, and the release of pro-inflammatory cytokines, which are closely related to chronic hyperglycemia. This review outlines the normal anatomy of the retina and how hyperglycemia can be involved in the neurodegeneration underlying this disease through over activation or inhibition of the mTOR pathway., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Casciano, Zauli, Rimondi, Mura, Previati, Busin and Zauli.)

The European culinary culture relies on a wide range of fermented products of plant origin, produced mostly through spontaneous fermentation. Unfortunately, this kind of fermentations is difficult to standardize. Therefore, the use of commercial starter cultures is becoming common to achieve more stable, reproducible, and predictable results. Among plant-based fermentation processes, that of the red beet ( Beta vulgaris L. var. conditiva ) is scarcely described in the scientific literature. In this work, we compared different types of fermentation methods of beetroot and evaluated the processes' micro-biological, physico-chemical, structural, and volatilome features. A multi-variate analysis was used to match the production of specific VOCs to each starter and to define the correlations between the process variables and volatilome. Overall, the results showed a successful lactic acid fermentation. The analysis of the volatilome clearly discriminated the metabolic profiles of the different fermentations. Among them, the sample fermented with the mixture was the one with the most complex and diversified volatilome. Furthermore, samples did not appear softened after fermentation. Although this work had its weaknesses, such as the limited number of samples and variety, it may pave the way for the standardization of artisanal fermentation procedures of red beetroot in order to improve the quality and safety of the derived food products.

The way of cooking vegetables could differently affect the phenolic profiles of foods and their impact on human colon microbiota. In this work, we investigated the stability and bioaccessibility as well as the impact and fate of dark purple eggplant (DPE) phenolic compounds in the gut microbiota after grilling or frying in comparison to the raw one. After cooking, DPE underwent a gastro-intestinal digestion along with a proximal colon fermentation using the short-term batch model MICODE (multi-unit in vitro colon gut model). During the process, the phenolic compounds profiles (through high-resolution mass spectrometry) and microbiomics (qPCR of 14 core taxa) analyses were performed. Results showed that thermal treatments increased the amount of extractable phenolic compounds as well as their bioaccessibility. The highest gastro-intestinal release was observed in fried DPE (2468.46 ± 13.64 μmol/100 g), followed by grilled DPE (1007. 96 ± 12.84 μmol/100 g) and raw DPE (900.93 ± 10.56 μmol/100 g). Mass spectrometry analysis confirmed that colonic bacteria were able to metabolize DPE phenolic compounds mainly to 3-(3'-hydroxyphenyl)propanoic acid. Furthermore, results indicated that frying was better than grilling in terms of fostering more the growth of beneficial bacterial taxa and limiting that of opportunistic taxa. For example, fried DPE determined an increase in abundance of Bifidobacteriaceae Lactobacillales of 2.66 and 3.80 times. This work is one of the first exploring how cooking methods can affect the phenolic composition of DPE and differently impact on the colon microbiota tuning and modifying the food functionalities., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Chenodeoxycholic acid and ursodeoxycholic acid (CDCA and UDCA, respectively) have been conjugated with paclitaxel (PTX) anticancer drugs through a high-yield condensation reaction. Bile acid-PTX hybrids (BA-PTX) have been investigated for their pro-apoptotic activity towards a selection of cancer cell lines as well as healthy fibroblast cells. Chenodeoxycholic-PTX hybrid (CDC-PTX) displayed cytotoxicity and cytoselectivity similar to PTX, whereas ursodeoxycholic-PTX hybrid (UDC-PTX) displayed some anticancer activity only towards HCT116 colon carcinoma cells. Pacific Blue (PB) conjugated derivatives of CDC-PTX and UDC-PTX (CDC-PTX-PB and UDC-PTX-PB, respectively) were also prepared via a multistep synthesis for evaluating their ability to enter tumor cells. CDC-PTX-PB and UDC-PTX-PB flow cytometry clearly showed that both CDCA and UDCA conjugation to PTX improved its incoming into HCT116 cells, allowing the derivatives to enter the cells up to 99.9%, respect to 35% in the case of PTX. Mean fluorescence intensity analysis of cell populations treated with CDC-PTX-PB and UDC-PTX-PB also suggested that CDC-PTX-PB could have a greater ability to pass the plasmatic membrane than UDC-PTX-PB. Both hybrids showed significant lower toxicity with respect to PTX on the NIH-3T3 cell line.

Following the One Health principles in food science, the challenge to valorize byproducts from the industrial sector is open. Hemp ( Cannabis sativa subsp. sativa ) is considered an important icon of sustainability and as an alternative food source. Hemp seed bran, in particular, is a byproduct of industrial hemp seed processing, which is not yet valorized. The success, and a wider market diffusion of hemp seed for food applications, is hindered by its unpleasant taste, which is produced by certain compounds that generally overwhelm the pleasant bouquet of the fresh product. This research concerns the exploration of hemp seed bran through fermentation using beneficial lactobacilli, focusing on the sensorial and bioactive traits of the products when they are subjected to bacterial transformation. By studying of the aromatic profile formation during the fermentation process the aim was to modulate it in order to reduce off-odors without affecting the presence of healthy volatile organic compounds (VOCs). Applying multivariate analyses, it was possible to target the contribution of processing parameters to the generation of flavoring and bioactive compounds. To conclude, the fermentation process proposed was able to reduce unpleasant VOCs, whilst at the same time keeping the healthy ones, and it also improved nutritional quality, depending on time and bacterial starters. The fermentation proposed was a sustainable biotechnological approach that fitted perfectly with the valorization of hemp byproducts from the perspective of a green-oriented industrial process that avoids synthetic masking agents.

Cultivation of asparagus ( A sparagus officinalis L.; Asp) for food and medicinal use has taken place since the early Roman Empire. Today, Asp represents a worldwide diffuse perennial crop. Lower portions of the spears represent a food industry waste product that can be used to extract bioactive molecules. In this study, aqueous extracts derived from the non-edible portion of the plant (hard stem) were prepared and characterized for chemical content. Furthermore, the biocompatibility and bioactivity of Asp aqueous extracts were assessed in vitro on normal fibroblasts and on breast cancer cell lines. Results showed no interference with fibroblast viability, while a remarkable cytostatic concentration-dependent activity, with significant G1/S cell cycle arrest, was specifically observed in breast cancer cells without apoptosis induction. Asp extracts were also shown to significantly inhibit cell migration. Further analyses showed that Asp extracts were characterized by specific pro-oxidant activity against tumoral cells, and, importantly, that their combination with menadione resulted in a significant enhancement of oxidants production with respect to menadione alone in breast cancer cells but not in normal cells. This selectivity of action on tumoral cells, together with the easiness of their preparation, makes the aqueous Asp extracts very attractive for further investigation in breast cancer research, particularly to investigate their role as possible co-adjuvant agents of clinical drug therapies.

Gluten free (GF) foods, designed and marketed for the needs of people who are unable to metabolize gluten, in recent years have aroused growing interest that has led to the conquest of important market segments, with a strongly growing trend. Given the low protein content of standard GF flours, it is particularly important to fortify GF foods, and to study the effect that this process exerts on functional and sensorial characteristics. In this work, fortification of GF bakery goods was done with the addition of Arthrospira platensis (spirulina) flour. Two different dough formulations (with and without fortification) were fermented by four different processes, including spontaneous, single strains and sourdough starters. The baked products were then subjected to "consumer's tests". During the process, fermentation performances, prebiotic activity, and the VOC (Volatile Organic Compound) profiles were analyzed and compared through robust multivariate statistics. The results obtained evidenced that fortification led to a product with more abundant (medium organic acids) and exclusive bioactives (thymol, borneol, and nicotinic acid), which were correlated to the prebiotic activity of spirulina breads. This work, for the first time indicates that spirulina can be used to fortify GF bakery, improving also its functional potential.

The perception and discriminating of odors are sensory activities that are an integral part of our daily life. The first brain region where odors are processed is the olfactory bulb (OB). Among the different cell populations that make up this brain area, interneurons play an essential role in this sensory activity. Moreover, probably because of their activity, they represent an exception compared to other parts of the brain, since OB interneurons are continuously generated in the postnatal and adult period. In this review, we will focus on periglomerular (PG) cells which are a class of interneurons found in the glomerular layer of the OB. These interneurons can be classified into distinct subtypes based on their neurochemical nature, based on the neurotransmitter and calcium-binding proteins expressed by these cells. Dopaminergic (DA) periglomerular cells and calretinin (CR) cells are among the newly generated interneurons and play an important role in the physiology of OB. In the OB, DA cells are involved in the processing of odors and the adaptation of the bulbar network to external conditions. The main role of DA cells in OB appears to be the inhibition of glutamate release from olfactory sensory fibers. Calretinin cells are probably the best morphologically characterized interneurons among PG cells in OB, but little is known about their function except for their inhibitory effect on noisy random excitatory signals arriving at the main neurons. In this review, we will mainly describe the electrophysiological properties related to the excitability profiles of DA and CR cells, with a particular view on the differences that characterize DA mature interneurons from cells in different stages of adult neurogenesis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Capsoni, Fogli Iseppe, Casciano and Pignatelli.)

The search for new fiber supplements that can claim to be "prebiotic" is expanding fast, as the role of prebiotics and intestinal microbiota in well-being has been well established. This work explored the prebiotic potential of a novel fiber plus D-Limonene supplement (FLS) in comparison to fructooligosaccharides (FOS) over distal colonic fermentation with the in vitro model MICODE (multi-unit in vitro colon gut model). During fermentation, volatilome characterization and core microbiota quantifications were performed, then correlations among volatiles and microbes were interpreted. The results indicated that FLS generated positive effects on the host gut model, determining: (i) eubiosis; (ii) increased abundance of beneficial bacteria, as Bifidobacteriaceae ; (iii) production of beneficial compounds, as n-Decanoic acid; (iv) reduction in detrimental bacteria, as Enterobaceteriaceae ; (v) reduction in detrimental compounds, as skatole. The approach that we followed permitted us to describe the prebiotic potential of FLS and its ability to steadily maintain the metabolism of colon microbiota over time. This aspect is two-faced and should be investigated further because if a fast microbial turnover and production of beneficial compounds is a hallmark of a prebiotic, the ability to reduce microbiota changes and to reduce imbalances in the productions of microbial metabolites could be an added value to FLS. In fact, it has been recently demonstrated that these aspects could serve as an adjuvant in metabolic disorders and cognitive decline.

Psoriasis is a chronic inflammatory disease of the skin associated with systemic and joint manifestations and accompanied by comorbidities, such as metabolic syndrome and increased risk of cardiovascular disease. Psoriasis has a strong genetic basis, but exacerbation requires additional signals that are still largely unknown. The clinical manifestations involve the interplay between dendritic and T cells in the dermis to generate a self-sustaining inflammatory loop around the TNFα/IL-23/IL-17 axis that forms the psoriatic plaque. In addition, in recent years, a critical role of keratinocytes in establishing the interplay that leads to psoriatic plaques' formation has re-emerged. In this review, we analyze the most recent evidence of the role of keratinocytes and danger associates molecular patterns, such as extracellular ATP in the generation of psoriatic skin lesions. Particular attention will be given to purinergic signaling in inflammasome activation and in the initiation of psoriasis. In this phase, keratinocytes' inflammasome may trigger early inflammatory pathways involving IL-1β production, to elicit the subsequent cascade of events that leads to dendritic and T cell activation. Since psoriasis is likely triggered by skin-damaging events and trauma, we can envisage that intracellular ATP, released by damaged cells, may play a role in triggering the inflammatory response underlying the pathogenesis of the disease by activating the inflammasome. Therefore, purinergic signaling in the skin could represent a new and early step of psoriasis; thus, opening the possibility to target single molecular actors of the purinome to develop new psoriasis treatments.

In B-chronic lymphocytic leukemia (B-CLL), the interaction between leukemic cells and the microenvironment promotes tumor cell survival. The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib is one of the first-in-class molecules for the treatment of B-CLL patients; however, the emerging mechanisms of resistance to ibrutinib call for new therapeutic strategies. The purpose of the current study was to investigate the ability of ibrutinib plus the MDM2-inhibitor nutlin-3 to counteract the tumor microenvironment protective effect. We observed that primary B-CLL cells cultivated in microenvironment mimicking conditions were protected from apoptosis by the up-regulation of c-MYC and of p53. In the same setting, combined treatments with ibrutinib plus nutlin-3 led to significantly higher levels of apoptosis compared to the single treatments, counteracting the c-MYC up-regulation. Moreover, the combination induced high p53 levels and a significant dissipation of the mitochondrial membrane potential, together with BAX cleavage in the more active p18 form and phospho-BAD down-regulation, that are key components of the mitochondrial apoptotic pathway, enhancing the apoptosis level. Our findings propose a new therapeutic strategy to overcome the tumor microenvironment protection involved in B-CLL resistance to drugs, with possible clinical implications also for other hematologic and solid tumors for which ibrutinib is considered a therapeutic option.

Plant-based drinks as a substitute for animal milk consumption are crucial products in the food industry. Soy and rice drinks are the most successful milk substitutes but are low in fiber and protein contents, respectively, whilst being rich in sugars. Generally, an improvement is foreseen; thus, apart from supplement addition, a natural occurring strategy is functionalizing the drinks by beneficial bacteria fermentation. The aim of this work is to develop novel plant-based drinks assessing different mixtures of soy and rice milks fermented with single or multi-strain probiotics (Lactobacillus fermentum, L. plantarum, L. helveticus, Bifidobacterium bifidum, and B. longum). The drinks were characterized to study bacterial performances, by means of culture-dependent and -independent techniques, and their volatilome, by means of solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS) analysis. Through multivariate analysis, these features were investigated and correlated to define accurate descriptors of the produced functional drinks. The results showed that combined drinks and multi-strain fermentation generated higher-value products. For example, combined drinks in comparison with single ones had a lower amount of toxic 2-acetyl-3,5-dimethylfuran and higher abundances of desirable compounds such as 2-butanone, 3-hydroxy and butanoic acid. Multivariate analysis of volatile metabolites and physiological parameters could offer a novel approach to assess the quality of functional plant-based drinks and result in a decisional tool for industrial applications.

In this work, an aquaponic cultivation system for Lactuca sativa (L.) and Chicorium intybus (L.) was compared to a hydroponic one, focusing on the main microbial populations related to food safety and their volatile compounds (VOCs), concluding with Spearman correlations among the microbes and VOCs. Different sections of both systems were sampled at the end of the commercial development of the plants. Plants cultivated in aquaponics were in general more contaminated than those from hydroponics, while for the cultivation waters a higher contamination of the hydroponics than aquaponics system was unexpectedly observed. Furthermore, the chicory exhibited higher levels of all microbial groups compared to lettuce grown under the same cultivation system. The results obtained also showed correlations between the distribution of some VOCs and microbial groups in the phyllosphere, while some examples of positive correlations between 2-nonanone (a positive phytostimulant compound) and anaerobic bacilli of the rhizosphere in lettuce were reported. So far, multivariate analysis of VOCs was able to discriminate on the basis of varieties but not on the cultivation systems. In conclusion, the microbial characteristics of the two ecosystems depended both on plant variety and cultivation method but further studies will need to deeply investigate the variables influencing the microbial quality of vegetable foods obtained by aquaponics. On the other hand, the analysis of the VOCs was more related to the microbial community of each plant variety considered, whatever the cultivation system. In precision agriculture, metabolomics may represent an opportunity to study the holobiome and through it the interactions between plants and their microbial populations, to possibly provide for a tool to assess the microbiological quality of vegetable foods obtained by aquaponic systems.