Alzheimer's disease (AD) has been defined as a multi-factorial disorder resulting from a complex array of networked cellular and molecular mechanisms. In particular, elevated levels of Aβ protein and its aggregation products in the presence of metal ions proved to be highly neurotoxic and therapeutic strategies aimed at preventing Aβ generation and oxidative stress may represent an effective approach for AD treatment. A recent paradigm for the treatment of complex diseases such as AD suggests the employment of multifunctional compounds, single chemical entities capable of simultaneously modulating different targets involved in the pathology. In this paper, the "pharmacophores combination" strategy was applied, connecting the main scaffold of the BACE-1 ligand 1 to that of the chalcone 2, as metal chelating pharmacophore, to obtain a small library of compounds. Conjugate 5 emerged as the most interesting derivative, proving to inhibit BACE-1 with low-micromolar potency, and showing neuroprotective effects. In particular, 5 proved to be able to protect from metal-associated oxidative stress by hampering intracellular Cu(2+)-induced ROS formation without any direct neurotoxic effect.

Spermatogenesis is a highly complicated biological process that occurs in the epithelium of the seminiferous tubules. It is regulated by a complex network of endocrine and paracrine factors and by juxtacrine testicular cross-talk. Sertoli cells (SC) play a key role in spermatogenesis due to their production of trophic, differentiation and immune-modulating factors, but many of the molecular pathways of SC action remain controversial and unclear. Over the last two decades, research has focused on extracellular vesicles as an important mechanism of intercellular communication. The aim of this study was to investigate the presence of extracellular vesicles (EVs) in SC and the modulation of their content in SC after FSH and testosterone stimulation. Highly purified porcine pre-pubertal Sertoli cells were isolated according to previously established methods. After 48 h of culture with FSH or FSH + testosterone stimulation, we identified sertolian EVs containing specific mRNAs. Proteomic analysis of EVs content identified 29 proteins under non-stimulatory conditions, most of which were related to receptor binding activity. FSH stimulation induced increases in inhibin-alpha, inhibin-beta, plakoglobin, haptoglobin, D-3-phosphoglycerate dehydrogenase and sodium/potassium-transporting ATPase in sertolian EVs. Testosterone stimulation enhanced the abundance of inhibin-alpha, inhibin-beta, tissue-type plasminogen activator, epidermal growth factor-like protein 8, elongating factor 1-gamma and D-3-phosphoglycerate dehydrogenase. These results are likely to help determine the unknown molecular secretion of Sertoli cells.

Platinum-based anticancer drugs cause neurotoxicity. In particular, oxaliplatin produces early-developing, painful, and cold-exacerbated paresthesias. However, the mechanism underlying these bothersome and dose-limiting adverse effects is unknown. We hypothesized that the transient receptor potential ankyrin 1 (TRPA1), a cation channel activated by oxidative stress and cold temperature, contributes to mechanical and cold hypersensitivity caused by oxaliplatin and cisplatin. Oxaliplatin and cisplatin evoked glutathione-sensitive relaxation, mediated by TRPA1 stimulation and the release of calcitonin gene-related peptide from sensory nerve terminals in isolated guinea pig pulmonary arteries. No calcium response was observed in cultured mouse dorsal root ganglion neurons or in naïve Chinese hamster ovary (CHO) cells exposed to oxaliplatin or cisplatin. However, oxaliplatin, and with lower potency, cisplatin, evoked a glutathione-sensitive calcium response in CHO cells expressing mouse TRPA1. One single administration of oxaliplatin produced mechanical and cold hyperalgesia in rats, an effect selectively abated by the TRPA1 antagonist HC-030031. Oxaliplatin administration caused mechanical and cold allodynia in mice. Both responses were absent in TRPA1-deficient mice. Administration of cisplatin evoked mechanical allodynia, an effect that was reduced in TRPA1-deficient mice. TRPA1 is therefore required for oxaliplatin-evoked mechanical and cold hypersensitivity, and contributes to cisplatin-evoked mechanical allodynia. Channel activation is most likely caused by glutathione-sensitive molecules, including reactive oxygen species and their byproducts, which are generated after tissue exposure to platinum-based drugs from cells surrounding nociceptive nerve terminals.

No commercial vaccine is yet available against Group A Streptococcus (GAS), major cause of pharyngitis and impetigo, with a high frequency of serious sequelae in low- and middle-income countries. Group A Carbohydrate (GAC), conjugated to an appropriate carrier protein, has been proposed as an attractive vaccine candidate. Here, we explored the possibility to use GAS Streptolysin O (SLO), SpyCEP and SpyAD protein antigens with dual role of antigen and carrier, to enhance the efficacy of the final vaccine and reduce its complexity. All protein antigens resulted good carrier for GAC, inducing similar anti-GAC IgG response to the more traditional CRM197 conjugate in mice. However, conjugation to the polysaccharide had a negative impact on the anti-protein responses, especially in terms of functionality as evaluated by an IL-8 cleavage assay for SpyCEP and a hemolysis assay for SLO. After selecting CRM197 as carrier, optimal conditions for its conjugation to GAC were identified through a Design of Experiment approach, improving process robustness and yield This work supports the development of a vaccine against GAS and shows how novel statistical tools and recent advancements in the field of conjugation can lead to improved design of glycoconjugate vaccines.

Nanoparticle systems are being explored for the display of carbohydrate antigens, characterized by multimeric presentation of glycan epitopes and special chemico-physical properties of nano-sized particles. Among them, outer membrane vesicles (OMVs) are receiving great attention, combining antigen presentation with the immunopotentiator effect of the Toll-like receptor agonists naturally present on these systems. In this context, we are testing Generalized Modules for Membrane Antigens (GMMA), OMVs naturally released from Gram-negative bacteria mutated to increase blebbing, as carrier for polysaccharides. Here, we investigated the impact of saccharide length, density, and attachment site on the immune response elicited by GMMA in animal models, using a variety of structurally diverse polysaccharides from different pathogens (i.e., Neisseria meningitidis serogroup A and C, Haemophilus influenzae type b, and streptococcus Group A Carbohydrate and Salmonella Typhi Vi). Anti-polysaccharide immune response was not affected by the number of saccharides per GMMA particle. However, lower saccharide loading can better preserve the immunogenicity of GMMA as antigen. In contrast, saccharide length needs to be optimized for each specific antigen. Interestingly, GMMA conjugates induced strong functional immune response even when the polysaccharides were linked to sugars on GMMA. We also verified that GMMA conjugates elicit a T-dependent humoral immune response to polysaccharides that is strictly dependent on the nature of the polysaccharide. The results obtained are important to design novel glycoconjugate vaccines using GMMA as carrier and support the development of multicomponent glycoconjugate vaccines where GMMA can play the dual role of carrier and antigen. In addition, this work provides significant insights into the mechanism of action of glycoconjugates.

GMMA are exosomes released from engineered Gram-negative bacteria resembling the composition of outer membranes. We applied the GMMA technology for the development of an O-Antigen (OAg) based vaccine against Shigella sonnei, the most epidemiologically relevant cause of shigellosis. S. sonnei OAg has been identified as a key antigen for protective immunity, and GMMA are able to induce anti-OAg-specific IgG response in animal models and healthy adults. The contribution of protein-specific antibodies induced upon vaccination with GMMA has never been fully elucidated. Anti-protein antibodies are induced in mice upon immunization with either OAg-negative and OAg-positive GMMA. Here we demonstrated that OAg chains shield the bacteria from anti-protein antibody binding and therefore anti-OAg antibodies were the main drivers of bactericidal activity against OAg-positive bacteria. Interestingly, antibodies that are not targeting the OAg are functional against OAg-negative bacteria. The immunodominant protein antigens were identified by proteomic analysis. Our study confirms a critical role of the OAg on the immune response induced by S. sonnei GMMA. However, little is known about OAg length and density regulation during infection and, therefore, protein exposure. Hence, the presence of protein antigens on S. sonnei GMMA represents an added value for GMMA vaccines compared to other OAg-based formulations.

Shigellosis is a leading cause of diarrheal disease in low-middle-income countries (LMICs). Effective vaccines will help to reduce the disease burden, exacerbated by increasing antibiotic resistance, in the most susceptible population represented by young children. A challenge for a broadly protective vaccine against shigellosis is to cover the most epidemiologically relevant serotypes among >50 Shigella serotypes circulating worldwide. The GMMA platform has been proposed as an innovative delivery system for Shigella O-antigens, and we have developed a 4-component vaccine against S. sonnei, S. flexneri 1b, 2a and 3a identified among the most prevalent Shigella serotypes in LMICs. Driven by the immunogenicity results obtained in clinic with a first-generation mono-component vaccine, a new S. sonnei GMMA construct was generated and combined with three S. flexneri GMMA in a 4-component Alhydrogel formulation (altSonflex1-2-3). This formulation was highly immunogenic, with no evidence of negative antigenic interference in mice and rabbits. The vaccine induced bactericidal antibodies also against heterologous Shigella strains carrying O-antigens different from those included in the vaccine. The Monocyte Activation Test used to evaluate the potential reactogenicity of the vaccine formulation revealed no differences compared to the S. sonnei mono-component vaccine, shown to be safe in several clinical trials in adults. A GLP toxicology study in rabbits confirmed that the vaccine was well tolerated. The preclinical study results support the clinical evaluation of altSonflex1-2-3 in healthy populations, and a phase 1-2 clinical trial is currently ongoing.

Molecular interactions between male and female factors during mating profoundly affect the reproductive behavior and physiology of female insects. In natural populations of the malaria mosquito Anopheles gambiae, blood-fed females direct nutritional resources towards oogenesis only when inseminated. Here we show that the mating-dependent pathway of egg development in these mosquitoes is regulated by the interaction between the steroid hormone 20-hydroxy-ecdysone (20E) transferred by males during copulation and a female Mating-Induced Stimulator of Oogenesis (MISO) protein. RNAi silencing of MISO abolishes the increase in oogenesis caused by mating in blood-fed females, causes a delay in oocyte development, and impairs the function of male-transferred 20E. Co-immunoprecipitation experiments show that MISO and 20E interact in the female reproductive tract. Moreover MISO expression after mating is induced by 20E via the Ecdysone Receptor, demonstrating a close cooperation between the two factors. Male-transferred 20E therefore acts as a mating signal that females translate into an increased investment in egg development via a MISO-dependent pathway. The identification of this male-female reproductive interaction offers novel opportunities for the control of mosquito populations that transmit malaria.

Over 18 million disease cases and half a million deaths worldwide are estimated to be caused annually by Group A Streptococcus. A vaccine to prevent GAS disease is urgently needed. SpyCEP (Streptococcus pyogenes Cell-Envelope Proteinase) is a surface-exposed serine protease that inactivates chemokines, impairing neutrophil recruitment and bacterial clearance, and has shown promising immunogenicity in preclinical models. Although SpyCEP structure has been partially characterized, a more complete and higher resolution understanding of its antigenic features would be desirable prior to large scale manufacturing. To address these gaps and facilitate development of this globally important vaccine, we performed immunogenicity studies with a safety-engineered SpyCEP mutant, and comprehensively characterized its structure by combining X-ray crystallography, NMR spectroscopy and molecular dynamics simulations. We found that the catalytically-inactive SpyCEP antigen conferred protection similar to wild-type SpyCEP in a mouse infection model. Further, a new higher-resolution crystal structure of the inactive SpyCEP mutant provided new insights into this large chemokine protease comprising nine domains derived from two non-covalently linked fragments. NMR spectroscopy and molecular simulation analyses revealed conformational flexibility that is likely important for optimal substrate recognition and overall function. These combined immunogenicity and structural data demonstrate that the full-length SpyCEP inactive mutant is a strong candidate human vaccine antigen. These findings show how a multi-disciplinary study was used to overcome obstacles in the development of a GAS vaccine, an approach applicable to other future vaccine programs. Moreover, the information provided may also facilitate the structure-based discovery of small-molecule therapeutics targeting SpyCEP protease inhibition.

Shigellosis represents a major public health problem worldwide. The morbidity of the disease, especially in children in developing countries, together with the increase of antimicrobial resistance make a vaccine against Shigella an urgent medical need. Several vaccines under development are targeting Shigella lipopolysaccharide (LPS), whose extreme diversity renders necessary the development of multivalent vaccines. Immunity against Shigella LPS can elicit antibodies capable of killing bacteria in a serotype-specific manner. Therefore, although a correlation of protection against shigellosis has not been established, demonstration of vaccine-elicited antibody bactericidal activity may provide one means of vaccine protection against Shigella. To facilitate Shigella vaccine development, we have set up a high-throughput serum bactericidal assay based on luminescence readout (L-SBA), which has been already used to determine the functionality of antibodies against S. sonnei in multiple clinical trials. Here we present the setup and intra-laboratory characterization of L-SBA against three epidemiologically relevant Shigella flexneri serotypes using human sera. We assessed the linearity, repeatability and reproducibility of the method, demonstrating high assay specificity to detect the activity of antibodies against each homologous strain without any heterologous aspecificity against species-related and non-species-related strains; this assay is ready to be used to determine bactericidal activity of clinical sera raised by multivalent vaccines and in sero-epidemiological studies.

(1) Background: PTTG1 sustains the invasiveness of several cancer types. We previously reported that in seminomas, PTTG1 was detected in the peripheral area of the tumor and in the leading infiltrative edge. Here, we investigate the PTTG1 role on the invasive properties of seminoma. (2) Methods: three seminoma cell lines were used as in vitro model. PTTG1 levels and localization were investigated by biochemical and immunofluorescence analyses. Wound-healing, Matrigel invasion assays, and zymography were applied to study migratory and invasive capability of the cell lines. RNA interference and overexpression experiments were performed to address the PTTG1 role in seminoma invasiveness. PTTG1 and its target MMP-2 were analyzed in human testicular tumors using the Atlas database. (3) Results: PTTG1 was highly and differentially expressed in the seminoma cell lines. Nuclear PTTG1 was positively correlated to the aggressive phenotype. Its modulation confirms these results. Atlas database analysis revealed that PTTG1 was localized in the nucleus in seminoma compared with non-seminoma tumors, and that MMP-2 levels were significantly higher in seminomas. (4) Conclusions: nuclear PTTG1 promotes invasiveness of seminoma cell lines. Atlas database supported these results. These data lead to the hypothesis that nuclear PTTG1 is an eligible prognostic factor in seminomas.

Sex profoundly affects cancer incidence and susceptibility to therapy, with sex hormones highly contributing to this disparity. Various studies and omics data suggest a relationship between sex and the oncosuppressor p53 circuitry, including its regulators MDM2 and MDM4. Association of this network with genetic variation underlies sex-related altered cancer risk, age of onset, and cancer sensitivity to therapy. Moreover, sex-related factors, mainly estrogenic hormones, can affect the levels and/or function of the p53 network both in hormone-dependent and independent cancer. Despite this evidence, preclinical and clinical studies aimed to evaluate p53 targeted therapy rarely consider sex and related factors. This review summarizes the studies reporting the relationship between sex and the p53 circuitry, including its associated regulators, MDM2 and MDM4, with particular emphasis on estrogenic hormones. Moreover, we reviewed the evaluation of sex/hormone in preclinical studies and clinical trials employing p53-target therapies, and discuss how patients' sex and hormonal status could impact these therapeutic approaches.

Salmonella enterica serovars Typhimurium and Enteritidis are the predominant causes of invasive non-typhoidal Salmonella (iNTS) disease. Considering the co-endemicity of S. Typhimurium and S. Enteritidis, a bivalent vaccine formulation against both pathogens is necessary for protection against iNTS disease, thus investigation of glycoconjugate combination is required. In the present work, we investigated the immune responses induced by S. Typhimurium and S. Enteritidis monovalent and bivalent glycoconjugate vaccines adjuvanted with aluminum hydroxide (alum) only or in combination with cytosine-phosphorothioate-guanine oligodeoxynucleotide (CpG). Humoral and cellular, systemic and local, immune responses were characterized in two different mouse strains. All conjugate vaccines elicited high levels of serum IgG against the respective O-antigens (OAg) with bactericidal activity. The bivalent conjugate vaccine induced systemic production of antibodies against both S. Typhimurium and S. Enteritidis OAg. The presence of alum or alum + CpG adjuvants in vaccine formulations significantly increased the serum antigen-specific antibody production. The alum + CpG bivalent vaccine formulation triggered the highest systemic anti-OAg antibodies and also a significant increase of anti-OAg IgG in intestinal washes and fecal samples, with a positive correlation with serum levels. These data demonstrate the ability of monovalent and bivalent conjugate vaccines against S. Typhimurium and S. Enteritidis to induce systemic and local immune responses in different mouse strains, and highlight the suitability of a bivalent glycoconjugate formulation, especially when adjuvanted with alum + CpG, as a promising candidate vaccine against iNTS disease.

Many tumor-related factors have shown the ability to affect metabolic pathways by paving the way for cancer-specific metabolic features. Here, we investigate the regulation of mTORC1 by MDM4, a p53-inhibitor with oncogenic or anti-survival activities depending on cell growth conditions.

Activation of wild-type p53 in response to genotoxic stress occurs through different mechanisms including protein conformation, posttranslational modifications, and nuclear localization, leading to DNA binding to sequence-specific promoters. Zinc ion plays a crucial role in stabilizing p53/DNA binding to induce canonical target genes. Mutant p53 proteins undergo protein misfolding that can be counteracted by zinc. However, whether zinc supplementation might have a beneficial antitumor effect in wild-type p53-carrying cells in combination with drugs, has not been addressed so far.

Shigellosis is the leading cause of diarrheal disease, especially in children of low- and middle-income countries, and is often associated with anti-microbial resistance. Currently, there are no licensed vaccines widely available against Shigella, but several candidates based on the O-antigen (OAg) portion of lipopolysaccharides are in development. We have proposed Generalized Modules for Membrane Antigens (GMMA) as an innovative delivery system for OAg, and a quadrivalent vaccine candidate containing GMMA from S. sonnei and three prevalent S. flexneri serotypes (1b, 2a and 3a) is moving to a phase II clinical trial, with the aim to elicit broad protection against Shigella. GMMA are able to induce anti-OAg-specific functional IgG responses in animal models and healthy adults. We have previously demonstrated that antibodies against protein antigens are also generated upon immunization with S. sonnei GMMA. In this work, we show that a quadrivalent Shigella GMMA-based vaccine is able to promote a humoral response against OAg and proteins of all GMMA types contained in the investigational vaccine. Proteins contained in GMMA provide T cell help as GMMA elicit a stronger anti-OAg IgG response in wild type than in T cell-deficient mice. Additionally, we observed that only the trigger of Toll-like Receptor (TLR) 4 and not of TLR2 contributed to GMMA immunogenicity. In conclusion, when tested in mice, GMMA of a quadrivalent Shigella vaccine candidate combine both adjuvant and carrier activities which allow an increase in the low immunogenic properties of carbohydrate antigens.

No vaccine to protect against an estimated 238,000 shigellosis deaths per year is widely available. S. sonnei is the most prevalent Shigella, and multiple serotypes of S. flexneri, which change regionally and globally, also cause significant disease. The leading Shigella vaccine strategies are based on the delivery of serotype specific O-antigens. A strategy to minimize the complexity of a broadly-protective Shigella vaccine is to combine components from S. sonnei with S. flexneri serotypes that induce antibodies with maximum cross-reactivity between different serotypes. We used the GMMA-technology to immunize animal models and generate antisera against 14 S. flexneri subtypes from 8 different serotypes that were tested for binding to and bactericidal activity against a panel of 11 S. flexneri bacteria lines. Some immunogens induced broadly cross-reactive antibodies that interacted with most of the S. flexneri in the panel, while others induced antibodies with narrower specificity. Most cross-reactivity could not be assigned to modifications of the O-antigen, by glucose, acetate or phosphoethanolamine, common to several of the S. flexneri serotypes. This allowed us to revisit the current dogma of cross-reactivity among S. flexneri serotypes suggesting that a broadly protective vaccine is feasible with limited number of appropriately selected components. Thus, we rationally designed a 4-component vaccine selecting GMMA from S. sonnei and S. flexneri 1b, 2a and 3a. The resulting formulation was broadly cross-reactive in mice and rabbits, inducing antibodies that killed all S. flexneri serotypes tested. This study provides the framework for a broadly-protective Shigella vaccine which needs to be verified in human trials.

Technology platforms are an important strategy to facilitate the design, development and implementation of vaccines to combat high-burden diseases that are still a threat for human populations, especially in low- and middle-income countries, and to address the increasing number and global distribution of pathogens resistant to antimicrobial drugs. Generalized Modules for Membrane Antigens (GMMA), outer membrane vesicles derived from engineered Gram-negative bacteria, represent an attractive technology to design affordable vaccines. Here, we show that GMMA, decorated with heterologous polysaccharide or protein antigens, leads to a strong and effective antigen-specific humoral immune response in mice. Importantly, GMMA promote enhanced immunogenicity compared to traditional formulations (e.g., recombinant proteins and glycoconjugate vaccines), without negative impact to the anti-GMMA immune response. Our findings support the use of GMMA as a "plug and play" technology for the development of effective combination vaccines targeting different bugs at the same time.

To investigate the role of adiposity in the associations between ultra-processed food (UPF) consumption and head and neck cancer (HNC) and oesophageal adenocarcinoma (OAC) in the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort.

The development of drugs with different pharmacological properties appears to be an innovative therapeutic approach for Alzheimer's disease. In this article, we describe a simple structural modification of AP2238, a first dual function lead, in particular the introduction of the catechol moiety performed in order to search for multi-target ligands. The new compound AP2469 retains anti-acetylcholinesterase (AChE) and beta-site amyloid precursor protein cleaving enzyme (BACE)1 activities compared to the reference, and is also able to inhibit Aβ 42 self-aggregation, Aβ 42 oligomer-binding to cell membrane and subsequently reactive oxygen species formation in both neuronal and microglial cells. The ability of AP2469 to interfere with Aβ 42 oligomer-binding to neuron and microglial cell membrane gives this molecule both neuroprotective and anti-inflammatory properties. These findings, together with its strong chain-breaking antioxidant performance, make AP2469 a potential drug able to modify the course of the disease.