“Tuning aggregative versus non-aggregative lectin binding with glycosylated nanoparticles by the nature of the polymer ligand”

Glycan–lectin interactions drive a diverse range of biological signaling and recognition processes. The display of glycans in multivalent format enables their intrinsically weak binding affinity to lectins to be overcome by the cluster glycoside effect, which results in a non-linear increase in binding affinity. As many lectins have multiple binding sites, upon interaction with glycosylated nanomaterials either aggregation or surface binding without aggregation can occur. Depending on the application area, either one of these responses are desirable (or undesirable) but methods to tune the aggregation state, independently from the overall extent/affinity of binding are currently missing. Herein, we use gold nanoparticles decorated with galactose-terminated polymer ligands, obtained by photo-initiated RAFT polymerization to ensure high end-group fidelity, to show the dramatic impact on agglutination behaviour due to the chemistry of the polymer linker. Poly(N-hydroxyethyl acrylamide) (PHEA)-coated gold nanoparticles, a polymer widely used as a non-ionic stabilizer, showed preference for aggregation with lectins compared to poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA)-coated nanoparticles which retained colloidal stability, across a wide range of polymer lengths and particle core sizes. Using biolayer interferometry, it was observed that both coatings gave rise to similar binding affinity and hence provided conclusive evidence that aggregation rate alone cannot be used to measure affinity between nanoparticle systems with different stabilizing linkers. This is significant, as turbidimetry is widely used to demonstrate glycomaterial activity, although this work shows the most aggregating may not be the most avid, when comparing different polymer backbones/coating. Overall, our findings underline the potential of PHPMA as the coating of choice for applications where aggregation upon lectin binding would be problematic, such as in vivo imaging or drug delivery

Sulfur Starvation in Extremophilic Microalga Galdieria sulphuraria: Can Glutathione Contribute to Stress Tolerance?

This study reports the effects of sulfur (S) deprivation in cultures of Galdieria sulphuraria (Cyanidiophyceae). Galdieria is a unicellular red alga that usually grows, forming biomats on rocks, in S-rich environments. These are volcanic areas, where S is widespread since H2S is the prevalent form of gas. The glutathione content in Galdieria sulphuraria is much higher than that found in the green algae and even under conditions of S deprivation for 7 days, it remains high. On the other hand, the S deprivation causes a decrease in the total protein content and a significant increase in soluble protein fraction. This suggests that in the conditions of S starvation, the synthesis of enzy-matic proteins, that metabolically support the cell in the condition of nutritional stress, could be up regulated. Among these enzymatic proteins, those involved in cell detoxification, due to the accumulation of ROS species, have been counted

Seismic vulnerability assessment of existing Italian hospitals: The case study of the national cancer institute “G. Pascale foundation” of Naples

Introduction: A large portion of the Italian built heritage is characterized by a significant seismic vulnerability since many structures were designed with outdated criteria, i.e., without accounting for seismic actions. This aspect is particularly relevant for strategic structures and infrastructures, whose functionalities are crucial in case of seismic events. Objective: The main aim of the present paper is to share the key findings related to the seismic vulnerability assessment of the National Institute for the Study and Treatment of Cancer (IRCCS) “Giovanni Pascale Foundation” in Naples. In particular, the main evidences could be easily extended to existing hospitals realized in the last century, with the main reference to: construction techniques, quality of constructional material, overt and convert seismic vulnerabilities and possible intervention strategies for risk mitigation. Methods: In the present paper, the assessment methodologies adopted for such a strategic hospital complex are provided, focusing in particular on: i. preliminary research of original design documents and on-site investigation for determining constructional details; ii. material tests on structural elements; iii. vulnerability seismic assessment by means of non-linear FE analyses (push-over and capacity spectrum method); iv. recommendations on retrofitting measures and cost estimations. Results: The conducted study puts into clear evidence the inadequacy of the investigated buildings to face the design seismic actions provided by the current Italian code and thus showed the significant seismic vulnerabilities affecting the Institute “G. Pascale Foundation” of Naples. Among these, particular attention has also been focused on the so-called intrinsic vulnerabilities, namely the ones not measurable explicitly and interesting non-structural elements (e.g., connection of shelves, stained glass windows, facilities, etc.). Conclusion: The presented case study highlights the strong seismic vulnerability affecting structures realized in the past century, despite their strategic functions. On the whole, the examined structures can be considered as representative of this building typology, and the adopted calculation criteria, as well as the assumptions of the assessment process, could be easily extended to similar case studies

Changes upon the gluten-free diet of HLA-DQ2 and TRAFD1 gene expression in peripheral blood of celiac disease patients

Background: Celiac disease (CD) is a chronic immuno-mediated enteropathy caused by dietary gluten in genetically susceptible individuals carrying HLA (Human Leukocytes Antigen) genes that encode for DQ2.5 and DQ8 molecules. TRAFD1 (TRAF-type zinc finger domain 1) is a gene recently found associated with CD and defined as a master regulator of IFNγ signalling and of MHC class I antigen processing/presentation. There is no specific drug therapy and the only effective treatment is the gluten-free diet (GFD). The great majority of celiac patients when compliant with GFD have a complete remission of symptoms and recovery of gut mucosa architecture and function. Until now, very few studies have investigated molecular differences occurring in CD patients upon the GFD therapy. Methods: We looked at the expression of both HLA DQ2.5 and TRAFD1 risk genes in adult patients with acute CD at the time of and in treated patients on GFD. Specifically, we measured by qPCR the HLA-DQ2.5 and TRAFD1 mRNAs on peripheral blood mononuclear cells (PBMC) from the two groups of patients. Results: When we compared the HLA-DQ mRNA expression, we didn't find significant variation between the two groups of patients, thus indicating that GFD patients have the same capability to present gliadin antigens to cognate T cells as patients with active disease. Conversely, TRAFD1 was more expressed in PBMC from treated CD subjects. Notably, TRAFD1 transcripts significantly increased in the patients analyzed longitudinally during the GFD, indicating a role in the downregulation of gluten-induced inflammatory pathways. Conclusion: Our study demonstrated that HLA-DQ2.5 and TRAFD1 molecules are two important mediators of anti-gluten immune response and inflammatory process

Glycogen Synthase Kinase 3: Ion Channels, Plasticity, and Diseases

Glycogen synthase kinase 3β (GSK3) is a multifaceted serine/threonine (S/T) kinase expressed in all eukaryotic cells. GSK3β is highly enriched in neurons in the central nervous system where it acts as a central hub for intracellular signaling downstream of receptors critical for neuronal function. Unlike other kinases, GSK3β is constitutively active, and its modulation mainly involves inhibition via upstream regulatory pathways rather than increased activation. Through an intricate converging signaling system, a fine-tuned balance of active and inactive GSK3β acts as a central point for the phosphorylation of numerous primed and unprimed substrates. Although the full range of molecular targets is still unknown, recent results show that voltage-gated ion channels are among the downstream targets of GSK3β. Here, we discuss the direct and indirect mechanisms by which GSK3β phosphorylates voltage-gated Na+ channels (Nav 1.2 and Nav 1.6) and voltage-gated K+ channels (Kv 4 and Kv 7) and their physiological effects on intrinsic excitability, neuronal plasticity, and behavior. We also present evidence for how unbalanced GSK3β activity can lead to maladaptive plasticity that ultimately renders neuronal circuitry more vulnerable, increasing the risk for developing neuropsy-chiatric disorders. In conclusion, GSK3β-dependent modulation of voltage-gated ion channels may serve as an important pharmacological target for neurotherapeutic development

Lovastatin induces apoptosis of k-ras-transformed thyroid cells via inhibition of ras farnesylation and by modulating redox state.

Transformation of thyroid cells with either K-ras or H-ras viral oncogenes produces cell types with different phenotype and different response to the inhibition of the prenylation pathway by 3-hydroxy-3-methylglutaryl-CoA reductase or farnesyltransferase inhibitors. These inhibitors induce apoptosis in K-ras-transformed FRTL-5 cells (FRTL-5-K-Ras) whereas cell cycle arrest is induced in H-ras-transformed FRTL-5 (FRTL-5-H-Ras). In FRTL-5-K-Ras cells, the product of K-ras gene is implicated in the scavenging of reactive oxygen species (ROS) through the activation of extracellular-signal-regulated kinase (ERK)1/2 kinases. We observed that lovastatin blocked ras activation through inhibition of farnesylation and induced apoptosis, increasing ROS levels through inhibition of ERK1/2 signaling and Mn-SOD expression. Lovastatin-induced apoptosis was due to intracellular ROS increase since both, the antioxidant compound pyrrolidinedithiocarbamate or the SOD-mimetic compound, antagonized apoptosis. Moreover, both p38 mitogen-activated protein kinase and nuclear factor kappaB pathways, activated as a consequence of high ROS levels, are involved in the apoptotic effect, indicating that cell death induced by lovastatin was dependent on oxidative stress. Lovastatin antitumor efficacy in K-ras-dependent thyroid tumors was further confirmed in vivo, proposing a new therapeutic strategy for those tumor diseases that are sustained by an inappropriate K-ras expression

Effect of Gliadin Stimulation on HLA-DQ2.5 Gene Expression in Macrophages from Adult Celiac Disease Patients

Macrophages play an important role in the pathogenesis of celiac disease (CD) because they are involved in both inflammatory reaction and antigen presentation. We analyzed the expression of CD-associated HLA-DQ2.5 risk alleles on macrophages isolated by two cohorts of adult patients, from the U.S. and Italy, at different stages of disease and with different genotypes. After isolating and differentiating macrophages from PBMC, we assessed the HLA genotype and quantified the HLA-DQ2.5 mRNAs by qPCR, before and after gliadin stimulation. The results confirmed the differences in expression between DQA1*05:01 and DQB1*02:01 predisposing alleles and the non-CD associated alleles, as previously shown on other types of APCs. The gliadin challenge confirmed the differentiation of macrophages toward a proinflammatory phenotype, but above all, it triggered an increase of DQA1*05:01 mRNA, as well as a decrease of the DQB1*02:01 transcript. Furthermore, we observed a decrease in the DRB1 genes expression and a downregulation of the CIITA transactivator. In conclusion, our findings provide new evidences on the non-coordinated regulation of celiac disease DQ2.5 risk genes and support the hypothesis that gliadin could interfere in the three-dimensional arrangement of chromatin at the HLA locus

Immuno-modulatory and anti-Inflammatory effects of dihydrogracilin A, a trpene derived from the marine sponge Dendrilla membranosa.

We assessed the immunomodulatory and anti-inflammatory effects of 9,11-dihydrogracilin A (DHG), a molecule derived from the Antarctic marine sponge Dendrilla membranosa. We used in vitro and in vivo approaches to establish DHG properties. Human peripheral blood mononuclear cells (PBMC) and human keratinocytes cell line (HaCaT cells) were used as in vitro system, whereas a model of murine cutaneous irritation was adopted for in vivo studies. We observed that DHG reduces dose dependently the proliferative response and viability of mitogen stimulated PBMC. In addition, DHG induces apoptosis as revealed by AnnexinV staining and downregulates the phosphorylation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-B), signal transducer and activator of transcription (STAT) and extracellular signal\u2013regulated kinase (ERK) at late time points. These effects were accompanied by down-regulation of interleukin 6 (IL-6) production, slight decrease of IL-10 and no inhibition of tumor necrosis factor-alpha (TNF-) secretion. To assess potential properties of DHG in epidermal inflammation we used HaCaT cells; this compound reduces cell growth, viability and migration. Finally, we adopted for the in vivo study the croton oil-induced ear dermatitis murine model of inflammation. Of note, topical use of DHG significantly decreased mouse ear edema. These results suggest that DHG exerts anti-inflammatory effects and its anti-edema activity in vivo strongly supports its potential therapeutic application in inflammatory cutaneous diseases