Anomalous small-angle X-ray scattering from charged soft matter

A review of recent applications of Anomalous Small-Angle X-ray Scattering (ASAXS) to charged soft matter systems is presented. Although the potential of ASAXS was realized in the eighties [1], applications to soft matter systems became feasible in recent years thanks to the technical developments at the synchrotron sources. Examples include both stiff chain and flexible polyelectrolytes, colloidal brush-like polyelectrolytes, DNA, RNA, and polysaccharides where the counterion profile could be determined with high precision and compared with theoretical models. In addition, ASAXS has also been found useful for microstructure characterization in soft materials. Finally, the present capability for ASAXS studies is illustrated by an example involving a surfactant micellar system

Interaction of mucins with bioinspired polymers and drug delivery particles

Mucins are glycoproteins with high molecular weight and an abundance of negatively charged oligosaccharide side chains, representing the main components in the mucous gels apart from water. Mucin structure consists of a flexible backbone (mainly serine and threonine residues) which serves as anchoring points for oligosaccharide side chains, and hydrophobic \u201cnaked domains\u201d enriched in cysteine residues. The latter can form inter-molecular bonds via disulphide links, promoting mucin association in solution. Therefore, mucins can establish adhesive interactions with particulates/biomacromolecules via electrostatic interactions, van der Waals forces, hydrophobic forces, hydrogen bonding, or chain entanglement. Mucosal drug delivery vehicles can either penetrate rapidly or establish prolonged contact. However, their development is of great challenge because little is still known about the interactions between mucin and other macromolecules. We are currently working on a comprehensive study of the interaction between mucin and macromolecules of interest for pharmaceutical developments by complementary techniques. To this scope, we employ biocompatible natural and synthetic polymers with different physical-chemical characteristics. Among them, linear polyamidoamines with amphoteric character are particularly interesting for their cyto-biocompatibility. It is indeed crucial to characterise such interactions not only in the bulk but also at the interface, since complexation between mucins and biomacromolecules takes place close to the cell membrane surface. Moreover, the strategy to overcome mucus barrier and achieve long retention time in the cell surface is to develop nano-agents which can effectively penetrate the mucus layer and accumulate at the epithelial surface. In this framework we present preliminary investigations in the bulk by small angle x-ray scattering (SAXS) and at the solid-liquid interface by employing quartz crystal microbalance (QCM-D)

Model cell membrane interaction with a bioinspired amphoteric polymer

We present recent investigation by means of nanoscale techniques on biocompatible linear polyamidoamines with amphoteric character, namely AGMA1 and ARGO7. These polymers have been shown of extremely promising and already proved medical interest, comprising their strong protection actions against virus infection, mainly papilloma and herpes and the extremely low toxicity of their DNA complexes, with respect to other used polymers such as PEI and protamine, applied in nanovector design for gene delivery. Our studies focus on the most important of these polymers, AGMA1, a prevailingly cationic 4-aminobutylguanidine-deriving PAA, whose mechanism of action is so far not fully understood. The current understanding is that its interaction with cell surfaces by means of glycosaminoglycans (HSPG) has a major role in its protective action against viruses. Yet, AGMA1 is active also against HPV-31, whose attachment does not appear to be dependent on HSPG. HPV-31, whose attachment does not appear to be dependent on HSPG. Therefore, AGMA1 binds other (as yet unidentified) receptors on the cell surface. As the known recipient is the HS carbohydrate moiety, other sugars rich membrane components have been proposed as probable AGMA1 target. Therefore, to shed a light on the mechanism of interaction of the polymer with sugar containing biologically relevant molecules, not HS, we have investigated AGMA1 in interaction with glycophyngolipids, Specifically, we studied multicomponent symmetric vesicles enriched in ganglioside GM1 built to mimic biological membrane domains, in the presence of AGMA1, At physiological pH, electrostatic effects should be the relevant interactions between GM1 and AGMA1. Taking advantage of the same mechanism we investigated the possibility of building lipid based core-shell particles to vehiculate AGMA1/siRNA complexes. Moreover, since it is probable that AGMA1 interacts with the barrier of mucus which cover the involved tissue we have extended our investigations also to mucin, constituting the biological barrier to the target tissues of the medical application of the polymers

Model cell membrane interaction with a bioinspired amphoteric polymer

We present recent investigation by means of nanoscale techniques on biocompatible linear polyamidoamines with amphoteric character, namely AGMA1 and ARGO7. These polymers have been shown of extremely promising and already proved medical interest, comprising their strong protection actions against virus infection, mainly papilloma and herpes and the extremely low toxicity of their DNA complexes, with respect to other used polymers such as PEI and protamine, applied in nanovector design for gene delivery. Our studies focus on the most important of these polymers, AGMA1, a prevailingly cationic 4-aminobutylguanidine-deriving PAA, whose mechanism of action is so far not fully understood. The current understanding is that its interaction with cell surfaces by means of glycosaminoglycans (HSPG) has a major role in its protective action against viruses. Yet, AGMA1 is active also against HPV-31, whose attachment does not appear to be dependent on HSPG. HPV-31, whose attachment does not appear to be dependent on HSPG. Therefore, AGMA1 binds other (as yet unidentified) receptors on the cell surface. As the known recipient is the HS carbohydrate moiety, other sugars rich membrane components have been proposed as probable AGMA1 target. Therefore, to shed a light on the mechanism of interaction of the polymer with sugar containing biologically relevant molecules, not HS, we have investigated AGMA1 in interaction with glycophyngolipids, Specifically, we studied multicomponent symmetric vesicles enriched in ganglioside GM1 built to mimic biological membrane domains, in the presence of AGMA1, At physiological pH, electrostatic effects should be the relevant interactions between GM1 and AGMA1. Taking advantage of the same mechanism we investigated the possibility of building lipid based core-shell particles to vehiculate AGMA1/siRNA complexes. Moreover, since it is probable that AGMA1 interacts with the barrier of mucus which cover the involved tissue we have extended our investigations also to mucin, constituting the biological barrier to the target tissues of the medical application of the polymers

Mucin Thin Layers : a Model for Mucus-Covered Tissues

The fate of macromolecules of biological or pharmacological interest that enter the mucus barrier is a current field of investigation. Studies of the interaction between the main constituent of mucus, mucins, and molecules involved in topical transmucoidal drug or gene delivery is a prerequisite for nanomedicine design. We studied the interaction of mucin with the bio-inspired arginine-derived amphoteric polymer D,L-ARGO7 by applying complementary techniques. Small angle X-ray scattering in bulk unveiled the formation of hundreds of nanometer-sized clusters, phase separated from the mucin mesh. Quartz microbalance with dissipation and neutron reflectometry measurements on thin mucin layers deposited on silica supports highlighted the occurrence of polymer interaction with mucin on the molecular scale. Rinsing procedures on both experimental set ups showed that interaction induces alteration of the deposited hydrogel. We succeeded in building up a new significant model for epithelial tissues covered by mucus, obtaining the deposition of a mucin layer 20 A\u30a thick on the top of a glycolipid enriched phospholipid single membrane, suitable to be investigated by neutron reflectometry. The model is applicable to unveil the cross structural details of mucus-covered epithelia in interaction with macromolecules within the A\u30a discreteness

Chitosan/glycosaminoglycan scaffolds for skin reparation

Burns and chronic wounds, often related to chronic diseases (as diabetes and cancer), are challenging lesions, difficult to heal. The prompt and full reconstitution of a functional skin is at the basis of the development of biopolymer-based scaffolds, representing a 3D substrate mimicking the dermal extracellular matrix. Aim of the work was to develop scaffolds intended for skin regeneration, according to: fabrication by electrospinning from aqueous polysaccharide solutions; prompt and easy treatment to obtain scaffolds insoluble in aqueous fluids; best performance in supporting wound healing. Three formulations were tested, based on chitosan (CH)and pullulan (P), associated with glycosaminoglycans (chondroitin sulfate - CS or hyaluronic acid \u2013 HA). A multidisciplinary approach has been used: chemico-physical characterization and preclinical evaluation allowed to obtain integrated information. This supports that CS gives distinctive properties and optimal features to the scaffold structure for promoting cell proliferation leading tissue reparation towards a complete skin restore

N6L pseudopeptide interferes with nucleophosmin protein-protein interactions and sensitizes leukemic cells to chemotherapy.

Abstract NPM1 is a multifunctional nucleolar protein implicated in several processes such as ribosome maturation and export, DNA damage response and apoptotic response to stress stimuli. The NPM1 gene is involved in human tumorigenesis and is found mutated in one third of acute myeloid leukemia patients, leading to the aberrant cytoplasmic localization of NPM1. Recent studies indicated that the N6L multivalent pseudopeptide, a synthetic ligand of cell–surface nucleolin, is also able to bind NPM1 with high affinity. N6L inhibits cell growth with different mechanisms and represents a good candidate as a novel anticancer drug for a number of malignancies of different histological origin. In this study we investigated whether N6L treatment could drive antitumor effect in acute myeloid leukemia cell lines. We found that N6L binds NPM1 at the N-terminal domain, co-localizes with cytoplasmic, mutated NPM1, and interferes with its protein-protein associations. N6L toxicity appears to be p53 dependent but interestingly, the leukemic cell line harbouring the mutated form of NPM1 is more resistant to treatment, suggesting that NPM1 cytoplasmic delocalization confers protection from p53 activation. Moreover, we show that N6L sensitizes AML cells to doxorubicin and cytarabine treatment. These studies suggest that N6L may be a promising option in combination therapies for acute myeloid leukemia treatment

miR-205-5p-mediated downregulation of ErbB/HER receptors in breast cancer stem cells results in targeted therapy resistance

The ErbB tyrosine kinase receptor family has been shown to have an important role in tumorigenesis, and the expression of its receptor members is frequently deregulated in many types of solid tumors. Various drugs targeting these receptors have been approved for cancer treatment. Particularly, in breast cancer, anti-Her2/EGFR molecules represent the standard therapy for Her2-positive malignancies. However, in a number of cases, the tumor relapses or progresses thus suggesting that not all cancer cells have been targeted. One possibility is that a subset of cells capable of regenerating the tumor, such as cancer stem cells (CSCs), may not respond to these therapeutic agents. Accumulating evidences indicate that miR-205-5p is significantly downregulated in breast tumors compared with normal breast tissue and acts as a tumor suppressor directly targeting oncogenes such as Zeb1 and ErbB3. In this study, we report that miR-205-5p is highly expressed in BCSCs and represses directly ERBB2 and indirectly EGFR leading to resistance to targeted therapy. Furthermore, we show that miR-205-5p directly regulates the expression of p63 which is in turn involved in the EGFR expression suggesting a miR-205/p63/EGFR regulation

Folding-competent and folding-defective forms of Ricin A chain have different fates following retrotranslocation from the endoplasmic reticulum

We report that a toxic polypeptide retaining the potential to refold upon dislocation from the endoplasmic reticulum (ER) to the cytosol (ricin A chain; RTA) and a misfolded version that cannot (termed RTAΔ), follow ER-associated degradation (ERAD) pathways in Saccharomyces cerevisiae that substantially diverge in the cytosol. Both polypeptides are dislocated in a step mediated by the transmembrane Hrd1p ubiquitin ligase complex and subsequently degraded. Canonical polyubiquitylation is not a prerequisite for this interaction because a catalytically inactive Hrd1p E3 ubiquitin ligase retains the ability to retrotranslocate RTA, and variants lacking one or both endogenous lysyl residues also require the Hrd1p complex. In the case of native RTA, we established that dislocation also depends on other components of the classical ERAD-L pathway as well as an ongoing ER–Golgi transport. However, the dislocation pathways deviate strikingly upon entry into the cytosol. Here, the CDC48 complex is required only for RTAΔ, although the involvement of individual ATPases (Rpt proteins) in the 19S regulatory particle (RP) of the proteasome, and the 20S catalytic chamber itself, is very different for the two RTA variants. We conclude that cytosolic ERAD components, particularly the proteasome RP, can discriminate between structural features of the same substrate

Breakthrough SARS-CoV-2 infections in MS patients on disease-modifying therapies

Background: Patients with multiple sclerosis (pwMS) treated with anti-CD20 or fingolimod showed a reduced humoral response to SARS-CoV-2 vaccines. Objective: In this study we aimed to monitor the risk of breakthrough SARS-CoV-2 infection in pwMS on different disease-modifying therapies (DMTs). Methods: Data on the number of vaccinated patients and the number of patients with a breakthrough infection were retrospectively collected in 27 Italian MS centers. We estimated the rate of breakthrough infections and of infection requiring hospitalization per DMT. Results: 19,641 vaccinated pwMS were included in the database. After a median follow-up of 8 months, we observed 137 breakthrough infections. Compared with other DMTs, the rate of breakthrough infections was significantly higher on ocrelizumab (0.57% vs 2.00%, risk ratio (RR) = 3.55, 95% CI = 2.74-4.58, p < 0.001) and fingolimod (0.58% vs 1.62%, RR = 2.65, 95% CI = 1.75-4.00, p < 0.001), while there were no significant differences in any other DMT group. In the ocrelizumab group the hospitalization rate was 16.7% versus 19.4% in the pre-vaccination era (RR = 0.86, p = 0.74) and it was 3.9% in all the other DMT groups versus 11.9% in the pre-vaccination period (RR = 0.33, p = 0.02). Conclusions: The risk of breakthrough SARS-CoV-2 infections is higher in patients treated with ocrelizumab and fingolimod, and the rate of severe infections was significantly reduced in all the DMTs excluding ocrelizumab