VP6-SUMO Self-Assembly as Nanocarriers for Gastrointestinal Delivery

High proteolytic degradation and poor absorption through epithelial barriers are major challenges to successful oral delivery of therapeutics. Nanoparticle platforms can enhance drug stability and extend the residence time in gastrointestinal (GI) tract. However, drug delivery systems are often inactivated in acidic environment of stomach or suffer poor absorption from intestinal cells due to the mucus layer. To overcome these issues we developed a drug delivery system constituted by a protein construct made by a Rotavirus capsid protein (VP6) and the small ubiquitin-like modifier SUMO. This chimeric construct allows specificity towards intestinal cells, the Rotavirus natural target, combined by an enhanced stability given by the eukaryotic protein transporter SUMO. Furthermore SUMO can act as a molecular switch that facilitates import/export of its ligand to the nucleus, the hypersensitive subcellular site target of many cell killing therapies. In this paper we show that SUMO-VP6 constructs self-assembly into stable nanocarriers. SUMO-VP6 nanocarriers display ideal features for drug delivery: a small size and high monodispersity, a high stability in different pH conditions and a high uptake in the nuclear and cytoplasmic compartment of intestinal cells. These features make SUMO-VP6 nanocarriers a promising novel system for oral delivery of poorly soluble drugs

Personalized bone reconstruction and regeneration in the treatment of craniosynostosis

Craniosynostosis (CS) is the second most prevalent craniofacial congenital malformation due to the premature fusion of skull sutures. CS care requires surgical treatment of variable complexity, aimed at resolving functional and cosmetic defects resulting from the skull growth constrain. Despite significant innovation in the management of CS, morbidity and mortality still exist. Residual cranial defects represent a potential complication and needdedicated management to drive a targeted bone regeneration while modulating suture ossification. To this aim, existing techniques are rapidly evolving and include the implementation of novel biomaterials, 3D printing and additive manufacturing techniques, and advanced therapies based on tissue engineering. This review aims at providing an exhaustive and up\u2010to\u2010date overview of the strategies in use to correct these congenital defects, focusing on the technological advances in the fields of biomaterials and tissue engineering implemented in pediatric surgical skull reconstruction, i.e., biodegradable bone fixation systems, biomimetic scaffolds, drug delivery systems, and cell\u2010based approaches

Structural determinants driving the binding process between PDZ domain of wild type human PALS1 protein and SLiM sequences of SARS-CoV E proteins

Short Linear Motifs (SLiMs) are functional protein microdomains that typically mediate interactions between a short linear region in one protein and a globular domain in another. Surface Plasmon Resonance assays have been performed to determine the binding affinity between PDZ domain of wild type human PALS1 protein and tetradecapeptides representing the SLiMs sequences of SARS-CoV-1 and SARS-CoV-2 E proteins (E-SLiMs). SARS-CoV-2 E-SLiM binds to the human target protein with a higher affinity compared to SARS-CoV-1, showing a difference significantly greater than previously reported using the F318W mutant of PALS1 protein and shorter target peptides. Moreover, molecular dynamics simulations have provided clear evidence of the structural determinants driving this binding process. Specifically, the Arginine 69 residue in the SARS-CoV-2 E-SLiM is the key residue able to both enhance the specific polar interaction with negatively charged pockets of the PALS1 PDZ domain and reduce significantly the mobility of the viral peptide. These experimental and computational data are reinforced by the comparison of the interaction between the PALS1 PDZ domain with the natural ligand CRB1, as well as the corresponding E-SLiMs of other coronavirus members such as MERS and OCF43. Our results provide a model at the molecular level of the strategies used to mimic the endogenous SLiM peptide in the binding of the tight junctions of the host cell, explaining one of the possible reasons of the severity of the infection and pulmonary inflammation by SARS-CoV-2

Correlation Between Metabolic Syndrome, Periodontitis and Reactive Oxygen Species Production. A Pilot Study.

BACKGROUND AND OBJECTIVE: Metabolic syndrome (MetS) is associated with an increased risk of periodontitis even if the mechanism is unknown. Since both MetS and periodontitis are characterized by an alteration of inflammation status, the aim of this pilot study was to determine if differences in ROS metabolism of phagocytes isolated from (A) patients with MetS, (B) patients with both MetS and mild periodontitis, (C) healthy subjects and (D) normal weight subjects with mild periodontitis, were present. METHODS: ROS metabolism was studied by a Chemiluminescence (CL) technique: the system was made up of luminol (100 nmol/L) and cells (1 × 105) in the presence or absence of stimulus constituted by opsonized zymosan (0.5 mg). The final volume (1.0 mL) was obtained using modified KRP buffer. ROS production was measured at 25°C for 2 h, using an LB 953 luminometer (Berthold, EG & G Co, Germany). All the experiments were performed in triplicate. STATISTICAL ANALYSIS: All results are mean ± standard deviation (SD). The group of means was compared by the analysis of variance "(ANOVA)". A value of p < 0.05 was considered significant. RESULTS: Results showed that basal ROS production (both from PMNs and from PBMs) of groups A, B and D was increased with respect to that obtained from group C (p <0.05). CONCLUSION: These results are congruent with literature data, although the actual clinical relevance of the phenomenon remains to be evaluated

Mother and Daughter Carrying of the Same Pathogenic Variant in FGFR2 with Discordant Phenotype

Craniosynostosis are a heterogeneous group of genetic conditions characterized by the premature fusion of the skull bones. The most common forms of craniosynostosis are Crouzon, Apert and Pfeiffer syndromes. They differ from each other in various additional clinical manifestations, e.g., syndactyly is typical of Apert and rare in Pfeiffer syndrome. Their inheritance is autosomal dominant with incomplete penetrance and one of the main genes responsible for these syndromes is FGFR2, mapped on chromosome 10, encoding fibroblast growth factor receptor 2. We report an FGFR2 gene variant in a mother and daughter who present with different clinical features of Crouzon syndrome. The daughter is more severely affected than her mother, as also verified by a careful study of the face and oral cavity. The c.1032G>A transition in exon 8, already reported as a synonymous p.Ala344 = variant in Crouzon patients, also activates a new donor splice site leading to the loss of 51 nucleotides and the in-frame removal of 17 amino acids. We observed lower FGFR2 transcriptional and translational levels in the daughter compared to the mother and healthy controls. A preliminary functional assay and a molecular modeling added further details to explain the discordant phenotype of the two patients

The Thermal Structural Transition of α-Crystallin Inhibits the Heat Induced Self-Aggregation

-crystallin, the major constituent of human lens, is a member of the heat-shock proteins family and it is known to have a quaternary structural transition at . The presence of calcium ions and/or temperature changes induce supramolecular self-aggregation, a process of relevance in the cataractogenesis. Here we investigate the potential effect of the bovine -crystallin's structural transition on the self-aggregation process. Along all the temperatures investigated, aggregation proceeds by forming intermediate molecular assemblies that successively aggregate in clusters. The final morphology of the aggregates, above and below , is similar, but the aggregation kinetics are completely different. The size of the intermediate molecular assemblies, and their repulsive energy barrier show a marked increase while crossing . Our results highlight the key role of heat modified form of -crystallin in protecting from aggregation and preserving the transparency of the lens under hyperthermic conditions

Engineered human nanoferritin bearing the drug genz-644282 for cancer therapy

Gastrointestinal tumors, including pancreatic and colorectal cancers, represent one of the greatest public health issues worldwide, leading to a million global deaths. Recent research demonstrated that the human heavy chain ferritin (HFt) can encapsulate different types of drugs in its cavity and can bind to its receptor, CD71, in several solid and hematological tumors, thus highlighting the potential use of ferritin for tumor-targeting therapies. Here, we describe the development and characterization of a novel nanomedicine based on the HFt that is named The-0504. In particular, this novel system is a nano-assembly comprising an engineered version of HFt that entraps about 80 molecules of a potent, wide-spectrum, non-camptothecin topoisomerase I inhibitor (Genz-644282). The-0504 can be produced by a standardized pre-industrial process as a pure and homogeneously formulated product with favourable lyophilization properties. The preliminary anticancer activity was evaluated in cultured cancer cells and in a mouse model of pancreatic cancer. Overall results reported here make The-0504 a candidate for further preclinical development against CD-71 expressing deadly tumors

Time-resolved dynamic light scattering as a method to monitor compaction during protein folding

The mechanisms and the dynamics of protein folding are subject of a still increasing number of theoretical and experimental studies. While spectroscopic methods are already used for many years to measure the folding rates and to monitor the formation of secondary and tertiary structure, kinetic measurements of the compactness are only beginning to emerge. Time-resolved dynamic light scattering (DLS) is a useful tool to follow the compaction during protein folding by measuring the hydrodynamic Stokes radius Rs. Additionally, changes in the state of association can be detected by simultaneous measurements of the scattering intensity. The usefulness of different techniques for time-resolved DLS measurements and the general limits for kinetic DLS experiments are discussed first. Then we describe the adaptation of a stopped-flow system (SFM-3) to a DLS apparatus, the particular data acquisition schemes, and the experimentally attainable limits. The feasibility of stopped-flow DLS is demonstrated by the results of folding investigations with ribonuclease A, phosphoglycerate kinase, and bovine α-lactalbumin. Refolding was initiated by denaturant dilution jumps, which were repeated up to 100 times in order to obtain a reasonable signal-to-noise ratio. Kinetic DLS experiments can be performed fairly with a time resolution of one second. The time resolution of 100ms is probably the attainable limit. The capabilities of time-resolved DLS and time-resolved small-angle X-ray scattering are compared

Structure of fibrin gels studied by elastic light scattering techniques: Dependence of fractal dimension, gel crossover length, fiber diameter, and fiber density on monomer concentration

The concentration dependence of the structure of fibrin gels, formed following fibrinogen activation by thrombin at a constant molar ratio, was investigated by means of elastic light scattering techniques. The scattered intensity distributions were measured in absolute units over a wave-vector range q of about three decades ( 3c 3 \u3c7 102 -3 \u3c7 105 cm-1). A set of gel-characterizing parameters were recovered by accurately fitting the data with a single function recently developed by us [F. Ferri et al., Phys. Rev. E 63, 031401 (2001)], based on a simple structural model. Accordingly, the gels can be described as random networks of fibers of average diameter d and density \u3c1, entangled together to form densely packed and spatially correlated blobs of mass fractal dimension Dm and average size (or crossover length) \u3be. As previously done for d, we show here that the recovered \u3be is also a good approximation of a weight average, namely, d 3c 1ad2w and \u3be 3c\u3bew. By varying the fibrinogen concentration cF between 0.034-0.81 mg/ml, gels with 100 65\u3be 65 10 \u3bcm, 100 64d 64200 nm, 1.2 64D m 641.4, and constant \u3c1 3c0.4 mg/ml were obtained. The power-law cF dependencies that we found for both \u3be and d are consistent with the model, provided that the blobs are allowed to partially overlap by a factor \u3b7 likewise scaling with cF (2 65\u3b7 651). Recasting the whole dataset on a single master curve provided further evidence of the similarity between the structures of all the gels, and confirmed the self-consistency of the model

Self-similarity properties of alpha-crystallin supramolecular aggregates.

The supramolecular aggregation of alpha-crystallin, the major protein of the eye lens, was investigated by means of static and dynamic light scattering. The aggregation was induced by generating heat-modified alpha-crystallin forms and by stabilizing the clusters with calcium ions. The kinetic pattern of the aggregation and the structural features of the clusters can be described according to the reaction limited cluster-cluster aggregation theory previously adopted for the study of colloidal particles aggregation systems. Accordingly, the average mass and the hydrodynamic radius of alpha-crystallin supramolecular aggregates grow exponentially in time. The structure factor of the clusters is typical of fractal aggregates. A fractal dimension df approximately 2.15 was determined, indicating a low probability of sticking together of the primitive aggregating particles. As a consequence, the slow-forming clusters assemble a rather compact structure. The basic units forming the fractal aggregates were found to have a radius about twice (approximately 17 nm) that of the native protein and 5.3 times its size, which is consistent with an intermediate molecular assembly corresponding to the already known high molecular weight forms of alpha-crystallin