Fibronectin Functionalization: A Way to Enhance Dynamic Cell Culture on Alginate/Hydroxyapatite Scaffolds.

Bone defects are a global health concern; bone tissue engineering (BTE) is the most promising alternative to reduce patient morbidity and overcome the inherent drawbacks of autograft and allograft bone. Three-dimensional scaffolds are pivotal in this field due to their potential to provide structural support and mimic the natural bone microenvironment. Following an already published protocol, a 3D porous structure consisting of alginate and hydroxyapatite was prepared after a gelation step and a freezing-drying step.

Alginate-Sr/Mg Containing Bioactive Glass Scaffolds: The Characterization of a New 3D Composite for Bone Tissue Engineering.

In bone regeneration, combining natural polymer-based scaffolds with Bioactive Glasses (BGs) is an attractive strategy to improve the mechanical properties of the structure, as well as its bioactivity and regenerative potential. Methods: For this purpose, a well-studied alginate/hydroxyapatite (Alg/HAp) porous scaffold was enhanced with an experimental bioglass (BGMS10), characterized by a high crystallization temperature and containing therapeutic ions such as strontium and magnesium. This resulted in an improved biological response compared to 45S5 Bioglass, the "gold" standard among BGs.

On the Cholesterol Raising Effect of Coffee Diterpenes Cafestol and 16--Methylcafestol: Interaction with Farnesoid X Receptor.

The diterpene cafestol represents the most potent cholesterol-elevating compound known in the human diet, being responsible for more than 80% of the effect of coffee on serum lipids, with a mechanism still not fully clarified. In the present study, the interaction of cafestol and 16--methylcafestol with the stabilized ligand-binding domain (LBD) of the Farnesoid X Receptor was evaluated by fluorescence and circular dichroism. Fluorescence quenching was observed with both cafestol and 16--methylcafestol due to an interaction occurring in the close environment of the tryptophan W454 residue of the protein, as confirmed by docking and molecular dynamics.

DNA-Directed Protein Anchoring on Oligo/Alkanethiol-Coated Gold Nanoparticles: A Versatile Platform for Biosensing Applications.

Herein, we report on a smart biosensing platform that exploits gold nanoparticles (AuNPs) functionalized through ssDNA self-assembled monolayers (SAM) and the DNA-directed immobilization (DDI) of DNA-protein conjugates; a novel, high-sensitivity optical characterization technique based on a miniaturized gel electrophoresis chip integrated with online thermal lens spectrometry (MGEC-TLS), for the high-sensitivity detection of antigen binding events. Specifically, we characterized the physicochemical properties of 20 nm AuNPs covered with mixed SAMs of thiolated single-stranded DNA and bio-repellent molecules, referred to as top-terminated oligo-ethylene glycol (TOEG6), demonstrating high colloidal stability, optimal binder surface density, and proper hybridization capacity. Further, to explore the design in the frame of cancer-associated antigen detection, complementary ssDNA fragments conjugated with a nanobody, called C8, were loaded on the particles and employed to detect the presence of the HER2-ECD antigen in liquid.

Antibody Affinity Maturation Using Computational Methods: From an Initial Hit to Small-Scale Expression of Optimized Binders.

Nanobodies (VHHs) are engineered fragments of the camelid single-chain immunoglobulins. The VHH domain contains the highly variable segments responsible for antigen recognition. VHHs can be easily produced as recombinant proteins.

Encapsulation of Trimethine Cyanine in Cucurbit[8]uril: Solution versus Solid-State Inclusion Behavior.

Inclusion of polymethine cyanine dyes in the cavity of macrocyclic receptors is an effective strategy to alter their absorption and emission behavior in aqueous solution. In this paper, the effect of the host-guest interaction between cucurbit[8]uril (CB[8]) and a model trimethine indocyanine (Cy3) on dye spectral properties and aggregation in water is investigated. Solution studies, performed by a combination of spectroscopic and calorimetric techniques, indicate that the addition of CB[8] disrupts Cy3 aggregates, leading to the formation of a 1 : 1 host-guest complex with an association constant of 1.

An anti-HER2 nanobody binds to its antigen HER2 via two independent paratopes.

High-resolution structural data of complexes between antibodies and membrane receptors still represent a demanding task. In this study, we used complementary sets of experimental data to obtain a structural model of the complex formed by the human epidermal growth factor receptor 2 (HER2) and its specific nanobody A10. First we identified by NMR the residues that bind or rearrange as a consequence of the complex formation.

Atomic Force Microscopy Investigation of the Interactions between the MCM Helicase and DNA.

The MCM (minichromosome maintenance) protein complex forms an hexameric ring and has a key role in the replication machinery of Eukaryotes and Archaea, where it functions as the replicative helicase opening up the DNA double helix ahead of the polymerases. Here, we present a study of the interaction between DNA and the archaeal MCM complex from by means of atomic force microscopy (AFM) single molecule imaging. We first optimized the protocol (surface treatment and buffer conditions) to obtain AFM images of surface-equilibrated DNA molecules before and after the interaction with the protein complex.

Effect of Humanizing Mutations on the Stability of the Llama Single-Domain Variable Region.

In vivo clinical applications of nanobodies (VHHs) require molecules that induce minimal immunoresponse and therefore possess sequences as similar as possible to the human VH domain. Although the relative sequence variability in llama nanobodies has been used to identify scaffolds with partially humanized signature, the transformation of the hallmarks in the framework2 still represents a major problem. We assessed a set of mutants in silico and experimentally to elucidate what is the contribution of single residues to the VHH stability and how their combinations affect the mutant nanobody stability.

Genomic integrity and mitochondrial metabolism defects in Warsaw syndrome cells: a comparison with Fanconi anemia.

Warsaw breakage syndrome (WABS), is caused by biallelic mutations of DDX11, a gene coding a DNA helicase. We have recently reported two affected sisters, compound heterozygous for a missense (p.Leu836Pro) and a frameshift (p.

A consensus protocol for the in silico optimisation of antibody fragments.

We present an in silico mutagenetic protocol for improving the binding affinity of single domain antibodies (or nanobodies, VHHs). The method iteratively attempts random mutations in the interacting region of the protein and evaluates the resulting binding affinity towards the target by scoring, with a collection of scoring functions, short explicit solvent molecular dynamics trajectories of the binder-target complexes. The acceptance/rejection of each attempted mutation is carried out by a consensus decision-making algorithm, which considers all individual assessments derived from each scoring function.

Comparative analysis of fusion tags used to functionalize recombinant antibodies.

Recombinant antibodies can be expressed as fusion constructs in combination with tags which simplify their engineering into reliable and homogeneous immunoreagents by allowing site-specific, 1:1 functionalization. Several tags and corresponding reagents for recombinant protein derivatization have been proposed but benchmarking surveys for the evaluation of their effect on the characteristics of recombinant antibodies have not been reported. In this work we evaluated the impact on expression yields, shelf-stability, thermostability and binding affinity of a set of C-terminal tags fused to the same anti-Her2 nanobody.

New insights into the GINS complex explain the controversy between existing structural models.

GINS is a key component of eukaryotic replicative forks and is composed of four subunits (Sld5, Psf1, Psf2, Psf3). To explain the discrepancy between structural data from crystallography and electron microscopy (EM), we show that GINS is a compact tetramer in solution as observed in crystal structures, but also forms a double-tetrameric population, detectable by EM. This may represent an intermediate step towards the assembly of two replicative helicase complexes at origins, moving in opposite directions within the replication bubble.

The tumor suppressor inhibitor of growth 4 binds double-stranded DNA through its disordered central region.

The tumor suppressor inhibitor of growth 4 (ING4) regulates chromatin structure by recruiting the histone acetyl transferase complex HBO1 to sites with histone H3 trimethylated at K4. ING4 dimerizes through its N-terminal domain and recognizes H3K4me3 by the C-terminal plant homeodomain (PHD). The central region of ING4 is disordered and contains the nuclear localization signal.

¹H, ¹⁵N, and ¹³C chemical shift assignments for the winged helix domains of two archeal MCM C-termini.

High-fidelity replication guarantees the stable inheritance of genetic information stored in the DNA of living organisms. The minichromosome maintenance (MCM) complex functions as replicative DNA-unwinding helicase and has been identified as one key player in the replication process of archea and eukarya. Despite the availability of considerable structural information on archeal MCMs, such information was missing for their C-terminal domain.

Structure and mechanism of hexameric helicases.

Hexameric helicases are responsible for many biological processes, ranging from DNA replication in various life domains to DNA repair, transcriptional regulation and RNA metabolism, and encompass superfamilies 3-6 (SF3-6).To harness the chemical energy from ATP hydrolysis for mechanical work, hexameric helicases have a conserved core engine, called ASCE, that belongs to a subdivision of the P-loop NTPases. Some of the ring helicases (SF4 and SF5) use a variant of ASCE known as RecA-like, while some (SF3 and SF6) use another variant known as AAA+ fold.

Cryo-electron microscopy reveals a novel DNA-binding site on the MCM helicase.

The eukaryotic MCM2-7 complex is recruited at origins of replication during the G1 phase and acts as the main helicase at the replication fork during the S phase of the cell cycle. To characterize the interplay between the MCM helicase and DNA prior to the melting of the double helix, we determined the structure of an archaeal MCM orthologue bound to a 5.6-kb double-stranded DNA segment, using cryo-electron microscopy.

Biochemical evidence of a physical interaction between Sulfolobus solfataricus B-family and Y-family DNA polymerases.

The hyper-thermophilic archaeon Sulfolobus solfataricus possesses two functional DNA polymerases belonging to the B-family (Sso DNA pol B1) and to the Y-family (Sso DNA pol Y1). Sso DNA pol B1 recognizes the presence of uracil and hypoxanthine in the template strand and stalls synthesis 3-4 bases upstream of this lesion ("read-ahead" function). On the other hand, Sso DNA pol Y1 is able to synthesize across these and other lesions on the template strand.