Biology subjects

Guerreiro, B. M.

Publications and source records attributed to Guerreiro, B. M..

4 recordsLinked to original sources

bioRxiv2025

Tosylation-mediated uronic acid knockout from cryoprotective FucoPol revealed the importance of polyanionicity in ice growth disruption

Negative formal charge is a critical structural trait for the expression of cryoprotection due to its strong ice growth disruption at the growing ice facet. Polyanionic polysaccharides containing uronic acids (UA) in their structure are therefore often associated with psychrophilic-like traits, such as defense against cryoinjury. However, their practical biological post-thaw benefit is convoluted amongst other contributing factors, including hydrophilic-hydrophobic balance, molecular weight, rheological properties, and structural conformation. Here, we leveraged ubiquitous (31.7% m.d.w.) and COOH-targeted (63.4% m.d.w.) leaving-group SN2 tosylation procedures, coupled with clean purification methodologies (high-MW membrane dialysis), to synthesize UA-free variants (FP-OTs18, FP-OTs96) of the cryoprotective, UA-containing polysaccharide FucoPol (FP). Tosylation was confirmed by ATR-FTIR (1602.3 cm-1 shift to 1627.9 cm-1) and 1H-NMR (brs,{delta} 7.92 ppm; brs, 7.53 ppm). Tosylated derivatives exhibited reduced solution conductivity ({Omega}=300 {micro}S/cm vs. ~800 {micro}S/cm), unchanged molecular weight (Mw), and retained biocompatibility. However, the substitution of hydrophilic, low-steric hindrance carboxyl groups with hydrophobic, high steric hindrance tosyl groups promoted a 30-40% reduction in zero-shear viscosity (0) and precluded the formation of a hypothermic gel-state due to loss of inter-chain non-covalent interactions, leading to a drastic loss of cryoprotective activity (10-70% of the original post-thaw viability observed for native FucoPol). The cryobiologically antagonistic effect of FP-OTs18 and FP-OTs96 reveals that UAs are a key player in polysaccharide-based cryopreservation, not only due to standalone beneficial polyanionicity, but also because their presence largely contributes to the expression of agonistic structural traits that elicit a cryoprotective response.

biochemistry↗

bioRxiv2025

Elucidation of the polysaccharide cryoprotection mechanism: kinetic inhibition, thermodynamic Gibbs-Thomson modulation and volumetric confinement

The emergence of gel-forming, ice-binding polysaccharides as potential candidates in cryobiology and the discovery of new structure-function relationships has fueled a knowledge convergence effort. Several polysaccharides have shown strong biological post-thaw benefits in cell cryopreservation despite some expressing contradictory ice growth anticipation, the main source of cryoinjury. The bio-based fucose-rich polysaccharide FucoPol, a current model under our scope of expertise, has further demonstrated crystal size reduction, thermal hysteresis, nucleation anticipation, nucleation stochastic narrowing and Gibbs-Thomson growth modulation effects, the latter similar to a type I antifreeze protein, in different thermodynamic settings: bulk vs. directional freezing; isobaric vs. isochoric systems; and sterile vs. biological media. Here, we undergo a critical reiteration of the consortium of findings over the years on cryoprotective polysaccharide research, rationalize several heuristic models to explain a dual nucleation behavior scenario and put forth a unifying theory to explain in which subset of conditions optimal cryoprotection may emerge from bio-based polysaccharides. We argue that gel-forming, ice-binding polysaccharides that show cryoprotective traits by anticipating nucleation and ice growth act by a combination of kinetic hampering of molecular diffusion (concentration effect); Gibbs-Thomson specific ice binding (templating effect) that induces growth modulation and size reduction; and indulge in the formation of a gel architecture of defined porosity (mesh size effect), the main initiator of a predominant pro-nucleation setting, increased stochastic determinism and the annihilation of large r* nuclei that elicits a small-nuclei survivorship bias. Classical Nucleation Theory formalisms support this hypothesis under the circumstance that a change in system state, initiated by a sol-gel transition near hypothermia, must exist to drive a meaningful shift in system energetics that explains the ANTI/PRO nucleation duality observed. The molecular fractioning of kinetically hindered bulk water into fractionally partitioned gel pores reduces system scale by a mesh size constant and enables the Gibbs-Thomson ice-binding affinity condition to be satisfied when r = rGT. A decreased nucleation energy barrier and maximal r* constraint thus invokes a predominant pro-nucleating system by enhancing nucleation susceptibility.

biophysics↗

bioRxiv2025

Polysaccharides in cryopreservation: multidimensional systematic review of extremophilic traits and the role of selective pressure in structure-function relationships

Cryopreservation of biological matter has accumulated apex biomedical interest for its potential in elongating the shelf-life of biological matter in a state of suspended animation. In Nature, extremophilic microorganisms have an outstanding ability of surviving in habitats where extreme cold, heat, salinity and acidity defy the established boundaries for life. Through Darwinian selective adaptation, they have developed biochemical defense strategies to counteract lethal stimuli. Here, we have compiled an extremophilic EPS structure-function relationship database (XPOL-DB) which aggregates reports on 145 extremophilic and mesophilic EPS, for a total of 128 biochemical and establishes the psychrophilic chemical profile for cold adaptation. Psychrophilic EPS are highly-branched, polyanionic, elongated structures of increased flexibility and molecular weight (16-300 MDa), with predominant expression of polar monomers (GalNAc, GalA, GlcNAc, GlcA) - compared to the linear, rigid, neutral thermophilic EPS. This critical analysis revealed the significant EPS similarity between psychrophiles and halophiles suggests ice growth and extreme salinity are rooted in a shared mechanism of physical membrane destabilization, for which similar chemical traits can dualistically imbue freeze and salt tolerance. Psychrophiles are exciting testbeds for the mapping of how extreme cold selects for cryobiological EPS adaptation; and can fuel reverse engineering efforts to design optimal bio-based, non-cytotoxic cryoprotectant polysaccharides.

molecular biology↗

bioRxiv2023

Fucose is an essential feature in cryoprotective polysaccharides

Biological cryopreservation often involves using a cryoprotective agent (CPA) to mitigate lethal physical stressors cells endure during freezing and thawing, but effective CPA concentrations are cytotoxic. Hence, natural polysaccharides have been studied as biocompatible alternatives. Our current investigation studied 26 natural polysaccharides as potential CPA, probing correlations between post-thaw metabolic viability (PTV) of cryopreserved Vero cells and monomeric composition. The best performing cryoprotective polysaccharides contained significant fucose amounts, resulting in average PTV 2.8-fold (up to 3.1-fold) compared to 0.8-fold and 2.2-fold for all non-cryoprotective and cryoprotective polysaccharides, respectively, outperforming the optimized commercial CryoStor CS5 formulation (2.6-fold). Stoichiometrically, a balance between fucose (18-35.7 mol%), uronic acids (UA) (13.5-26 mol%) and high molecular weight (MW > 1 MDa) generated optimal PTV. To deconvolute multiple variable effects, principal component analysis (PCA) coupled to K-means clustering was performed. Two major mechanisms of action explained PTV variability: a charge-dependent effect of contrasting charged uronic acid and neutral monomer compositions, and a MW-scaled charge-independent mechanism exclusively attributed to fucose. Ultimately, our research showed the critical role neutral fucose plays in enhancing cellular cryopreservation outcomes, disputing previous assumptions of polyanionicity being the sole governing predictor of cryoprotection, highlighting the potential of fucose-rich polyanionic polysaccharides.

cell biology↗