Tardigrades, also known as water bears, make up a phylum of small but extremely hardy animals, renowned for their ability to survive extreme stresses, including desiccation. How tardigrades survive desiccation is one of the enduring mysteries of animal physiology. Here we show that CAHS D, an intrinsically disordered protein belonging to a unique family of proteins possessed only by tardigrades, undergoes a liquid-to-gel phase transition in a concentration dependent manner. Unlike other gelling proteins, such as gelatin, our data support a mechanism in which gel formation of CAHS D is driven by intermolecular β-β interactions. We find that gel formation corresponds with strong coordination of water and slowing of water diffusion. The degree of water coordination correlates with the ability of CAHS D to protect lactate dehydrogenase from unfolding when dried. This implies that the mechanism for unfolding protection can be attributed to a combination of hydration and slowed molecular motion. Conversely, rapid diffusion leading to efficient molecular shielding appears to be the predominant mechanism preventing protein aggregation. Our study demonstrates that distinct mechanisms are required for holistic protection during desiccation, and that protectants, such as CAHS D, can act as molecular ‘Swiss Army Knives’ capable of providing protection through several different mechanisms simultaneously.

Current cancer research is limited by the availability of reliable in vivo and in vitro models that are able to reproduce the fundamental hallmarks of cancer. Animal experimentation is of paramount importance in the progress of research, but it is becoming more evident that it has several limitations due to the numerous differences between animal tissues and real, in vivo human tissues. 3D bioprinting techniques have become an attractive tool for many basic and applied research fields. Concerning cancer, this technology has enabled the development of three-dimensional in vitro tumor models that recreate the characteristics of real tissues and look extremely promising for studying cancer cell biology. As 3D bioprinting is a relatively recently developed technique, there is still a lack of characterization of the chemical cellular microenvironment of 3D bioprinted constructs. In this work, we fabricated a cervical tumor model obtained by 3D bioprinting of HeLa cells in an alginate-based matrix. Characterization of the spheroid population obtained as a function of culturing time was performed by phase-contrast and confocal fluorescence microscopies. Scanning electrochemical microscopy and platinum nanoelectrodes were employed to characterize oxygen concentrations—a fundamental characteristic of the cellular microenvironment—with a high spatial resolution within the 3D bioprinted cervical tumor model; we also demonstrated that the diffusion of a molecular model of drugs in the 3D bioprinted construct, in which the spheroids were embedded, could be measured quantitatively over time using scanning electrochemical microscopy.

In this paper, we describe a new method to obtain D₂O/H₂O exchange in photosynthetic reaction centres from Rhodobacter sphaeroides. The method is characterized by: (i) a very high efficiency of the isotopic replacement; (ii) an extremely low amount of D₂O needed; (iii) the short time required for dehydration and D₂O rehydration; (iv) the possibility of controlling concomitantly the hydration state of the sample. The proposed method can be applied to other proteins.

Fullerenes are considered excellent photosensitizers, being highly suitable for photodynamic therapy (PDT). A lack of water solubility and low biocompatibility are, in many instances, still hampering the full exploitation of their potential in nanomedicine. Here, we used human serum albumin (HSA) to disperse fullerenes by binding up to five fullerene cages inside the hydrophobic cavities. Albumin was bioconjugated with folic acid to specifically address the folate receptors that are usually overexpressed in several solid tumors. Concurrently, tetramethylrhodamine isothiocyanate, TRITC, a tag for imaging, was conjugated to C₆₀@HSA in order to build an effective phototheranostic platform. The in vitro experiments demonstrated that: (i) HSA disperses C₆₀ molecules in a physiological environment, (ii) HSA, upon C₆₀ binding, maintains its biological identity and biocompatibility, (iii) the C₆₀@HSA complex shows a significant visible-light-induced production of reactive oxygen species, and (iv) folate bioconjugation improves both the internalization and the PDT-induced phototoxicity of the C₆₀@HSA complex in HeLa cells.

AlgMa bioink obtainedviaa green synthetic route and a dual crosslinking process is characterized by rheology to study the effect of crosslinking on cell viability and to fabricate bioprinted tissue models with spatially controlled stiffness.