In vivo label-free tissue histology through a microstructured imaging window.

Tissue histopathology, based on hematoxylin and eosin (H&E) staining of thin tissue slices, is the gold standard for the evaluation of the immune reaction to the implant of a biomaterial. It is based on lengthy and costly procedures that do not allow longitudinal studies. The use of non-linear excitation microscopy in vivo, largely label-free, has the potential to overcome these limitations. With this purpose, we develop and validate an implantable microstructured device for the non-linear excitation microscopy assessment of the immune reaction to an implanted biomaterial label-free. The microstructured device, shaped as a matrix of regular 3D lattices, is obtained by two-photon laser polymerization. It is subsequently implanted in the chorioallantoic membrane (CAM) of embryonated chicken eggs for 7 days to act as an intrinsic 3D reference frame for cell counting and identification. The histological analysis based on H&E images of the tissue sections sampled around the implanted microstructures is compared to non-linear excitation and confocal images to build a cell atlas that correlates the histological observations to the label-free images. In this way, we can quantify the number of cells recruited in the tissue reconstituted in the microstructures and identify granulocytes on label-free images within and outside the microstructures. Collagen and microvessels are also identified by means of second-harmonic generation and autofluorescence imaging. The analysis indicates that the tissue reaction to implanted microstructures is like the one typical of CAM healing after injury, without a massive foreign body reaction. This opens the path to the use of similar microstructures coupled to a biomaterial, to image in vivo the regenerating interface between a tissue and a biomaterial with label-free non-linear excitation microscopy. This promises to be a transformative approach, alternative to conventional histopathology, for the bioengineering and the validation of biomaterials in in vivo longitudinal studies.

Human gut epithelium features recapitulated in MINERVA 2.0 millifluidic organ-on-a-chip device.

We developed an innovative millifluidic organ-on-a-chip device, named MINERVA 2.0, that is optically accessible and suitable to serial connection. In the present work, we evaluated MINERVA 2.0 as millifluidic gut epithelium-on-a-chip by using computational modeling and biological assessment. We also tested MINERVA 2.0 in a serially connected configuration prodromal to address the complexity of multiorgan interaction. Once cultured under perfusion in our device, human gut immortalized Caco-2 epithelial cells were able to survive at least up to 7 days and form a three-dimensional layer with detectable tight junctions (occludin and zonulin-1 positive). Functional layer development was supported by measurable trans-epithelial resistance and FITC-dextran permeability regulation, together with mucin-2 expression. The dynamic culturing led to a specific transcriptomic profile, assessed by RNASeq, with a total of 524 dysregulated transcripts (191 upregulated and 333 downregulated) between static and dynamic condition. Overall, the collected results suggest that our gut-on-a-chip millifluidic model displays key gut epithelium features and, thanks to its modular design, may be the basis to build a customizable multiorgan-on-a-chip platform.

3D photopolymerized microstructured scaffolds influence nuclear deformation, nucleo/cytoskeletal protein organization, and gene regulation in mesenchymal stem cells.

Mechanical stimuli from the extracellular environment affect cell morphology and functionality. Recently, we reported that mesenchymal stem cells (MSCs) grown in a custom-made 3D microscaffold, the Nichoid, are able to express higher levels of stemness markers. In fact, the Nichoid is an interesting device for autologous MSC expansion in clinical translation and would appear to regulate gene activity by altering intracellular force transmission. To corroborate this hypothesis, we investigated mechanotransduction-related nuclear mechanisms, and we also treated spread cells with a drug that destroys the actin cytoskeleton. We observed a roundish nuclear shape in MSCs cultured in the Nichoid and correlated the nuclear curvature with the import of transcription factors. We observed a more homogeneous euchromatin distribution in cells cultured in the Nichoid with respect to the Flat sample, corresponding to a standard glass coverslip. These results suggest a different gene regulation, which we confirmed by an RNA-seq analysis that revealed the dysregulation of 1843 genes. We also observed a low structured lamina mesh, which, according to the implemented molecular dynamic simulations, indicates reduced damping activity, thus supporting the hypothesis of low intracellular force transmission. Also, our investigations regarding lamin expression and spatial organization support the hypothesis that the gene dysregulation induced by the Nichoid is mainly related to a reduction in force transmission. In conclusion, our findings revealing the Nichoid's effects on MSC behavior is a step forward in the control of stem cells via mechanical manipulation, thus paving the way to new strategies for MSC translation to clinical applications.

Bone Marrow Mesenchymal Stem Cells Expanded Inside the Nichoid Micro-Scaffold: a Focus on Anti-Inflammatory Response.

Purpose: Mesenchymal stem cells (MSCs) represent a promising source for stem cell therapies in numerous diseases, including pediatric respiratory system diseases. Characterized by low immunogenicity, high anti-inflammatory, and immunoregulatory features, MSCs demonstrated an excellent therapeutic profile in numerous in vitro and preclinical models. MSCs reside in a specialized physiologic microenvironment, characterized by a unique combination of biophysical, biochemical, and cellular properties. The exploitation of the 3D micro-scaffold Nichoid, which simulates the native niche, enhanced the anti-inflammatory potential of stem cells through mechanical stimulation only, overcoming the limitation of biochemical and xenogenic growth factors application. Materials and Methods: In this work, we expanded pediatric bone marrow MSCs (BM-MSCs) inside the Nichoid and performed a complete cellular characterization with different approaches including viability assays, immunofluorescence analyses, RNA sequencing, and gene expression analysis. Results: We demonstrated that BM-MSCs inside the scaffold remain in a stem cell quiescent state mimicking the condition of the in vivo environment. Moreover, the gene expression profile of these cells shows a significant up-regulation of genes involved in immune response when compared with the flat control. Conclusion: The significant changes in the expression profile of anti-inflammatory genes could potentiate the therapeutic effect of BM-MSCs, encouraging the possible clinical translation for the treatment of pediatric congenital and acquired pulmonary disorders, including post-COVID lung manifestations. Lay Summary: Regenerative medicine is the research field integrating medicine, biology, and biomedical engineering. In this context, stem cells, which are a fundamental cell source able to regenerate tissues and restore damage in the body, are the key component for a regenerative therapeutic approach. When expanded outside the body, stem cells tend to differentiate spontaneously and lose regenerative potential due to external stimuli. For this reason, we exploit the scaffold named Nichoid, which mimics the in vivo cell niche architecture. In this scaffold, mesenchymal stem cells "feel at home" due to the three-dimensional mechanical stimuli, and our findings could be considered as an innovative culture system for the in vitro expansion of stem cells for clinical translation. Future Perspective: The increasing demand of safe and effective cell therapies projects our findings toward the possibility of improving cell therapies based on the use of BM-MSCs, particularly for their clinical translation in lung diseases.

Patients' Stem Cells Differentiation in a 3D Environment as a Promising Experimental Tool for the Study of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease (NDD) that affects motor neurons, causing weakness, muscle atrophy and spasticity. Unfortunately, there are only symptomatic treatments available. Two important innovations in recent years are three-dimensional (3D) bioprinting and induced pluripotent stem cells (iPSCs). The aim of this work was to demonstrate the robustness of 3D cultures for the differentiation of stem cells for the study of ALS. We reprogrammed healthy and sALS peripheral blood mononuclear cells (PBMCs) in iPSCs and differentiated them in neural stem cells (NSCs) in 2D. NSCs were printed in 3D hydrogel-based constructs and subsequently differentiated first in motor neuron progenitors and finally in motor neurons. Every step of differentiation was tested for cell viability and characterized by confocal microscopy and RT-qPCR. Finally, we tested the electrophysiological characteristics of included NSC34. We found that NSCs maintained good viability during the 3D differentiation. Our results suggest that the hydrogel does not interfere with the correct differentiation process or with the electrophysiological features of the included cells. Such evidence confirmed that 3D bioprinting can be considered a good model for the study of ALS pathogenesis.

RNA Molecular Signature Profiling in PBMCs of Sporadic ALS Patients: HSP70 Overexpression Is Associated with Nuclear SOD1.

Superoxide dismutase 1 (SOD1) is one of the causative genes associated with amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder. SOD1 aggregation contributes to ALS pathogenesis. A fraction of the protein is localized in the nucleus (nSOD1), where it seems to be involved in the regulation of genes participating in the oxidative stress response and DNA repair. Peripheral blood mononuclear cells (PBMCs) were collected from sporadic ALS (sALS) patients (n = 18) and healthy controls (n = 12) to perform RNA-sequencing experiments and differential expression analysis. Patients were stratified into groups with "high" and "low" levels of nSOD1. We obtained different gene expression patterns for high- and low-nSOD1 patients. Differentially expressed genes in high nSOD1 form a cluster similar to controls compared to the low-nSOD1 group. The pathways activated in high-nSOD1 patients are related to the upregulation of HSP70 molecular chaperones. We demonstrated that, in this condition, the DNA damage is reduced, even under oxidative stress conditions. Our findings highlight the importance of the nuclear localization of SOD1 as a protective mechanism in sALS patients.

On-chip magnetophoretic capture in a model of malaria-infected red blood cells.

The search for new rapid diagnostic tests for malaria is a priority for developing an efficient strategy to fight this endemic disease, which affects more than 3 billion people worldwide. In this study, we characterize systematically an easy-to-operate lab-on-chip, designed for the magnetophoretic capture of malaria-infected red blood cells (RBCs). The method relies on the positive magnetic susceptibility of infected RBCs with respect to blood plasma. A matrix of nickel posts fabricated in a silicon chip placed face down is aimed at attracting infected cells, while healthy cells sediment on a glass slide under the action of gravity. Using a model of infected RBCs, that is, erythrocytes with methemoglobin, we obtained a capture efficiency of about 70% after 10 min in static conditions. By proper agitation, the capture efficiency reached 85% after just 5 min. Sample preparation requires only a 1:10 volume dilution of whole blood, previously treated with heparin, in a phosphate-buffered solution. Nonspecific attraction of untreated RBCs was not observed in the same time interval.

Whole transcriptomic analysis of mesenchymal stem cells cultured in Nichoid micro-scaffolds.

Mesenchymal stem cells (MSCs) are known to be ideal candidates for clinical applications where not only regenerative potential but also immunomodulation ability is fundamental. Over the last years, increasing efforts have been put into the design and fabrication of 3D synthetic niches, conceived to emulate the native tissue microenvironment and aiming at efficiently controlling the MSC phenotype in vitro. In this panorama, our group patented an engineered microstructured scaffold, called Nichoid. It is fabricated through two-photon polymerization, a technique enabling the creation of 3D structures with control of scaffold geometry at the cell level and spatial resolution beyond the diffraction limit, down to 100 nm. The Nichoid's capacity to maintain higher levels of stemness as compared to 2D substrates, with no need for adding exogenous soluble factors, has already been demonstrated in MSCs, neural precursors, and murine embryonic stem cells. In this work, we evaluated how three-dimensionality can influence the whole gene expression profile in rat MSCs. Our results show that at only 4 days from cell seeding, gene activation is affected in a significant way, since 654 genes appear to be differentially expressed (392 upregulated and 262 downregulated) between cells cultured in 3D Nichoids and in 2D controls. The functional enrichment analysis shows that differentially expressed genes are mainly enriched in pathways related to the actin cytoskeleton, extracellular matrix (ECM), and, in particular, cell adhesion molecules (CAMs), thus confirming the important role of cell morphology and adhesions in determining the MSC phenotype. In conclusion, our results suggest that the Nichoid, thanks to its exclusive architecture and 3D cell adhesion properties, is not only a useful tool for governing cell stemness but could also be a means for controlling immune-related MSC features specifically involved in cell migration.

Pectin-based bioinks for 3D models of neural tissue produced by a pH-controlled kinetics.

Introduction: In the view of 3D-bioprinting with cell models representative of neural cells, we produced inks to mimic the basic viscoelastic properties of brain tissue. Moving from the concept that rheology provides useful information to predict ink printability, this study improves and expands the potential of the previously published 3D-reactive printing approach by introducing pH as a key parameter to be controlled, together with printing time. Methods: The viscoelastic properties, printability, and microstructure of pectin gels crosslinked with CaCO3 were investigated and their composition was optimized (i.e., by including cell culture medium, HEPES buffer, and collagen). Different cell models representative of the major brain cell populations (i.e., neurons, astrocytes, microglial cells, and oligodendrocytes) were considered. Results and Discussion: The outcomes of this study propose a highly controllable method to optimize the printability of internally crosslinked polysaccharides, without the need for additives or post-printing treatments. By introducing pH as a further parameter to be controlled, it is possible to have multiple (pH-dependent) crosslinking kinetics, without varying hydrogel composition. In addition, the results indicate that not only cells survive and proliferate following 3D-bioprinting, but they can also interact and reorganize hydrogel microstructure. Taken together, the results suggest that pectin-based hydrogels could be successfully applied for neural cell culture.

The nuclear import of the transcription factor MyoD is reduced in mesenchymal stem cells grown in a 3D micro-engineered niche.

Smart biomaterials are increasingly being used to control stem cell fate in vitro by the recapitulation of the native niche microenvironment. By integrating experimental measurements with numerical models, we show that in mesenchymal stem cells grown inside a 3D synthetic niche both nuclear transport of a myogenic factor and the passive nuclear diffusion of a smaller inert protein are reduced. Our results also suggest that cell morphology modulates nuclear proteins import through a partition of the nuclear envelope surface, which is a thin but extremely permeable annular portion in cells cultured on 2D substrates. Therefore, our results support the hypothesis that in stem cell differentiation, the nuclear import of gene-regulating transcription factors is controlled by a strain-dependent nuclear envelope permeability, probably related to the reorganization of stretch-activated nuclear pore complexes.

Nonlinear Optical Microscopy: From Fundamentals to Applications in Live Bioimaging.

A recent challenge in the field of bioimaging is to image vital, thick, and complex tissues in real time and in non-invasive mode. Among the different tools available for diagnostics, nonlinear optical (NLO) multi-photon microscopy allows label-free non-destructive investigation of physio-pathological processes in live samples at sub-cellular spatial resolution, enabling to study the mechanisms underlying several cellular functions. In this review, we discuss the fundamentals of NLO microscopy and the techniques suitable for biological applications, such as two-photon excited fluorescence (TPEF), second and third harmonic generation (SHG-THG), and coherent Raman scattering (CRS). In addition, we present a few of the most recent examples of NLO imaging employed as a label-free diagnostic instrument to functionally monitor in vitro and in vivo vital biological specimens in their unperturbed state, highlighting the technological advantages of multi-modal, multi-photon NLO microscopy and the outstanding challenges in biomedical engineering applications.

Characterization of Mesenchymal Stem Cell Differentiation within Miniaturized 3D Scaffolds through Advanced Microscopy Techniques.

Three-dimensional culture systems and suitable substrates topographies demonstrated to drive stem cell fate in vitro by mechanical conditioning. For example, the Nichoid 3D scaffold remodels stem cells and shapes nuclei, thus promoting stem cell expansion and stemness maintenance. However, the mechanisms involved in force transmission and in biochemical signaling at the basis of fate determination are not yet clear. Among the available investigation systems, confocal fluorescence microscopy using fluorescent dyes enables the observation of cell function and shape at the subcellular scale in vital and fixed conditions. Contrarily, nonlinear optical microscopy techniques, which exploit multi-photon processes, allow to study cell behavior in vital and unlabeled conditions. We apply confocal fluorescence microscopy, coherent anti-Stokes Raman scattering (CARS), and second harmonic generation (SHG) microscopy to characterize the phenotypic expression of mesenchymal stem cells (MSCs) towards adipogenic and chondrogenic differentiation inside Nichoid scaffolds, in terms of nuclear morphology and specific phenotypic products, by comparing these techniques. We demonstrate that the Nichoid maintains a rounded nuclei during expansion and differentiation, promoting MSCs adipogenic differentiation while inhibiting chondrogenesis. We show that CARS and SHG techniques are suitable for specific estimation of the lipid and collagenous content, thus overcoming the limitations of using unspecific fluorescent probes.

Bioengineering tools to speed up the discovery and preclinical testing of vaccines for SARS-CoV-2 and therapeutic agents for COVID-19.

This review provides an update for the international research community on the cell modeling tools that could accelerate the understanding of SARS-CoV-2 infection mechanisms and could thus speed up the development of vaccines and therapeutic agents against COVID-19. Many bioengineering groups are actively developing frontier tools that are capable of providing realistic three-dimensional (3D) models for biological research, including cell culture scaffolds, microfluidic chambers for the culture of tissue equivalents and organoids, and implantable windows for intravital imaging. Here, we review the most innovative study models based on these bioengineering tools in the context of virology and vaccinology. To make it easier for scientists working on SARS-CoV-2 to identify and apply specific tools, we discuss how they could accelerate the discovery and preclinical development of antiviral drugs and vaccines, compared to conventional models.

Lamin A/C Mechanotransduction in Laminopathies.

Mechanotransduction translates forces into biological responses and regulates cell functionalities. It is implicated in several diseases, including laminopathies which are pathologies associated with mutations in lamins and lamin-associated proteins. These pathologies affect muscle, adipose, bone, nerve, and skin cells and range from muscular dystrophies to accelerated aging. Although the exact mechanisms governing laminopathies and gene expression are still not clear, a strong correlation has been found between cell functionality and nuclear behavior. New theories base on the direct effect of external force on the genome, which is indeed sensitive to the force transduced by the nuclear lamina. Nuclear lamina performs two essential functions in mechanotransduction pathway modulating the nuclear stiffness and governing the chromatin remodeling. Indeed, A-type lamin mutation and deregulation has been found to affect the nuclear response, altering several downstream cellular processes such as mitosis, chromatin organization, DNA replication-transcription, and nuclear structural integrity. In this review, we summarize the recent findings on the molecular composition and architecture of the nuclear lamina, its role in healthy cells and disease regulation. We focus on A-type lamins since this protein family is the most involved in mechanotransduction and laminopathies.

Natural and Synthetic Polymers for Bone Scaffolds Optimization.

Bone tissue is the structural component of the body, which allows locomotion, protects vital internal organs, and provides the maintenance of mineral homeostasis. Several bone-related pathologies generate critical-size bone defects that our organism is not able to heal spontaneously and require a therapeutic action. Conventional therapies span from pharmacological to interventional methodologies, all of them characterized by several drawbacks. To circumvent these effects, tissue engineering and regenerative medicine are innovative and promising approaches that exploit the capability of bone progenitors, especially mesenchymal stem cells, to differentiate into functional bone cells. So far, several materials have been tested in order to guarantee the specific requirements for bone tissue regeneration, ranging from the material biocompatibility to the ideal 3D bone-like architectural structure. In this review, we analyse the state-of-the-art of the most widespread polymeric scaffold materials and their application in in vitro and in vivo models, in order to evaluate their usability in the field of bone tissue engineering. Here, we will present several adopted strategies in scaffold production, from the different combination of materials, to chemical factor inclusion, embedding of cells, and manufacturing technology improvement.

Advantages and limitations of a supernegative GFP in facilitating MyoD intracellular tracking.

Despite intracellular molecular dynamics being fundamental to understand pathological, biomechanical or biochemical events, several processes are still not clear because of the difficulty of monitoring and measuring these phenomena. To engineer an effective fluorescent tool useful to improve protein intracellular tracking studies, we fused a supernegative green fluorescent protein, (-30)GFP, to a myogenic transcription factor, MyoD. The (-30)GFP-MyoD was able to pass the plasma membrane when complexed with cationic lipids. Fluorescence confocal microscopy showed the protein delivery in just 3 hours with high levels of protein transduction efficiency. Confocal acquisitions also confirmed the maintenance of the MyoD nuclear localization. To examine how the supernegative GFP influenced MyoD activity, we did gene expression analyses, which showed an inhibitory effect of (-30)GFP on transcription factor function. This negative effect was possibly due to a charge-driven interference mechanism, as suggested by further investigations by molecular dynamics simulations. Summarizing these results, despite the functional limitations related to the charge structural characteristics that specifically affected MyoD function, we found (-30)GFP is a suitable fluorescent label for improving protein intracellular tracking studies, such as nucleocytoplasmic transport in mechanotransduction.

Mechanical regulation of nucleocytoplasmic translocation in mesenchymal stem cells: characterization and methods for investigation.

Mesenchymal stem cells (MSCs) have immune-modulatory and tissue-regenerative properties that make them a suitable and promising tool for cell-based therapy application. Since the bio-chemo-mechanical environment influences MSC fate and behavior, the understanding of the mechanosensors involved in the transduction of mechanical inputs into chemical signals could be pivotal. In this context, the nuclear pore complex is a molecular machinery that is believed to have a key role in force transmission and in nucleocytoplasmic shuttling regulation. To fully understand the nuclear pore complex role and the nucleocytoplasmic transport dynamics, recent advancements in fluorescence microscopy provided the possibility to study passive and facilitated nuclear transports also in mechanically stimulated cell culture conditions. Here, we review the current available methods for the investigation of nucleocytoplasmic shuttling, including photo-perturbation-based approaches, fluorescence correlation spectroscopy, and single-particle tracking techniques. For each method, we analyze the advantages, disadvantages, and technical limitations. Finally, we summarize the recent knowledge on mechanical regulation of nucleocytoplasmic translocation in MSC, the relevant progresses made so far, and the future perspectives in the field.

The Effect of Cell Morphology on the Permeability of the Nuclear Envelope to Diffusive Factors.

A recent advance in understanding stem cell differentiation is that the cell is able to translate its morphology, i.e., roundish or spread, into a fate decision. We hypothesize that strain states in the nuclear envelope (NE) cause changes in the structure of the nuclear pore complexes. This induces significant changes in the NE's permeability to the traffic of the transcription factors involved in stem cell differentiation which are imported into the nucleus by passive diffusion. To demonstrate this, we set up a numerical model of the transport of diffusive molecules through the nuclear pore complex (NPC), on the basis of the NPC deformation. We then compared the prediction of the model for two different cell configurations with roundish and spread nuclear topologies with those measured on cells cultured in both configurations. To measure the geometrical features of the NPC, using electron tomography we reconstructed three-dimensional portions of the envelope of cells cultured in both configurations. We found non-significant differences in both the shape and size of the transmembrane ring of single pores with envelope deformation. In the numerical model, we thus assumed that the changes in pore complex permeability, caused by the envelope strains, are due to variations in the opening configuration of the nuclear basket, which in turn modifies the porosity of the pore complex mainly on its nuclear side. To validate the model, we cultured cells on a substrate shaped as a spatial micro-grid, called the "nichoid," which is nanoengineered by two-photon laser polymerization, and induces a roundish nuclear configuration in cells adhering to the nichoid grid, and a spread configuration in cells adhering to the flat substrate surrounding the grid. We then measured the diffusion through the nuclear envelope of an inert green-fluorescent protein, by fluorescence recovery after photobleaching (FRAP). Finally, we compared the diffusion times predicted by the numerical model for roundish vs. spread cells, with the measured times. Our data show that cell stretching modulates the characteristic time needed for the nuclear import of a small inert molecule, GFP, and the model predicts a faster import of diffusive molecules in the spread compared to roundish cells.

Live-cell p53 single-molecule binding is modulated by C-terminal acetylation and correlates with transcriptional activity.

Live-cell microscopy has highlighted that transcription factors bind transiently to chromatin but it is not clear if the duration of these binding interactions can be modulated in response to an activation stimulus, and if such modulation can be controlled by post-translational modifications of the transcription factor. We address this question for the tumor suppressor p53 by combining live-cell single-molecule microscopy and single cell in situ measurements of transcription and we show that p53-binding kinetics are modulated following genotoxic stress. The modulation of p53 residence times on chromatin requires C-terminal acetylation-a classical mark for transcriptionally active p53-and correlates with the induction of transcription of target genes such as CDKN1a. We propose a model in which the modification state of the transcription factor determines the coupling between transcription factor abundance and transcriptional activity by tuning the transcription factor residence time on target sites.Both transcription binding kinetics and post-translational modifications of transcription factors are thought to play a role in the modulation of transcription. Here the authors use single-molecule tracking to directly demonstrate that p53 acetylation modulates promoter residence time and transcriptional activity.

Temperature and pressure effects on GFP mutants: explaining spectral changes by molecular dynamics simulations and TD-DFT calculations.

By combining spectroscopic measurements under high pressure with molecular dynamics simulations and quantum mechanics calculations we investigate how sub-angstrom structural perturbations are able to tune protein function. We monitored the variations in fluorescence output of two green fluorescent protein mutants (termed Mut2 and Mut2Y, the latter containing the key T203Y mutation) subjected to pressures up to 600 MPa, at various temperatures in the 280-320 K range. By performing 150 ns molecular dynamics simulations of the protein structures at various pressures, we evidenced subtle changes in conformation and dynamics around the light-absorbing chromophore. Such changes explain the measured spectral tuning in the case of the sizable 120 cm(-1) red-shift observed for pressurized Mut2Y, but absent in Mut2. Previous work [Barstow et al., Proc. Natl. Acad. Sci. U. S. A., 2008, 105, 13362] on pressure effects on GFP also involved a T203Y mutant. On the basis of cryocooling X-ray crystallography, the pressure-induced fluorescence blue shift at low temperature (77 K) was attributed to key changes in relative conformation of the chromophore and Tyr203 phenol ring. At room temperature, however, a red shift was observed at high pressure, analogous to the one we observe in Mut2Y. Our investigation of structural variations in compressed Mut2Y also explains their result, bridging the gap between low-temperature and room-temperature high-pressure effects.