DeLA-DrugSelf: Empowering multi-objective de novo design through SELFIES molecular representation.

In this paper, we introduce DeLA-DrugSelf, an upgraded version of DeLA-Drug [J. Chem. Inf. Model. 62 (2022) 1411-1424], which incorporates essential advancements for automated multi-objective de novo design. Unlike its predecessor, which relies on SMILES notation for molecular representation, DeLA-DrugSelf employs a novel and robust molecular representation string named SELFIES (SELF-referencing Embedded String). The generation process in DeLA-DrugSelf not only involves substitutions to the initial string representing the starting query molecule but also incorporates insertions and deletions. This enhancement makes DeLA-DrugSelf significantly more adept at executing data-driven scaffold decoration and lead optimization strategies. Remarkably, DeLA-DrugSelf explicitly addresses the SELFIES-related collapse issue, considering only collapse-free compounds during generation. These compounds undergo a rigorous quality metrics evaluation, highlighting substantial advancements in terms of drug-likeness, uniqueness, and novelty compared to the molecules generated by the previous version of the algorithm. To evaluate the potential of DeLA-DrugSelf as a mutational operator within a genetic algorithm framework for multi-objective optimization, we employed a fitness function based on Pareto dominance. Our objectives focused on target-oriented properties aimed at optimizing known cannabinoid receptor 2 (CB2R) ligands. The results obtained indicate that DeLA-DrugSelf, available as a user-friendly web platform (https://www.ba.ic.cnr.it/softwareic/delaself/), can effectively contribute to the data-driven optimization of starting bioactive molecules based on user-defined parameters.

Influence of Intramineral Proteins on the Growth of Carbonate Crystals Using as a Scaffold Membranes of Ratite Birds and Crocodiles Eggshells.

The lack of information on structural basis where proteins are involved, as well as the biomineralization processes of different systems such as bones, diatom frustules, and eggshells, have intrigued scientists from different fields for decades. This scientific curiosity has led to the use of methodologies that help understand the mechanism involved in the formation of these complex structures. Therefore, this work focuses on the use of eggshell membranes from different species of ratites (emu and ostrich) and reptiles (two species of crocodiles) as a model to differentiate biocalcification and biosilicification by introducing calcium phosphate or silica inside the membrane fiber mantles. We performed this to obtain information about the process of eggshell formation as well as the changes that occur in the membrane during crystal formation. In order to identify and understand the early processes leading to the formation of the microstructures present in the eggshell, we decided to carry out the synthesis of silica-carbonate of calcium, barium, and strontium called biomorph in the presence of intramineral proteins. This was carried out to evaluate the influence of these proteins on the formation of specific structures. We found that the proteins on untreated membranes, present a structural growth similar to those observed in the inner part of the eggshell, while in treated membranes, the structures formed present a high similarity with those observed in the outer and intermediate part of the eggshell. Finally, a topographic and molecular analysis of the biomorphs and membranes was performed by scanning electron microscopy (SEM), Raman and Fourier-transform Infrared (FTIR) spectroscopies.

A conformational rearrangement of the SARS-CoV-2 host protein sigma-1 is required for antiviral activity: insights from a combined in-silico/in-vitro approach.

The development of effective drugs to treat coronavirus infections remains a significant challenge for the scientific community. Recent evidence reports on the sigma-1 receptor (S1R) as a key druggable host protein in the SARS-CoV-1 and SARS-CoV-2 interactomes and shows a potent antiviral activity against SARS-CoV-2 for the S1R antagonist PB28. To improve PB28 activity, we designed and tested a series of its analogues and identified a compound that is fourfold more potent against SARS-CoV-2 than PB28 itself. Interestingly, we found no direct correlation between S1R affinity and SARS-CoV-2 antiviral activity. Building on this, we employed comparative induced fit docking and molecular dynamics simulations to gain insights into the possible mechanism that occurs when specific ligand-protein interactions take place and that may be responsible for the observed antiviral activity. Our findings offer a possible explanation for the experimental observations, provide insights into the S1R conformational changes upon ligand binding and lay the foundation for the rational design of new S1R ligands with potent antiviral activity against SARS-CoV-2 and likely other viruses.

The crystal structure of the EspB-EspK virulence factor-chaperone complex suggests an additional type VII secretion mechanism in Mycobacterium tuberculosis.

Pathogenic species from the Mycobacterium genus are responsible for a number of adverse health conditions in humans and animals that threaten health security and the economy worldwide. Mycobacteria have up to five specialized secretion systems (ESX-1 to ESX-5) that transport virulence factors across their complex cell envelope to facilitate manipulation of their environment. In pathogenic species, these virulence factors influence the immune system's response and are responsible for membrane disruption and contributing to cell death. While structural details of these secretion systems have been recently described, gaps still remain in the structural understanding of the secretion mechanisms of most substrates. Here, we describe the crystal structure of Mycobacterium tuberculosis ESX-1 secretion-associated substrate EspB bound to its chaperone EspK. We found that EspB interacts with the C-terminal domain of EspK through its helical tip. Furthermore, cryogenic electron microscopy, size exclusion chromatography analysis, and small-angle X-ray scattering experiments show that EspK keeps EspB in its secretion-competent monomeric form and prevents its oligomerization. The structure presented in this study suggests an additional secretion mechanism in ESX-1, analogous to the chaperoning of proline-glutamate (PE)-proline-proline-glutamate (PPE) proteins by EspG, where EspK facilitates the secretion of EspB in Mycobacterium species.

A Comparative Molecular Dynamics Study of Selected Point Mutations in the Shwachman-Bodian-Diamond Syndrome Protein SBDS.

The Shwachman-Diamond Syndrome (SDS) is an autosomal recessive disease whose majority of patients display mutations in a ribosome assembly protein named Shwachman-Bodian-Diamond Syndrome protein (SBDS). A specific therapy for treating this rare disease is missing, due to the lack of knowledge of the molecular mechanisms responsible for its pathogenesis. Starting from the observation that SBDS single-point mutations, localized in different domains of the proteins, are responsible for an SDS phenotype, we carried out the first comparative Molecular Dynamics simulations on three SBDS mutants, namely R19Q, R126T and I212T. The obtained 450-ns long trajectories were compared with those returned by both the open and closed forms of wild type SBDS and strongly indicated that two distinct conformations (open and closed) are both necessary for the proper SBDS function, in full agreement with recent experimental observations. Our study supports the hypothesis that the SBDS function is governed by an allosteric mechanism involving domains I and III and provides new insights into SDS pathogenesis, thus offering a possible starting point for a specific therapeutic option.

X-ray Characterization of Conformational Changes of Human Apo- and Holo-Transferrin.

Human serum transferrin (Tf) is a bilobed glycoprotein whose function is to transport iron through receptor-mediated endocytosis. The mechanism for iron release is pH-dependent and involves conformational changes in the protein, thus making it an attractive system for possible biomedical applications. In this contribution, two powerful X-ray techniques, namely Macromolecular X-ray Crystallography (MX) and Small Angle X-ray Scattering (SAXS), were used to study the conformational changes of iron-free (apo) and iron-loaded (holo) transferrin in crystal and solution states, respectively, at three different pH values of physiological relevance. A crystallographic model of glycosylated apo-Tf was obtained at 3.0 Å resolution, which did not resolve further despite many efforts to improve crystal quality. In the solution, apo-Tf remained mostly globular in all the pH conditions tested; however, the co-existence of closed, partially open, and open conformations was observed for holo-Tf, which showed a more elongated and flexible shape overall.

The structure and flexibility analysis of the Arabidopsis synaptotagmin 1 reveal the basis of its regulation at membrane contact sites.

Non-vesicular lipid transfer at ER and plasma membrane (PM) contact sites (CS) is crucial for the maintenance of membrane lipid homeostasis. Extended synaptotagmins (E-Syts) play a central role in this process as they act as molecular tethers of ER and PM and as lipid transfer proteins between these organelles. E-Syts are proteins constitutively anchored to the ER through an N-terminal hydrophobic segment and bind the PM via a variable number of C-terminal C2 domains. Synaptotagmins (SYTs) are the plant orthologous of E-Syts and regulate the ER-PM communication in response to abiotic stress. Combining different structural and biochemical techniques, we demonstrate that the binding of SYT1 to lipids occurs through a Ca2+-dependent lipid-binding site and by a site for phosphorylated forms of phosphatidylinositol, thus integrating two different molecular signals in response to stress. In addition, we show that SYT1 displays three highly flexible hinge points that provide conformational freedom to facilitate lipid extraction, protein loading, and subsequent transfer between PM and ER.

The effect of chemical structure of carboxylate molecules on hydroxyapatite nanoparticles. A structural and morphological study.

Being the most abundant non-macromolecular organic component of bone, the role of citrate (Cit) in hydroxyapatite (HA) crystallization is of high relevance. In this work we have investigated the influence of hydroxycitrate (CitOH) and glutarate (Glr) on HA crystallization in terms of particle growth, composition, and morphology in comparison to Cit. CitOH and Glr have been selected for this work because they share the same backbone structure of Cit but bear different functional groups in the central region. Our data has revealed that CitOH strongly inhibits HA crystallization more efficiently than Cit. CitOH-HA nanoparticles are composed of platy, elongated particles similar to those of Cit-HA but they are ca. twice smaller and have a lower crystal order. On the other hand, Glr does not inhibit HA crystallization as Cit, but leads to the formation of OCP platelets that convert with maturation time to HA nanorods with larger aspect ratio than Cit-HA. In comparison to Cit-HA samples, Glr-HA nanoparticles have bigger dimensions, and higher structural order. Overall, our data reveal that the central carboxyl group of Cit is involved in the selective binding with HA crystal surface and in regulating HA crystal growth. The results of this work highlight new possibilities to control the formation of HA for designing advanced bioactive materials and give new insights on the role of the structure of Cit in regulating the HA morphology.

X-ray ptychographic mode of self-assembled CdSe/CdS octapod-shaped nanocrystals in thick polymers.

This work describes the application of X-ray ptychography for the inspection of complex assemblies of highly anisotropic nanocrystals embedded in a thick polymer matrix. More specifically, this case deals with CdSe/CdS octapods, with pod length L = 39 ± 2 nm and pod diameter D = 12 ± 2 nm, dispersed in free-standing thick films (24 ± 4 µm) of polymethyl methacrylate and polystyrene, with different molecular weights. Ptychography is the only imaging method available to date that can be used to study architectures made by these types of nanocrystals in thick polymeric films, as any other alternative direct method, such as scanning/transmission electron microscopy, can be definitively ruled out as a result of the large thickness of the free-standing films. The electron density maps of the investigated samples are reconstructed by combining iterative difference map algorithms and a maximum likelihood optimization algorithm. In addition, post image processing techniques are applied to both reduce noise and provide a better visualization of the material morphological details. Through this process, at a final resolution of 27 nm, the reconstructed maps allow us to visualize the intricate network of octapods inside the polymeric matrices.

Table-top combined scanning X-ray small angle scattering and transmission microscopies of lipid vesicles dispersed in free-standing gel.

A mm thick free-standing gel containing lipid vesicles made of 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC) was studied by scanning Small Angle X-ray Scattering (SAXS) and X-ray Transmission (XT) microscopies. Raster scanning relatively large volumes, besides reducing the risk of radiation damage, allows signal integration, improving the signal-to-noise ratio (SNR), as well as high statistical significance of the dataset. The persistence of lipid vesicles in gel was demonstrated, while mapping their spatial distribution and concentration gradients. Information about lipid aggregation and packing, as well as about gel density gradients, was obtained. A posteriori confirmation of lipid presence in well-defined sample areas was obtained by studying the dried sample, featuring clear Bragg peaks from stacked bilayers. The comparison between wet and dry samples allowed it to be proved that lipids do not significantly migrate within the gel even upon drying, whereas bilayer curvature is lost by removing water, resulting in lipids packed in ordered lamellae. Suitable algorithms were successfully employed for enhancing transmission microscopy sensitivity to low absorbing objects, and allowing full SAXS intensity normalization as a general approach. In particular, data reduction includes normalization of the SAXS intensity against the local sample thickness derived from absorption contrast maps. The proposed study was demonstrated by a room-sized instrumentation, although equipped with a high brilliance X-ray micro-source, and is expected to be applicable to a wide variety of organic, inorganic, and multicomponent systems, including biomaterials. The employed routines for data reduction and microscopy, including Gaussian filter for contrast enhancement of low absorbing objects and a region growing segmentation algorithm to exclude no-sample regions, have been implemented and made freely available through the updated in-house developed software SUNBIM.

Exploring the role of elongation Factor-Like 1 (EFL1) in Shwachman-Diamond syndrome through molecular dynamics.

Shwachman-Diamond Syndrome (SDS) is an autosomal recessive disorder whose patients present mutations in two ribosome assembly proteins, the Shwachman-Bodian-Diamond Syndrome protein (SBDS) and the Elongation Factor-Like 1 (EFL1). Due to the lack of knowledge of the molecular mechanisms responsible for SDS pathogenesis, current therapy is nonspecific and focuses only at alleviating the symptoms. Building on the recent observation that EFL1 single-point mutations clinically manifest as SDS-like phenotype, we carried out comparative Molecular Dynamics (MD) simulations on three mutants, T127A, M882K and R1095Q and wild type EFL1. As supported by small angle X-ray scattering experiments, the obtained data improve the static EFL1 model resulting from the Cryo-electron microscopy and clearly show that all the mutants experience a peculiar rotation, around the hinge region, of domain IV with respect to domains I and II leading to a different conformation respect to that of wild type protein. This study supports the notion that EFL1 function is governed by an allosteric mechanism involving the concerted action of GTPase domain (domain I) and the domain IV and can help point towards new approaches to SDS treatment.Communicated by Ramaswamy H. Sarma.

Structural Insights into the Intracellular Region of the Human Magnesium Transport Mediator CNNM4.

The four member family of "Cyclin and Cystathionine ß-synthase (CBS) domain divalent metal cation transport mediators", CNNMs, are the least-studied mammalian magnesium transport mediators. CNNM4 is abundant in the brain and the intestinal tract, and its abnormal activity causes Jalili Syndrome. Recent findings show that suppression of CNNM4 in mice promotes malignant progression of intestinal polyps and is linked to infertility. The association of CNNM4 with phosphatases of the regenerating liver, PRLs, abrogates its Mg2+-efflux capacity, thus resulting in an increased intracellular Mg2+ concentration that favors tumor growth. Here we present the crystal structures of the two independent intracellular domains of human CNNM4, i.e., the Bateman module and the cyclic nucleotide binding-like domain (cNMP). We also derive a model structure for the full intracellular region in the absence and presence of MgATP and the oncogenic interacting partner, PRL-1. We find that only the Bateman module interacts with ATP and Mg2+, at non-overlapping sites facilitating their positive cooperativity. Furthermore, both domains dimerize autonomously, where the cNMP domain dimer forms a rigid cleft to restrict the Mg2+ induced sliding of the inserting CBS1 motives of the Bateman module, from a twisted to a flat disk shaped dimer.

A biophysical and structural study of two chitinases from Agave tequilana and their potential role as defense proteins.

Plant chitinases are enzymes that have several functions, including providing protection against pathogens. Agave tequilana is an economically important plant that is poorly studied. Here, we identified a chitinase from short reads of the A. tequilana transcriptome (AtChi1). A second chitinase, differing by only six residues from the first, was isolated from total RNA of plants infected with Fusarium oxysporum (AtChi2). Both enzymes were overexpressed in Escherichia coli and analysis of their sequences indicated that they belong to the class I glycoside hydrolase family19, whose members exhibit two domains: a carbohydrate-binding module and a catalytic domain, connected by a flexible linker. Activity assays and thermal shift experiments demonstrated that the recombinant Agave enzymes are highly thermostable acidic endochitinases with Tm values of 75 °C and 71 °C. Both exhibit a molecular mass close to 32 kDa, as determined by MALDI-TOF, and experimental pIs of 3.7 and 3.9. Coupling small-angle x-ray scattering information with homology modeling and docking simulations allowed us to structurally characterize both chitinases, which notably show different interactions in the binding groove. Even when the six different amino acids are all exposed to solvent in the loops located near the linker and opposite to the binding site, they confer distinct kinetic parameters against colloidal chitin and similar affinity for (GlnNAc)6, as shown by isothermal titration calorimetry. Interestingly, binding is more enthalpy-driven for AtChi2. Whereas the physiological role of these chitinases remains unknown, we demonstrate that they exhibit important antifungal activity against chitin-rich fungi such as Aspergillus sp. DATABASE: SAXS structural data are available in the SASBDB database with accession numbers SASDDE7 and SASDDA6. ENZYMES: Chitinases (EC3.2.1.14).

Interaction of the GTPase Elongation Factor Like-1 with the Shwachman-Diamond Syndrome Protein and Its Missense Mutations.

The Shwachman-Diamond Syndrome (SDS) is a disorder arising from mutations in the genes encoding for the Shwachman-Bodian-Diamond Syndrome (SBDS) protein and the GTPase known as Elongation Factor Like-1 (EFL1). Together, these proteins remove the anti-association factor eIF6 from the surface of the pre-60S ribosomal subunit to promote the formation of mature ribosomes. SBDS missense mutations can either destabilize the protein fold or affect surface epitopes. The molecular alterations resulting from the latter remain largely unknown, although some evidence suggest that binding to EFL1 may be affected. We further explored the effect of these SBDS mutations on the interaction with EFL1, and showed that all tested mutations disrupted the binding to EFL1. Binding was either severely weakened or almost abolished, depending on the assessed mutation. In higher eukaryotes, SBDS is essential for development, and lack of the protein results in early lethality. The existence of patients whose only source of SBDS consists of that with surface missense mutations highlights the importance of the interaction with EFL1 for their function. Additionally, we studied the interaction mechanism of the proteins in solution and demonstrated that binding consists of two independent and cooperative events, with domains 2⁻3 of SBDS directing the initial interaction with EFL1, followed by docking of domain 1. In solution, both proteins exhibited large flexibility and consisted of an ensemble of conformations, as demonstrated by Small Angle X-ray Scattering (SAXS) experiments.

Coherent Diffraction Imaging in Transmission Electron Microscopy for Atomic Resolution Quantitative Studies of the Matter.

The paper focuses on the development of electron coherent diffraction imaging in transmission electron microscopy, made in the, approximately, last ten years in our collaborative research group, to study the properties of materials at atomic resolution, overcoming the limitations due to the aberrations of the electron lenses and obtaining atomic resolution images, in which the distribution of the maxima is directly related to the specimen atomic potentials projected onto the microscope image detector. Here, it is shown how augmented coherent diffraction imaging makes it possible to achieve quantitative atomic resolution maps of the specimen atomic species, even in the presence of low atomic number atoms within a crystal matrix containing heavy atoms. This aim is achieved by: (i) tailoring the experimental set-up, (ii) improving the experimental data by properly treating parasitic diffused intensities to maximize the measure of the significant information, (iii) developing efficient methods to merge the information acquired in both direct and reciprocal spaces, (iv) treating the dynamical diffused intensities to accurately measure the specimen projected potentials, (v) improving the phase retrieval algorithms to better explore the space of solutions. Finally, some of the future perspectives of coherent diffraction imaging in a transmission electron microscope are given.

Relationship between nano/micro structure and physical properties of TiO2-sodium caseinate composite films.

Films obtained by casting, starting from conventional emulsions (CE), nanoemulsions (NE) or their gels, which led to different structures, with the aim of explore the relationship between structure and physical properties, were prepared. Sodium caseinate was used as the matrix, glycerol as plasticizer, glucono-delta-lactone as acidulant to form the gels, and TiO2 nanoparticles as reinforcement to improve physical behavior. Structural characterization was performed by SAXS and WAXS (Small and Wide Angle X-ray Scattering, respectively), combined with confocal and scanning electron microscopy. The results demonstrate that the incorporation of the lipid phase does not notably modify the mechanical properties of the films compared to solution films. Films from NE were more stable against oil release than those from CE. Incorporation of TiO2 improved mechanical properties as measured by dynamical mechanical analysis (DMA) and uniaxial tensile tests. TiO2 macroscopic spatial distribution homogeneity and the nanostructure character of NE films were confirmed by mapping the q-dependent scattering intensity in scanning SAXS experiments. SAXS microscopies indicated a higher intrinsic homogeneity of NE films compared to CE films, independently of the TiO2 load. NE-films containing structures with smaller and more homogeneously distributed building blocks showed greater potential for food applications than the films prepared from sodium caseinate solutions, which are the best known films.

Interdomain conformational flexibility underpins the activity of UGGT, the eukaryotic glycoprotein secretion checkpoint.

Glycoproteins traversing the eukaryotic secretory pathway begin life in the endoplasmic reticulum (ER), where their folding is surveyed by the 170-kDa UDP-glucose:glycoprotein glucosyltransferase (UGGT). The enzyme acts as the single glycoprotein folding quality control checkpoint: it selectively reglucosylates misfolded glycoproteins, promotes their association with ER lectins and associated chaperones, and prevents premature secretion from the ER. UGGT has long resisted structural determination and sequence-based domain boundary prediction. Questions remain on how this single enzyme can flag misfolded glycoproteins of different sizes and shapes for ER retention and how it can span variable distances between the site of misfold and a glucose-accepting N-linked glycan on the same glycoprotein. Here, crystal structures of a full-length eukaryotic UGGT reveal four thioredoxin-like (TRXL) domains arranged in a long arc that terminates in two ß-sandwiches tightly clasping the glucosyltransferase domain. The fold of the molecule is topologically complex, with the first ß-sandwich and the fourth TRXL domain being encoded by nonconsecutive stretches of sequence. In addition to the crystal structures, a 15-Å cryo-EM reconstruction reveals interdomain flexibility of the TRXL domains. Double cysteine point mutants that engineer extra interdomain disulfide bridges rigidify the UGGT structure and exhibit impaired activity. The intrinsic flexibility of the TRXL domains of UGGT may therefore endow the enzyme with the promiscuity needed to recognize and reglucosylate its many different substrates and/or enable reglucosylation of N-linked glycans situated at variable distances from the site of misfold.

Mutations in EFL1, an SBDS partner, are associated with infantile pancytopenia, exocrine pancreatic insufficiency and skeletal anomalies in aShwachman-Diamond like syndrome.

BACKGROUND: For the final step of the maturation of the ribosome, the nascent 40S and 60S subunits are exported from the nucleus to the cell cytoplasm. To prevent premature association of these ribosomal subunits, eukaryotic initiation factor 6 (eIF6) binds the 60S subunit within the nucleus. Its release in the cytoplasm requires the interaction of EFL1 and SDBS proteins. In Shwachman-Diamond syndrome (SDS), a defective SDBS protein prevents eIF6 eviction, inhibiting its recycle to the nucleus and subsequent formation of the active 80S ribosome. OBJECTIVE: This study aims to identify the molecular basis of an SDS-like disease, manifested by pancytopenia, exocrine pancreatic insufficiency and skeletal abnormalities in six patients from three unrelated families. METHODS: Whole exome analysis was used for mutation identification. Fluorescence microscopy studies assessed the localisation of Tif6-GFP, the yeast eIF6 homologue, in yeast WT and mutant cells. Human and yeast EFL1 proteins, WT and mutants, were expressed in Saccharomyces cerevisiae BCY123 strain, and circular dichroism and small-angle X-ray scattering were used to assess the folding and flexibility of these proteins. Green malachite colorimetric assay was performed to determine the GTPase activity of WT and Efl1 mutants. RESULTS: Four patients were homozygous for p.R1095Q variant and two patients were homozygous for p.M882K variant in EFL1. Residue R1095 and M882 are conserved across species. Neither the GTPase activity of the mutant proteins nor its activation by the SDBD protein or the 60S ribosomal subunit were affected. Complementation of efl1Δ yeast cells with the EFL1 mutants rescued the slow growth phenotype. Nonetheless, Tif6-GFP was relocalised to the cytoplasm in mutant yeast cells in contrast to its nuclear localisation in WT cells. CONCLUSIONS: Mutations in EFL1 clinically manifest as SDS-like phenotype. Similar to the molecular pathology of SDS, mutant EFL1 proteins do not promote the release of cytoplasmic Tif6 from the 60S subunit, likely preventing the formation of mature ribosomes.

Scanning Small- and Wide-Angle X-ray Scattering Microscopy Selectively Probes HA Content in Gelatin/Hydroxyapatite Scaffolds for Osteochondral Defect Repair.

This study is aimed at investigating the structure of a scaffold made of bovine gelatin and hydroxyapatite for bone tissue engineering purposes. In particular, the detailed characterization of such a material has a great relevance because of its application in the healing process of the osteochondral defect that consists of a damage of cartilage and injury of the adjacent subchondral bone, significantly compromising millions of patient's quality of life. Two different techniques exploiting X-ray radiation, with table-top setups, are used: microtomography (micro-CT) and microdiffraction. Micro-CT characterizes the microstructure in the three dimensions at the micrometer scale spatial resolution, whereas microdiffraction provides combined structural/morphological information at the atomic and nanoscale, in two dimensional microscopy images with a hundred micrometer spatial resolution. The combination of these two techniques allowed an appropriate structural characterization for the purpose of validating the engineering approach used for the realization of the hydroxyapatite gradient across the scaffold, with properties close to the natural model.

Ptychographic Imaging of Branched Colloidal Nanocrystals Embedded in Free-Standing Thick Polystyrene Films.

Research on composite materials is facing, among others, the challenging task of incorporating nanocrystals, and their superstructures, in polymer matrices. Electron microscopy can typically image nanometre-scale structures embedded in thin polymer films, but not in films that are micron size thick. Here, X-ray Ptychography was used to visualize, with a resolution of a few tens of nanometers, how CdSe/CdS octapod-shaped nanocrystals self-assemble in polystyrene films of 24 ± 4 µm, providing a unique means for non-destructive investigation of nanoparticles distribution and organization in thick polymer films.