Cooling rate uncovers epimer-dependent supramolecular organization of carbohydrate amphiphiles.

We show distinct CH-π interactions and assembly pathways for the amphiphile N-(fluorenylmethoxycarbonyl)-galactosamine and its epimer N-(fluorenylmethoxycarbonyl)-glucosamine. These differences result in the formation of supramolecular nanofibrous systems with opposite chirality. Our results showcase the importance of the carbohydrates structural diversity for their specific biointeractions and the opportunity that their ample interactome offers for synthesis of versatile and tunable supramolecular (bio) materials.

The Potential of the Fibronectin Inhibitor Arg-Gly-Asp-Ser in the Development of Therapies for Glioblastoma.

Glioblastoma (GBM) is the most lethal and common malignant primary brain tumor in adults. An important feature that supports GBM aggressiveness is the unique composition of its extracellular matrix (ECM). Particularly, fibronectin plays an important role in cancer cell adhesion, differentiation, proliferation, and chemoresistance. Thus, herein, a hydrogel with mechanical properties compatible with the brain and the ability to disrupt the dynamic and reciprocal interaction between fibronectin and tumor cells was produced. High-molecular-weight hyaluronic acid (HMW-HA) functionalized with the inhibitory fibronectin peptide Arg-Gly-Asp-Ser (RGDS) was used to produce the polymeric matrix. Liposomes encapsulating doxorubicin (DOX) were also included in the hydrogel to kill GBM cells. The resulting hydrogel containing liposomes with therapeutic DOX concentrations presented rheological properties like a healthy brain. In vitro assays demonstrated that unmodified HMW-HA hydrogels only caused GBM cell killing after DOX incorporation. Conversely, RGDS-functionalized hydrogels displayed per se cytotoxicity. As GBM cells produce several proteolytic enzymes capable of disrupting the peptide-HA bond, we selected MMP-2 to illustrate this phenomenon. Therefore, RGDS internalization can induce GBM cell apoptosis. Importantly, RGDS-functionalized hydrogel incorporating DOX efficiently damaged GBM cells without affecting astrocyte viability, proving its safety. Overall, the results demonstrate the potential of the RGDS-functionalized hydrogel to develop safe and effective GBM treatments.

Clinical Reasoning: A 24-Year-Old Pregnant Woman With Headache and Behavioral Change Progressing to Coma.

The broad differential diagnosis associated with progressive subacute encephalopathy can be intimidating, especially in a young, pregnant woman. In this case, a 24-year-old woman at 21 weeks of gestation presented with persistent, drug-resistant fronto-parietal headache, with subsequent progressive development of psychomotor lentification and inappropriate behavior. Physical examination was normal, as were routine laboratory parameters and CT findings, and these symptoms were initially interpreted in the context of chronic depression. Later, the patient developed generalized dystonia and fever, with rapid clinical deterioration, depression of consciousness and, eventually, progression to coma. This case emphasizes the complexity and challenges involved in the diagnostic approach to a patient with progressive subacute encephalopathy framed by worsening CNS symptoms. It highlights the clinical considerations and complementary investigation of various etiologies, in a step-by-step approach, ultimately leading to the final diagnosis. Early recognition and appropriate treatment of these conditions can lead to more favorable outcomes, particularly in gestating patients, where prompt intervention is crucial, and where critical decisions may have to be made regarding pregnancy and the safety of treatment options.

Fucoidan-Coated Silica Nanoparticles Promote the Differentiation of Human Mesenchymal Stem Cells into the Osteogenic Lineage.

Silica nanoparticles (SiNPs) are widely used in biomedical applications, such as cancer therapy/diagnosis or tissue engineering and regenerative medicine. Herein, we synthesized SiNPs and modified them with sulfonic acid groups (by organosilylation followed by oxidation) or a sulfated polysaccharide (i.e., fucoidan, a seaweed biopolymer, by using electrostatic surface immobilization) due to the known capacity of the sulfonic/sulfate moieties to stabilize proteins and promote stem cell differentiation toward the osteogenic lineage. The developed pristine and functionalized nanoparticles were characterized by dynamic light scattering (DLS), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and X-ray photoelectron spectroscopy (XPS), showing the monodisperse size distribution (between 360 and 450 nm) and the success of the coating/functionalization with fucoidan or sulfonic groups. The developed SiNPs (at a concentration of 50 µg/mL) were assessed through their contact with SaOs2 cells evidencing their cytocompatibility. Furthermore, the osteogenic differentiation of bmMSCs was evaluated by the quantification of ALP activity, as well as the expression profile of osteogenic-related genes, such as Runx2, ALP, and OP. We found that the coating of the SiNPs with fucoidan induced the osteogenic differentiation of bmMSCs, being an effective mediator of bone regeneration.

Glycopeptide-Based Supramolecular Hydrogels Induce Differentiation of Adipose Stem Cells into Neural Lineages.

We applied a bottom-up approach to develop biofunctional supramolecular hydrogels from an aromatic glycodipeptide. The self-assembly of the glycopeptide was induced by either temperature manipulation (heating-cooling cycle) or solvent (DMSO to water) switch. The sol-gel transition was salt-triggered in cell culture media and resulted in gels with the same chemical compositions but different mechanical properties. Human adipose derived stem cells (hASCs) cultured on these gels under basal conditions (i.e., without differentiation factors) overexpressed neural markers, such as GFAP, Nestin, MAP2, and ßIII-tubulin, confirming the differentiation into neural lineages. The mechanical properties of the gels influenced the number and distribution of the adhered cells. A comparison with gels obtained from the nonglycosylated peptide showed that glycosylation is crucial for the biofunctionality of the hydrogels by capturing and preserving essential growth factors, e.g., FGF-2.

Revisiting predictions of movie economic success: random Forest applied to profits.

Previous studies have employed machine learning tools to classify films according to success to guide a reduction in the degree of uncertainty of film production. We revisited the literature to contribute to three relevant issues in classifying films according to economic success. First, we explored the differences between the results of the shortest or longest samples in terms of time to study possible changes in patterns of consumption mainly due to technological changes and between total and wide-released films. Second, we used profits free of price inflation as measures of economic success instead of the usual box office nominal revenues. Third, we employed a smaller set of features, only the ones available at the time of production, to help producers maneuver contingencies since little or nothing can be done by the time a film is in the theaters. We followed the literature to choose the classifiers - Random Forest, Support Vector Machine, and Neural Network - and designed sub-datasets to model and compare the performance of our results. Our dataset includes all films with budgets disclosed at the Box Office Mojo website, resulting in 3167 movies released at theaters worldwide between 1980 and 2019. The Random Forest results outperform previous similar studies with different sampling in time, including results for a less usual larger sample, with the best data sample about 97% both in accuracy and F1-score. Supplementary Information: The online version contains supplementary material available at 10.1007/s11042-023-15169-4.

A new species of Neofavolus (Polyporales, Basidiomycota) from Brazil.

Neofavolus teixeirae sp. nov. (Basidiomycota) is described and illustrated based on specimens collected from a reforestation area in southeastern Brazil. This new species is characterized by a lateral stipe up to 1.3 cm long, lacerate and angular pores measuring 0.5-2 (-2.5) per mm, and cylindrical to subcylindrical basidiospores. Phylogenetic analyses of the ITS and LSU regions confirmed its phylogenetic placement and taxonomic identity. A key to Neofavolus species is presented.

The Influence of Feeder Cell-Derived Extracellular Matrix Density on Thymic Epithelial Cell Culture.

The thymus is responsible for the selection and development of T cells, having an essential role in the establishment of adaptive immunity. Thymic epithelial cells (TECs) are key players in T cell development interacting with thymocytes in the thymic 3D environment. Feeder-layer cells have been frequently used as platforms for the successful establishment of TEC cultures. Nevertheless, the role of the feeder cell-derived extracellular matrix (ECM) on TEC cultures was not previously reported. Therefore, this work aimed at assessing the effect of the ECM produced by feeder cells cultured at two different densities on the establishment of TEC culture. Due to the high surface area and porosity, electrospun fibrous meshes were used to support ECM deposition. The feeder cell-derived ECM was efficiently recovered after decellularization, maintaining the composition of major proteins. All the decellularized matrices were permeable and showed an increase in surface mechanical properties after decellularization. TEC cultures confirmed that the ECM density impacts cellular performance, with higher densities showing a decreased cellular activity. Our findings provide evidence that feeder cell-derived ECM is a suitable substrate for TEC culture and can potentially be applied in thymus bioengineering.

Cell-Laden Marine Gelatin Methacryloyl Hydrogels Enriched with Ascorbic Acid for Corneal Stroma Regeneration.

Corneal pathologies from infectious or noninfectious origin have a significant impact on the daily lives of millions of people worldwide. Despite the risk of organ rejection or infection, corneal transplantation is currently the only effective treatment. Finding safe and innovative strategies is the main goal of tissue-engineering-based approaches. In this study, the potential of gelatin methacryloyl (GelMA) hydrogels produced from marine-derived gelatin and loaded with ascorbic acid (as an enhancer of the biological activity of cells) was evaluated for corneal stromal applications. Marine GelMA was synthesized with a methacrylation degree of 75%, enabling effective photocrosslinking, and hydrogels with or without ascorbic acid were produced, encompassing human keratocytes. All the produced formulations exhibited excellent optical and swelling properties with easy handling as well as structural stability and adequate degradation rates that may allow proper extracellular matrix remodeling by corneal stromal cells. Formulations loaded with 0.5 mg/mL of ascorbic acid enhanced the biological performance of keratocytes and induced collagen production. These results suggest that, in addition to marine-derived gelatin being suitable for the synthesis of GelMA, the hydrogels produced are promising biomaterials for corneal regeneration applications.

Bladder Wall Stiffness after Cystectomy in Bladder Cancer Patients: A Preliminary Study.

Bladder cancer (BlCa), specifically urothelial carcinomas, is a heterogeneous disease that derives from the urothelial lining. Two main classes of BlCa are acknowledged: the non-muscle invasive BlCa and the muscle-invasive BlCa; the latter constituting an aggressive disease which invades locally and metastasizes systemically. Distinguishing the specific microenvironment that cancer cells experience between mucosa and muscularis propria layers can help elucidate how these cells acquire invasive capacities. In this work, we propose to measure the micromechanical properties of both mucosa and muscularis propria layers of the bladder wall of BlCa patients, using atomic force microscopy (AFM). To do that, two cross-sections of both the macroscopically normal urinary bladder wall and the bladder wall adjacent to the tumor were collected and immediately frozen, prior to AFM samples analysis. The respective "twin" formalin-fixed paraffin-embedded tissue fragments were processed and later evaluated for histopathological examination. H&E staining suggested that tumors promoted the development of muscle-like structures in the mucosa surrounding the neoplastic region. The average Young's modulus (cell stiffness) in tumor-adjacent specimens was significantly higher in the muscularis propria than in the mucosa. Similarly, the tumor-free specimens had significantly higher Young's moduli in the muscularis propria than in the urothelium. Young's moduli were higher in all layers of tumor-adjacent tissues when compared with tumor-free samples. Here we provide insights into the stiffness of the bladder wall layers, and we show that the presence of tumor in the surrounding mucosa leads to an alteration of its smooth muscle content. The quantitative assessment of stiffness range here presented provides essential data for future research on BlCa and for understanding how the biomechanical stimuli can modulate cancer cells' capacity to invade through the different bladder layers.

Efficacy of molecular and nano-therapies on brain tumor models in microfluidic devices.

The three-dimensional (3D) organization of cells affects their mobility, proliferation, and overall response to treatment. Spheroids, organoids, and microfluidic chips are used in cancer research to reproduce in vitro the complex and dynamic malignant microenvironment. Herein, single- and double-channel microfluidic devices are used to mimic the spatial organization of brain tumors and investigate the therapeutic efficacy of molecular and nano anti-cancer agents. Human glioblastoma multiforme (U87-MG) cells were cultured into a Matrigel matrix embedded within the microfluidic devices and exposed to different doses of free docetaxel (DTXL), docetaxel-loaded spherical polymeric nanoparticles (DTXL-SPN), and the aromatic N-glucoside N-(fluorenylmethoxycarbonyl)-glucosamine-6-phosphate (Fmoc-Glc6P). We observed that in the single-channel microfluidic device, brain tumor cells are more susceptible to DTXL treatment as compared to conventional cell monolayers (50-fold lower IC50 values). In the double-channel device, the cytotoxicity of free DTXL and DTXL-SPN is comparable, but significantly lowered as compared to the single-channel configuration. Finally, the administration of 500 µM Fmoc-Glc6P in the double-channel microfluidic device shows a 50 % U87-MG cell survival after only 24 h, and no deleterious effect on human astrocytes over 72 h. Concluding, the proposed microfluidic chips can be used to reproduce the 3D complex spatial arrangement of solid tumors and to assess the anti-cancer efficacy of therapeutic compounds administrated in situ or systemically.

Cell-surface contacts determine volume and mechanical properties of human embryonic kidney 293 T cells.

Alterations in the organization of the cytoskeleton precede the escape of adherent cells from the framework of cell-cell and cell-matrix interactions into suspension. With cytoskeletal dynamics being linked to cell mechanical properties, many studies elucidated this relationship under either native adherent or suspended conditions. In contrast, tethered cells that mimic the transition between both states have not been the focus of recent research. Using human embryonic kidney 293 T cells we investigated all three conditions in the light of alterations in cellular shape, volume, as well as mechanical properties and relate these findings to the level, structure, and intracellular localization of filamentous actin (F-actin). For cells adhered to a substrate, our data shows that seeding density affects cell size but does not alter their elastic properties. Removing surface contacts leads to cell stiffening that is accompanied by changes in cell shape, and a reduction in cellular volume but no alterations in F-actin density. Instead, we observe changes in the organization of F-actin indicated by the appearance of blebs in the semi-adherent state. In summary, our work reveals an interplay between molecular and mechanical alterations when cells detach from a surface that is mainly dominated by cell morphology.

3D Biocomposites Comprising Marine Collagen and Silica-Based Materials Inspired on the Composition of Marine Sponge Skeletons Envisaging Bone Tissue Regeneration.

Ocean resources are a priceless repository of unique species and bioactive compounds with denouement properties that can be used in the fabrication of advanced biomaterials as new templates for supporting the cell culture envisaging tissue engineering approaches. The collagen of marine origin can be sustainably isolated from the underrated fish processing industry by-products, while silica and related materials can be found in the spicules of marine sponges and diatoms frustules. Aiming to address the potential of biomaterials composed from marine collagen and silica-based materials in the context of bone regeneration, four different 3D porous structure formulations (COL, COL:BG, COL:D.E, and COL:BS) were fabricated by freeze-drying. The skins of Atlantic cod (Gadus morhua) were used as raw materials for the collagen (COL) isolation, which was successfully characterized by SDS-PAGE, FTIR, CD, and amino acid analyses, and identified as a type I collagen, produced with a 1.5% yield and a preserved characteristic triple helix conformation. Bioactive glass 45S5 bioglass® (BG), diatomaceous earth (D.E.) powder, and biosilica (BS) isolated from the Axinella infundibuliformis sponge were chosen as silica-based materials, which were obtained as microparticles and characterized by distinct morphological features. The biomaterials revealed microporous structures, showing a porosity higher than 85%, a mean pore size range of 138-315 µm depending on their composition, with 70% interconnectivity which can be favorable for cell migration and ensure the needed nutrient supply. In vitro, biological assays were conducted by culturing L929 fibroblast-like cells, which confirmed not only the non-toxic nature of the developed biomaterials but also their capability to support cell adhesion and proliferation, particularly the COL:BS biomaterials, as observed by calcein-AM staining upon seven days of culture. Moreover, phalloidin and DAPI staining revealed well-spread cells, populating the entire construct. This study established marine collagen/silica biocomposites as potential scaffolds for tissue engineering, setting the basis for future studies, particularly envisaging the regeneration of non-load-bearing bone tissues.

Switching in the expression pattern of actin isoforms marks the onset of contractility and distinct mechanodynamic behavior during cardiomyocyte differentiation.

Differentiation of cardiac progenitor cells (CPC) into cardiomyocytes is a fundamental step in cardiogenesis, which is marked by changes in gene expression responsible for remodeling of the cytoskeleton and in altering the mechanical properties of cells. Here we have induced the differentiation of CPC derived from human pluripotent stem cells into immature cardiomyocytes (iCM) which we compare with more differentiated cardiomyocytes (mCM). Using atomic force microscopy and real-time deformability cytometry, we describe the mechanodynamic changes that occur during the differentiation process and link our findings to protein expression data of cytoskeletal proteins. Increased levels of cardiac-specific markers as well as evolution of cytoskeletal morphology and contractility parameters correlated with the expected extent of cell differentiation that was accompanied by hypertrophic growth of cells. These changes were associated with switching in the balance of the different actin isoforms where ß-actin is predominantly found in CPC, smooth muscle α-actin is dominant in iCM cells and sarcomeric α-actin is found in significantly higher levels in mCM. We link these cytoskeletal changes to differences in mechano-dynamic behavior of cells that translate to changes in Young's modulus that depend on the cell adherence. Our results demonstrate that the intracellular balance of actin isoform expression can be used as a sensitive ruler to determine the stage of differentiation during early phases of cardiomyocyte differentiation that correlates with an increased expression of sarcomeric proteins and is accompanied by changes in cellular elasticity.

Nanosurgical Manipulation of Titin and Its M-Complex.

Titin is a multifunctional filamentous protein anchored in the M-band, a hexagonally organized supramolecular lattice in the middle of the muscle sarcomere. Functionally, the M-band is a framework that cross-links myosin thick filaments, organizes associated proteins, and maintains sarcomeric symmetry via its structural and putative mechanical properties. Part of the M-band appears at the C-terminal end of isolated titin molecules in the form of a globular head, named here the "M-complex", which also serves as the point of head-to-head attachment of titin. We used high-resolution atomic force microscopy and nanosurgical manipulation to investigate the topographical and internal structure and local mechanical properties of the M-complex and its associated titin molecules. We find that the M-complex is a stable structure that corresponds to the transverse unit of the M-band organized around the myosin thick filament. M-complexes may be interlinked into an M-complex array that reflects the local structural and mechanical status of the transversal M-band lattice. Local segments of titin and the M-complex could be nanosurgically manipulated to achieve extension and domain unfolding. Long threads could be pulled out of the M-complex, suggesting that it is a compact supramolecular reservoir of extensible filaments. Nanosurgery evoked an unexpected volume increment in the M-complex, which may be related to its function as a mechanical spacer. The M-complex thus displays both elastic and plastic properties which support the idea that the M-band may be involved in mechanical functions within the muscle sarcomere.

The Retropepsin-Type Protease APRc as a Novel Ig-Binding Protein and Moonlighting Immune Evasion Factor of Rickettsia.

Rickettsiae are obligate intracellular Gram-negative bacteria transmitted by arthropod vectors. Despite their reduced genomes, the function(s) of the majority of rickettsial proteins remains to be uncovered. APRc is a highly conserved retropepsin-type protease, suggested to act as a modulator of other rickettsial surface proteins with a role in adhesion/invasion. However, APRc's function(s) in bacterial pathogenesis and virulence remains unknown. This study demonstrates that APRc targets host serum components, combining nonimmune immunoglobulin (Ig)-binding activity with resistance to complement-mediated killing. We confirmed nonimmune human IgG binding in extracts of different rickettsial species and intact bacteria. Our results revealed that the soluble domain of APRc is capable of binding to human (h), mouse, and rabbit IgG and different classes of human Ig (IgG, IgM, and IgA) in a concentration-dependent manner. APRc-hIgG interaction was confirmed with total hIgG and normal human serum. APRc-hIgG displayed a binding affinity in the micromolar range. We provided evidence of interaction preferentially through the Fab region and confirmed that binding is independent of catalytic activity. Mapping the APRc region responsible for binding revealed the segment between amino acids 157 and 166 as one of the interacting regions. Furthermore, we demonstrated that expression of the full-length protease in Escherichia coli is sufficient to promote resistance to complement-mediated killing and that interaction with IgG contributes to serum resistance. Our findings position APRc as a novel Ig-binding protein and a novel moonlighting immune evasion factor of Rickettsia, contributing to the arsenal of virulence factors utilized by these intracellular pathogens to aid in host colonization. IMPORTANCE Many Rickettsia organisms are pathogenic to humans, causing severe infections, like Rocky Mountain spotted fever and Mediterranean spotted fever. However, immune evasion mechanisms and pathogenicity determinants in rickettsiae are far from being resolved. We provide evidence that the highly conserved rickettsial retropepsin-type protease APRc displays nonimmune immunoglobulin (Ig)-binding activity and participates in serum resistance. APRc emerges then as a novel Ig-binding protein from Gram-negative bacteria and the first to be identified in Rickettsia. Bacterial surface proteins capable of Ig binding are known to be multifunctional and key players in immune evasion. We demonstrate that APRc is also a novel moonlighting protein, exhibiting different actions on serum components and acting as a novel evasin. This work strengthens APRc as a virulence factor in Rickettsia and its significance as a potential therapeutic target. Our findings significantly contribute to a deeper understanding of the virulence strategies used by intracellular pathogens to subvert host immune responses.

Functional Gallic Acid-Based Dendrimers as Synthetic Nanotools to Remodel Amyloid-ß-42 into Noncytotoxic Forms.

The self-assembly of amyloid-ß (Aß) generates cytotoxic oligomers linked to the onset and progression of Alzheimer's disease (AD). As many fundamental molecular pathways that control Aß aggregation are yet to be unraveled, an important strategy to control Aß cytotoxicity is the development of bioactive synthetic nanotools capable of interacting with the heterogeneous ensemble of Aß species and remodel them into noncytotoxic forms. Herein, the synthesis of nanosized, functional gallic acid (Ga)-based dendrimers with a precise number of Ga at their surface is described. It is shown that these Ga-terminated dendrimers interact by H-bonding with monomeric/oligomeric Aß species at their Glu, Ala, and Asp residues, promoting their remodeling into noncytotoxic aggregates in a process controlled by the Ga units. The multivalent presentation of Ga on the dendrimer surface enhances their ability to interact with Aß, inhibiting the primary and secondary nucleation of Aß fibrillization and disrupting the Aß preformed fibrils.

Micropatterned gellan gum-based hydrogels tailored with laminin-derived peptides for skeletal muscle tissue engineering.

The efficacy of current therapies for skeletal muscle disorders/injuries are limited urging the need for new treatments. Skeletal muscle tissue engineered platforms represent a promising tool to shed light on the pathophysiology of skeletal muscle disorders/injuries and to investigate the efficacy of new therapies. Herein, we developed a skeletal muscle platform composed of aligned and differentiated myoblasts on micropatterned gellan gum (GG)-based hydrogels tailored with a laminin-derived peptide. To this aim, the binding of murine skeletal muscle cells (C2C12) to different laminin-derived peptides (CIKVAVS (V), KNRLTIELEVRTC (T), and RKRLQVQLSIRTC (Q)) and the binding of laminin-derived peptides to chemically functionalized GG was studied. C2C12-binding to peptide V, T and Q was 10%, 48% and 25%, whereas the peptide tethering to GG was 60%, 40% and 31%, respectively. Peptide-biofunctionalized hydrogels prepared with different polymer content showed different mechanics and peptide exposure at hydrogel surface. Cellular adhesion was detected in all hydrogel formulations, but spreading and differentiation was only promoted in peptide Q-biofunctionalized hydrogels and preferably in stiffer hydrogels. Myoblast alignment was promoted in micropatterned hydrogel surfaces. Overall, the engineered skeletal muscle herein proposed can be further explored as a platform to better understand skeletal muscle disorders/injuries and to screen new therapies.

Expanding the Conformational Landscape of Minimalistic Tripeptides by Their O-Glycosylation.

We report on the supramolecular self-assembly of tripeptides and their O-glycosylated analogues, in which the carbohydrate moiety is coupled to a central serine or threonine flanked by phenylalanine residues. The substitution of serine with threonine introduces differential side-chain interactions, which results in the formation of aggregates with different morphology. O-glycosylation decreases the aggregation propensity because of rebalancing of the π interactions. The glycopeptides form aggregates with reduced stiffness but increased thermal stability. Our results demonstrate that the designed minimalistic glycopeptides retain critical functional features of glycoproteins and therefore are promising tools for elucidation of molecular mechanisms involved in the glycoprotein interactome. They can also serve as an inspiration for the design of functional glycopeptide-based biomaterials.