Qualitative Metabolite Profiling of Orchis purpurea Huds. by GC and UHPLC/MS Approaches.

Orchids are experiencing wide success in ornamental, medicinal, and food fields. The reason for their success is correlated with both their morphology and metabolomics, the latter linked to their taste and biological effects. Despite many orchids having already been the subject of chemotaxonomic works, some of them are still untapped, like the case of Orchis purpurea. O. purpurea is one of the most common species of the genus Orchis, present in hedgerows, verges, and light woodland, where it is one of the few herbaceous plants able to be unpleasant to herbivorous animals. Essential oil from roots, stems, leaves, and flowers were analyzed via GC/MS analyses, revealing the presence of 70 compounds, with a clear prevalence of coumarin. The high concentration of this metabolite may explain the resistance of O. purpurea to herbivores, being associated with appetite-suppressing properties and a bitter taste. Non-volatile fractions were analyzed via UHPLC-MS analysis revealing the presence of hydroxycinnamic acid derivatives, polyphenols, and glycosidic compounds, probably responsible for their color and fragrance. Taken together, the herein presented results shed light on both the defensive strategy and the chemotaxonomy of O. purpurea.

Digested Cinnamon (Cinnamomum verum J. Presl) Bark Extract Modulates Claudin-2 Gene Expression and Protein Levels under TNFα/IL-1ß Inflammatory Stimulus.

Epigenetic changes, host-gut microbiota interactions, and environmental factors contribute to inflammatory bowel disease (IBD) onset and progression. A healthy lifestyle may help to slow down the chronic or remitting/relapsing intestinal tract inflammation characteristic of IBD. In this scenario, the employment of a nutritional strategy to prevent the onset or supplement disease therapies included functional food consumption. Its formulation consists of the addition of a phytoextract enriched in bioactive molecules. A good candidate as an ingredient is the Cinnamon verum aqueous extract. Indeed, this extract, subjected to a process of gastrointestinal digestion simulation (INFOGEST), exhibits beneficial antioxidant and anti-inflammatory properties in an in vitro model of the inflamed intestinal barrier. Here, we deepen the study of the mechanisms related to the effect of digested cinnamon extract pre-treatment, showing a correlation between transepithelial electrical resistance (TEER) decrement and alterations in claudin-2 expression under Tumor necrosis factor-α/Interleukin-1ß (TNF-α/IL-1) ß cytokine administration. Our results show that pre-treatment with cinnamon extract prevents TEER loss by claudin-2 protein level regulation, influencing both gene transcription and autophagy-mediated degradation. Hence, cinnamon polyphenols and their metabolites probably work as mediators in gene regulation and receptor/pathway activation, leading to an adaptive response against renewed insults.

LC-MS and GC-MS Data Fusion Metabolomics Profiling Coupled with Multivariate Analysis for the Discrimination of Different Parts of Faustrime Fruit and Evaluation of Their Antioxidant Activity.

The comparative chemical composition of different part of Faustrime fruits (peels, pulp, albedo, and seeds) extracted with different solvents was determined by GC-MS and UHPLC-HRMS QTof. The obtained data were also combined for their in vitro antioxidant activity by multivariate analysis to define a complex fingerprint of the fruit. The principal component analysis model showed the significative occurrence of volatile organic compounds as α-bisabolol and α-trans-bergamotol in the pulp and albedo, hexanoic acid in the seeds, and several coumarins and phenolics in the peels. The higher radical scavenging activity of the pulp was related to the incidence of citric acid in partial least square regression.

Antioxidant and Anti-Inflammatory Effect of Cinnamon (Cinnamomum verum J. Presl) Bark Extract after In Vitro Digestion Simulation.

Cinnamon bark is widely used for its organoleptic features in the food context and growing evidence supports its beneficial effect on human health. The market offers an increasingly wide range of food products and supplements enriched with cinnamon extracts which are eliciting beneficial and health-promoting properties. Specifically, the extract of Cinnamomum spp. is rich in antioxidant, anti-inflammatory and anticancer biomolecules. These include widely reported cinnamic acid and some phenolic compounds, such asproanthocyanidins A and B, and kaempferol. These molecules are sensitive to physical-chemical properties (such as pH and temperature) and biological agents that act during gastric digestion, which could impair molecules' bioactivity. Therefore, in this study, the cinnamon's antioxidant and anti-inflammatory bioactivity after simulated digestion was evaluated by analyzing the chemical profile of the pure extract and digested one, as well as the cellular effect in vitro models, such as Caco2 and intestinal barrier. The results showed that the digestive process reduces the total content of polyphenols, especially tannins, while preserving other bioactive compounds such as cinnamic acid. At the functional level, the digested extract maintains an antioxidant and anti-inflammatory effect at the cellular level.

Generation and maturation of human iPSC-derived 3D organotypic cardiac microtissues in long-term culture.

Cardiovascular diseases remain the leading cause of death worldwide; hence there is an increasing focus on developing physiologically relevant in vitro cardiovascular tissue models suitable for studying personalized medicine and pre-clinical tests. Despite recent advances, models that reproduce both tissue complexity and maturation are still limited. We have established a scaffold-free protocol to generate multicellular, beating human cardiac microtissues in vitro from hiPSCs-namely human organotypic cardiac microtissues (hOCMTs)-that show some degree of self-organization and can be cultured for long term. This is achieved by the differentiation of hiPSC in 2D monolayer culture towards cardiovascular lineage, followed by further aggregation on low-attachment culture dishes in 3D. The generated hOCMTs contain multiple cell types that physiologically compose the heart and beat without external stimuli for more than 100 days. We have shown that 3D hOCMTs display improved cardiac specification, survival and metabolic maturation as compared to standard monolayer cardiac differentiation. We also confirmed the functionality of hOCMTs by their response to cardioactive drugs in long-term culture. Furthermore, we demonstrated that they could be used to study chemotherapy-induced cardiotoxicity. Due to showing a tendency for self-organization, cellular heterogeneity, and functionality in our 3D microtissues over extended culture time, we could also confirm these constructs as human cardiac organoids (hCOs). This study could help to develop more physiologically-relevant cardiac tissue models, and represent a powerful platform for future translational research in cardiovascular biology.

Optimization of ultrasound-assisted extraction of naturally occurring glucosinolates from by-products of Camelina sativa L. and their effect on human colorectal cancer cell line.

The food waste generated by small and medium agro-industrial enterprises requires appropriate management and valorization in order to decrease environmental problems and recover high-value products, respectively. In this study, the Camelina sativa seed by-product was used as a source of glucosinolates. To begin, the chemical profile of the extract obtained using an international organization for standardization (ISO) procedure was determined by UPLC-HRMS/MS analysis. In addition, an extraction method based on ultrasound-assisted extraction was developed as an alternative and green method to recover glucosinolates. Main parameters that affect extraction efficiency were optimized using a response surface design. Under optimized conditions, the extract showed an improvement in extraction yield with a reduction in organic solvent amount compared to those obtained using the ISO procedure. Finally, the extract obtained with the ultrasound-assisted method was purified, tested on human colorectal cancer cell lines, and showed promising results.

Characterization of the Biological Activities of a New Polyphenol-Rich Extract from Cinnamon Bark on a Probiotic Consortium and Its Action after Enzymatic and Microbial Fermentation on Colorectal Cell Lines.

Cinnamon polyphenols are known as health-promoting agents. However, their positive impact depends on the extraction method and their bioaccessibility after digestion. In this work, cinnamon bark polyphenols were extracted in hot water and subjected to an in vitro enzymatic digestion. After a preliminary characterization of total polyphenols and flavonoids (respectively 520.05 ± 17.43 µgGAeq/mg and 294.77 ± 19.83 µgCATeq/mg powder extract), the extract antimicrobial activity was evidenced only against Staphylococcus aureus and Bacillus subtilis displaying a minimum inhibition growth concentration value of 2 and 1.3 mg/mL, respectively, although it was lost after in vitro extract digestion. The prebiotic potential was evaluated on probiotic Lactobacillus and Bifidobacterium strains highlighting a high growth on the in vitro digested cinnamon bark extract (up to 4 × 108 CFU/mL). Thus, the produced SCFAs and other secondary metabolites were extracted from the broth cultures and determined via GC-MSD analyses. The viability of healthy and tumor colorectal cell lines (CCD841 and SW480) was assayed after the exposition at two different concentrations (23 and 46 µgGAeq/mL) of the cinnamon extract, its digested, and the secondary metabolites produced in presence of cinnamon extract or its digested, showing positive protective effects against a tumorigenic condition.

Evidence for discrete modes of YAP1 signaling via mRNA splice isoforms in development and diseases.

Yes-associated protein 1 (YAP1) is a transcriptional co-activator downstream of Hippo pathway. The pathway exerts crucial roles in organogenesis and its dysregulation is associated with the spreading of different cancer types. YAP1 gene encodes for multiple protein isoforms, whose specific functions are not well defined. We demonstrate the splicing of isoform-specific mRNAs is controlled in a stage- and tissue-specific fashion. We designed expression vectors encoding for the most-represented isoforms of YAP1 with either one or two WW domains and studied their specific signaling activities in YAP1 knock-out cell lines. YAP1 isoforms display both common and unique functions and activate distinct transcriptional programs, as the result of their unique protein interactomes. By generating TEAD-based transcriptional reporter cell lines, we demonstrate individual YAP1 isoforms display unique effects on cell proliferation and differentiation. Finally, we illustrate the complexity of the regulation of Hippo-YAP1 effector in physiological and in pathological conditions of the heart.

Enzymatic Hydrolysate of Cinnamon Waste Material as Feedstock for the Microbial Production of Carotenoids.

In the context of the global need to move towards circular economies, microbial cell factories can be employed thanks to their ability to use side-stream biomasses from the agro-industrial sector to obtain additional products. The valorization of residues allows for better and complete use of natural resources and, at the same time, for the avoidance of waste management to address our needs. In this work, we focused our attention on the microbial valorization of cinnamon waste material after polyphenol extraction (C-PEW) (Cinnamomum verum J.Presl), generally discarded without any additional processing. The sugars embedded in C-PEW were released by enzymatic hydrolysis, more compatible than acid hydrolysis with the subsequent microbial cultivation. We demonstrated that the yeast Rhodosporidium toruloides was able to grow and produce up to 2.00 (±0.23) mg/L of carotenoids in the resulting hydrolysate as a sole carbon and nitrogen source despite the presence of antimicrobial compounds typical of cinnamon. To further extend the potential of our finding, we tested other fungal cell factories for growth on the same media. Overall, these results are opening the possibility to develop separate hydrolysis and fermentation (SHF) bioprocesses based on C-PEW and microbial biotransformation to obtain high-value molecules.

Tumor in 3D: In Vitro Complex Cellular Models to Improve Nanodrugs Cancer Therapy.

Nanodrugs represent novel solutions to reshuffle repurposed drugs for cancer therapy. They might offer different therapeutic options by combining targeted drug delivery and imaging in unique platforms. Such nanomaterials are deemed to overcome the limitations of currently available treatments, ultimately improving patients' life quality. However, despite these promises being made for over three decades, the poor clinical translation of nanoparticle- based therapies calls for deeper in vit.. and in vivo investigations. Translational issues arise very early during the development of nanodrugs, where complex and more reliable cell models are often replaced by easily accessible and convenient 2D monocultures. This is particularly true in the field of cancer therapy. In fact, 2D monocultures provide poor information about the real impact of the nanodrugs in a complex living organism, especially given the poor mimicry of the solid Tumors Microenvironment (TME). The dense and complex extracellular matrix (ECM) of solid tumors dramatically restricts nanoparticles efficacy, impairing the successful implementation of nanodrugs in medical applications. Herein, we propose a comprehensive guideline of the 3D cell culture models currently available, including their potential and limitations for the evaluation of nanodrugs activity. Advanced culture techniques, more closely resembling the physiological conditions of the TME, might give a better prediction of the reciprocal interactions between cells and nanoparticles and eventually help reconsider the use of old drugs for new applications.

YAP regulates cell mechanics by controlling focal adhesion assembly.

Hippo effectors YAP/TAZ act as on-off mechanosensing switches by sensing modifications in extracellular matrix (ECM) composition and mechanics. The regulation of their activity has been described by a hierarchical model in which elements of Hippo pathway are under the control of focal adhesions (FAs). Here we unveil the molecular mechanism by which cell spreading and RhoA GTPase activity control FA formation through YAP to stabilize the anchorage of the actin cytoskeleton to the cell membrane. This mechanism requires YAP co-transcriptional function and involves the activation of genes encoding for integrins and FA docking proteins. Tuning YAP transcriptional activity leads to the modification of cell mechanics, force development and adhesion strength, and determines cell shape, migration and differentiation. These results provide new insights into the mechanism of YAP mechanosensing activity and qualify this Hippo effector as the key determinant of cell mechanics in response to ECM cues.

Stable phenotype and function of immortalized Lin-Sca-1+ cardiac progenitor cells in long-term culture: a step closer to standardization.

Putative cardiac progenitor cells (CPCs) have been identified in the myocardium and are regarded as promising candidates for cardiac cell-based therapies. Although two distinct populations of CPCs reached the clinical setting, more detailed studies are required to portray the optimal cell type and therapeutic setting to drive robust cell engraftment and cardiomyogenesis after injury. Owing to the scarcity of the CPCs and the need for reproducibility, the generation of faithful cellular models would facilitate this scrutiny. Here, we evaluate whether immortalized Lin(-)Sca-1(+) CPCs (iCPC(Sca-1)) represent their native-cell counterpart, thereby constituting a robust in vitro model system for standardized investigation in the cardiac field. iCPC(Sca-1) were established in vitro as plastic adherent cells endowed with robust self-renewal capacity while preserving a stable phenotype in long-term culture. iCPC(Sca-1) differentiated into cardiomyocytic-, endothelial-, and smooth muscle-like cells when subjected to appropriate stimuli. The cell line consistently displayed features of Lin(-)Sca-1(+) CPCs in vitro, as well as in vivo after intramyocardial delivery in the onset of myocardial infarction (MI). Transplanted iCPC(Sca-1) significantly attenuated the functional and anatomical alterations caused by MI while promoting neovascularization. iCPC(Sca-1) are further shown to engraft, establish functional connections, and differentiate in loco into cardiomyocyte- and vasculature-like cells. These data validate iCPC(Sca-1) as an in vitro model system for Lin(-)Sca-1(+) progenitors and for systematic dissection of mechanisms underlying CPC subsets engraftment/differentiation in vivo. Moreover, iCPC(Sca-1) can be regarded as a ready-to-use CPCs source for pre-clinical bioengineering studies toward the development of novel strategies for restoration of the damaged myocardium.

Towards the generation of patient-specific patches for cardiac repair.

Cardiovascular diseases represent the main cause of morbidity and mortality worldwide. Millions of people are affected by such diseases in the industrialized countries, with hundreds of thousands new cases diagnosed every year. Among cardiac diseases, heart failure is the most common end-stage pathology, leading to impaired cardiac output and cardiac performance as a result of the irreversible loss of contractile cardiomyocytes. Tissue engineering holds the promise to provide personalized solutions to the problem of cardiac muscle repair. Indeed, the identification of little reservoirs of stem and progenitor cells within every body district opened new perspectives to the setup of patient-specific protocols for cardiac diseases. Nonetheless, the results of the first pre-clinical and clinical trials in which adult stem/progenitor cells were adopted pointed at the route of delivery to the injured organ as well as at the cell source as the main issues for cardiac tissue engineers. In fact, when adult stem cells were directly injected into the myocardium or delivered through bloodstream to the heart, no or few cells could be found engrafted within host tissue few days after the administration. Renewed enthusiasm was generated by the techniques set up to enrich cardiomyocytes obtained by embryonic stem cells and by the recent disclosure of the protocols to obtain reprogrammed pluripotent cells or reprogrammed cardiomyocytes out of patients' own somatic cells. In this context, additional efforts to setup efficient systems to deliver stem cells to the injured site are required. The application of forefront technologies to fabricate synthetic and hybrid scaffolds to be employed as cell delivery systems and the acknowledgement that surface physical, mechanical, chemical properties can exert specific effects on stem cells per se prompted new enthusiasm in the field. In this respect, a cardiac-specific scaffold should be able to comply with cardiac muscle architecture, be deformable as to indulge and possibly sustain cardiac contraction. As expected, such a scaffold should favor stem cell electromechanical coupling with host tissue, while promoting the vascularization of the newly-formed tissue.

Self-renewal and multipotency coexist in a long-term cultured adult rat dental pulp stem cell line: an exception to the rule?

The stemness state is characterized by self-renewal and differentiation properties. However, stem cells are not able to preserve these characteristics in long-term culture because of the intrinsic fragility of their phenotype easily undergoing senescence or neoplastic transformation. Furthermore, although isolated from the same original tissue using similar protocols, adult stem cells can display dissimilar phenotypes and important cell clone/species contamination. Finally, the lack of a clear standardization contributes to complicate the comprehension about the stemness condition. In this context, cell lines displaying a particularly stable phenotype must be identified to define one or multiple benchmarks against which other stem cell lines could be reliably assessed. The present paper demonstrates that it is possible to isolate from the rat dental pulp a stem cell line (MUR-1) that does not display neoplastic transformation in long-term culture. MUR-1 cells stably express a broad range of stemness markers and are able to differentiate into adipogenic, osteogenic, chondrogenic, neurogenic, and cardiomyogenic lineages independently of the culture passages. Moreover, serial in vitro passages have not changed their immunophenotype, proliferation capacity, or differentiation potential. The uniqueness of these characteristics candidates MUR-1 as a model to reliably improve the understanding of the mechanisms governing the stem cell fate in the same as well as in other stem cell populations.

Human cardiac progenitor cell grafts as unrestricted source of supernumerary cardiac cells in healthy murine hearts.

Human heart harbors a population of resident progenitor cells that can be isolated by stem cell antigen-1 antibody and expanded in culture. These cells can differentiate into cardiomyocytes in vitro and contribute to cardiac regeneration in vivo. However, when directly injected as single cell suspension, less than 1%-5% survive and differentiate. Among the major causes of this failure are the distressing protocols used to culture in vitro and implant progenitor cells into damaged hearts. Human cardiac progenitors obtained from the auricles of patients were cultured as scaffoldless engineered tissues fabricated using temperature-responsive surfaces. In the engineered tissue, progenitor cells established proper three-dimensional intercellular relationships and were embedded in self-produced extracellular matrix preserving their phenotype and multipotency in the absence of significant apoptosis. After engineered tissues were leant on visceral pericardium, a number of cells migrated into the murine myocardium and in the vascular walls, where they integrated in the respective textures. The study demonstrates the suitability of such an approach to deliver stem cells to the myocardium. Interestingly, the successful delivery of cells in murine healthy hearts suggests that myocardium displays a continued cell cupidity that is strictly regulated by the limited release of progenitor cells by the adopted source. When an unregulated cell source is added to the system, cells are delivered to the myocardium. The exploitation of this novel concept may pave the way to the setup of new protocols in cardiac cell therapy.

Multiscale three-dimensional scaffolds for soft tissue engineering via multimodal electrospinning.

A novel (scalable) electrospinning process was developed to fabricate bio-inspired multiscale three-dimensional scaffolds endowed with a controlled multimodal distribution of fiber diameters and geared towards soft tissue engineering. The resulting materials finely mingle nano- and microscale fibers together, rather than simply juxtaposing them, as is commonly found in the literature. A detailed proof of concept study was conducted on a simpler bimodal poly(epsilon-caprolactone) (PCL) scaffold with modes of fiber distribution at 600 nm and 3.3 microm. Three conventional unimodal scaffolds with mean diameters of 300 nm and 2.6 and 5.2 microm, respectively, were used as controls to evaluate the new materials. Characterization of the microstructure (i.e. porosity, fiber distribution and pore structure) and mechanical properties (i.e. stiffness, strength and failure mode) indicated that the multimodal scaffold had superior mechanical properties (Young's modulus approximately 40MPa and strength approximately 1MPa) in comparison with the controls, despite the large porosity ( approximately 90% on average). A biological assessment was conducted with bone marrow stromal cell type (mesenchymal stem cells, mTERT-MSCs). While the new material compared favorably with the controls with respect to cell viability (on the outer surface), it outperformed them in terms of cell colonization within the scaffold. The latter result, which could neither be practically achieved in the controls nor expected based on current models of pore size distribution, demonstrated the greater openness of the pore structure of the bimodal material, which remarkably did not come at the expense of its mechanical properties. Furthermore, nanofibers were seen to form a nanoweb bridging across neighboring microfibers, which boosted cell motility and survival. Lastly, standard adipogenic and osteogenic differentiation tests served to demonstrate that the new scaffold did not hinder the multilineage potential of stem cells.

Thick soft tissue reconstruction on highly perfusive biodegradable scaffolds.

The lack of a vascular network and poor perfusion is what mostly prevents three-dimensional (3D) scaffolds from being used in organ repair when reconstruction of thick tissues is needed. Highly-porous scaffolds made of poly(L-lactic acid) (PLLA) are prepared by directional thermally induced phase separation (dTIPS) starting from 1,4-dioxane/PLLA solutions. The influence of polymer concentration and temperature gradient, in terms of imposed intensity and direction, on pore size and distribution is studied by comparison with scaffolds prepared by isotropic TIPS. The processing parameters are optimized to achieve an overall porosity for the 3D scaffolds of about 93% with a degree of interconnectivity of 91%. The resulting pore network is characterized by the ordered repetition of closely packed dendrite-like cavities, each one showing stacks of 20 microm large side lamellar branches departing from 70 microm diameter vertical backbones, strongly resembling the vascular patterns. The in vitro biological responses after 1 and 2 weeks are evaluated from mesenchymal (bone marrow stromal) cells (MSC) static culturing. A novel vacuum-based deep-seeding method is set up to improve uniform cell penetration down to scaffold thicknesses of over 1 mm. Biological screenings show significant 3D scaffold colonization even after 18 h, while cellular retention is observed up to 14 d in vitro (DIV). Pore architecture-driven cellular growth is accompanied by cell tendency to preserve their multi-potency towards differentiation. Confluent tissues as thick as 1 mm were reconstructed taking advantage of the large perfusion enhanced by the highly porous microstructure of the engineered scaffolds, which could successfully serve for applications aimed at vascular nets and angiogenesis.

Criticality of the biological and physical stimuli array inducing resident cardiac stem cell determination.

The replacement of injured cardiac contractile cells with stem cell-derived functionally efficient cardiomyocytes has been envisaged as the resolutive treatment for degenerative heart diseases. Nevertheless, many technical issues concerning the optimal procedures to differentiate and engraft stem cells remain to be answered before heart cell therapy could be routinely used in clinical practice. So far, most studies have been focused on evaluating the differentiative potential of different growth factors without considering that only the synergistic cooperation of biochemical, topographic, chemical, and physical factors could induce stem cells to adopt the desired phenotype. The present study demonstrates that the differentiation of cardiac progenitor cells to cardiomyocytes does not occur when cells are challenged with soluble growth factors alone, but requires strictly controlled procedures for the isolation of a progenitor cell population and the artifactual recreation of a microenvironment critically featured by a fine-tuned combination of specific biological and physical factors. Indeed, the scaffold geometry and stiffness are crucial in enhancing growth factor differentiative effects on progenitor cells. The exploitation of this concept could be essential in setting up suitable procedures to fabricate functionally efficient engineered tissues. Disclosure of potential conflicts of interest is found at the end of this article.