Building new cardiac vasculature and myocardium: where are we at?

PURPOSE OF REVIEW: This review describes the latest advances in cell therapy, biomaterials and 3D bioprinting for the treatment of cardiovascular disease. RECENT FINDINGS: Cell therapies offer the greatest benefit for patients suffering from chronic ischemic and nonischemic cardiomyopathy. Rather than replacing lost cardiomyocytes, the effects of most cell therapies are mediated by paracrine signalling, mainly through the induction of angiogenesis and immunomodulation. Cell preconditioning, or genetic modifications are being studied to improve the outcomes. Biomaterials offer stand-alone benefits such as bioactive cues for cell survival, proliferation and differentiation, induction of vascularization or prevention of further cardiomyocyte death. They also provide mechanical support or electroconductivity, and can be used to deliver cells, growth factors or drugs to the injured site. Apart from classical biomaterial manufacturing techniques, 3D bioprinting offers greater spatial control over biomaterial deposition and higher resolution of the details, including hollow vessel-like structures. SUMMARY: Cell therapy induces mainly angiogenesis and immunomodulation. The ability to induce direct cardiomyocyte regeneration to replace the lost cardiomyocytes is, however, still missing until embryonic or induced pluripotent stem cell use becomes available. Cell therapy would benefit from combinatorial use with biomaterials, as these can prolong cell retention and survival, offer additional mechanical support and provide inherent bioactive cues. Biomaterials can also be used to deliver growth factors, drugs, and other molecules. 3D bioprinting is a high-resolution technique that has great potential in cardiac therapy.

Increased c-di-GMP Levels Lead to the Production of Alginates of High Molecular Mass in Azotobacter vinelandii.

Azotobacter vinelandii produces the linear exopolysaccharide alginate, a compound of significant biotechnological importance. The biosynthesis of alginate in A. vinelandii and Pseudomonas aeruginosa has several similarities but is regulated somewhat differently in the two microbes. Here, we show that the second messenger cyclic dimeric GMP (c-di-GMP) regulates the production and the molecular mass of alginate in A. vinelandii The hybrid protein MucG, containing conserved GGDEF and EAL domains and N-terminal HAMP and PAS domains, behaved as a c-di-GMP phosphodiesterase (PDE). This activity was found to negatively affect the amount and molecular mass of the polysaccharide formed. On the other hand, among the diguanylate cyclases (DGCs) present in A. vinelandii, AvGReg, a globin-coupled sensor (GCS) DGC that directly binds to oxygen, was identified as the main c-di-GMP-synthesizing contributor to alginate production. Overproduction of AvGReg in the parental strain phenocopied a ΔmucG strain with regard to alginate production and the molecular mass of the polymer. MucG was previously shown to prevent the synthesis of high-molecular-mass alginates in response to reduced oxygen transfer rates (OTRs). In this work, we show that cultures exposed to reduced OTRs accumulated higher levels of c-di-GMP; this finding strongly suggests that at least one of the molecular mechanisms involved in modulation of alginate production and molecular mass by oxygen depends on a c-di-GMP signaling module that includes the PAS domain-containing PDE MucG and the GCS DGC AvGReg.IMPORTANCE c-di-GMP has been widely recognized for its essential role in the production of exopolysaccharides in bacteria, such as alginate produced by Pseudomonas and Azotobacter spp. This study reveals that the levels of c-di-GMP also affect the physical properties of alginate, favoring the production of high-molecular-mass alginates in response to lower OTRs. This finding opens up new alternatives for the design of tailor-made alginates for biotechnological applications.

Cyclic di-GMP-Mediated Regulation of Extracellular Mannuronan C-5 Epimerases Is Essential for Cyst Formation in Azotobacter vinelandii.

The genus Azotobacter, belonging to the Pseudomonadaceae family, is characterized by the formation of cysts, which are metabolically dormant cells produced under adverse conditions and able to resist desiccation. Although this developmental process has served as a model for the study of cell differentiation in Gram-negative bacteria, the molecular basis of its regulation is still poorly understood. Here, we report that the ubiquitous second messenger cyclic dimeric GMP (c-di-GMP) is critical for the formation of cysts in Azotobacter vinelandii Upon encystment induction, the levels of c-di-GMP increased, reaching a peak within the first 6 h. In the absence of the diguanylate cyclase MucR, however, the levels of this second messenger remained low throughout the developmental process. A. vinelandii cysts are surrounded by two alginate layers with variable proportions of guluronic residues, which are introduced into the final alginate chain by extracellular mannuronic C-5 epimerases of the AlgE1 to AlgE7 family. Unlike in Pseudomonas aeruginosa, MucR was not required for alginate polymerization in A. vinelandii Conversely, MucR was necessary for the expression of extracellular alginate C-5 epimerases; therefore, the MucR-deficient strain produced cyst-like structures devoid of the alginate capsule and unable to resist desiccation. Expression of mucR was partially dependent on the response regulator AlgR, which binds to two sites in the mucR promoter, enhancing mucR transcription. Together, these results indicate that the developmental process of A. vinelandii is controlled through a signaling module that involves activation by the response regulator AlgR and c-di-GMP accumulation that depends on MucR.IMPORTANCEA. vinelandii has served as an experimental model for the study of the differentiation processes to form metabolically dormant cells in Gram-negative bacteria. This work identifies c-di-GMP as a critical regulator for the production of alginates with specific contents of guluronic residues that are able to structure the rigid laminated layers of the cyst envelope. Although allosteric activation of the alginate polymerase complex Alg8-Alg44 by c-di-GMP has long been recognized, our results show a previously unidentified role during the polymer modification step, controlling the expression of extracellular alginate epimerases. Our results also highlight the importance of c-di-GMP in the control of the physical properties of alginate, which ultimately determine the desiccation resistance of the differentiated cell.

Two-component system CbrA/CbrB controls alginate production in Azotobacter vinelandii.

Azotobacter vinelandii, belonging to the Pseudomonadaceae family, is a free-living bacterium that has been considered to be a good source for the production of bacterial polymers such as alginate. In A. vinelandii the synthesis of this polymer is regulated by the Gac/Rsm post-transcriptional regulatory system, in which the RsmA protein binds to the mRNA of the biosynthetic algD gene, inhibiting translation. In several Pseudomonas spp. the two-component system CbrA/CbrB has been described to control a variety of metabolic and behavioural traits needed for adaptation to changing environmental conditions. In this work, we show that the A. vinelandii CbrA/CbrB two-component system negatively affects alginate synthesis, a function that has not been described in Pseudomonas aeruginosa or any other Pseudomonas species. CbrA/CbrB was found to control the expression of some alginate biosynthetic genes, mainly algD translation. In agreement with this result, the CbrA/CbrB system was necessary for optimal rsmA expression levels. CbrA/CbrB was also required for maximum accumulation of the sigma factor RpoS. This last effect could explain the positive effect of CbrA/CbrB on rsmA expression, as we also showed that one of the promoters driving rsmA transcription was RpoS-dependent. However, although inactivation of rpoS increased alginate production by almost 100 %, a cbrA mutation increased the synthesis of this polymer by up to 500 %, implying the existence of additional CbrA/CbrB regulatory pathways for the control of alginate production. The control exerted by CbrA/CbrB on the expression of the RsmA protein indicates the central role of this system in regulating carbon metabolism in A. vinelandii.

Association models for binding of molecules to nanostructures.

The interaction between nanoparticles and molecules plays a key role in determining the activity and performance of a given nanostructure. These interactions are pivotal for a variety of applications including drug delivery, surface manipulation for targeted therapies, and catalysis. However, to this day, gathering precise association parameters for the interaction of the molecules with nanostructures remains elusive and mostly imprecise. In this review, we present a critical discussion of the most commonly used techniques and models intended for determining the association of molecules with nanoparticles. Particular emphasis has been put on discussing the limitations and pitfalls related to determining association constants in this tutorial review.

The signaling protein MucG negatively affects the production and the molecular mass of alginate in Azotobacter vinelandii.

Azotobacter vinelandii is a soil bacterium that produces the polysaccharide alginate. In this work, we identified a miniTn5 mutant, named GG9, which showed increased alginate production of higher molecular mass, and increased expression of the alginate biosynthetic genes algD and alg8 when compared to its parental strain. The miniTn5 was inserted within ORF Avin07920 encoding a hypothetical protein. Avin07910, located immediately downstream and predicted to form an operon with Avin07920, encodes an inner membrane multi-domain signaling protein here named mucG. Insertional inactivation of mucG resulted in a phenotype of increased alginate production of higher molecular mass similar to that of mutant GG9. The MucG protein contains a periplasmic and putative HAMP and PAS domains, which are linked to GGDEF and EAL domains. The last two domains are potentially involved in the synthesis and degradation, respectively, of bis-(3'-5')-cyclic dimeric GMP (c-di-GMP), a secondary messenger that has been reported to be essential for alginate production. Therefore, we hypothesized that the negative effect of MucG on the production of this polymer could be explained by the putative phosphodiesterase activity of the EAL domain. Indeed, we found that alanine replacement mutagenesis of the MucG EAL motif or deletion of the entire EAL domain resulted in increased alginate production of higher molecular mass similar to the GG9 and mucG mutants. To our knowledge, this is the first reported protein that simultaneous affects the production of alginate and its molecular mass.

Spherical silver nanoparticles in the detection of thermally denatured collagens.

We have developed a rapid colorimetric method to determine the concentration of denatured collagen in solution, which is based on the collagen-silver nanoparticle corona formation. Using the proposed method, the lowest detectable concentration of denatured collagen protein in a solution of pure collagen was 14.7, 8.5, and 8.6 µg mL(-1) for porcine (PCOL), rat tail (RCOL), and type I human recombinant (HCOL) collagen, respectively.

Use of photosensitive molecules in the crosslinking of biopolymers: applications and considerations in biomaterials development.

The development of diverse types of biomaterials has significantly contributed to bringing new biomedical strategies to treat clinical conditions. Applications of these biomaterials can range from mechanical support and protection of injured tissues to joint replacement, tissue implants, and drug delivery systems. Among the strategies commonly used to prepare biomaterials, the use of electromagnetic radiation to initiate crosslinking stands out. The predominance of photo-induced polymerization methods relies on a fast, efficient, and straightforward process that can be easily adjusted to clinical needs. This strategy consists of irradiating the components that form the material with photons in the near ultraviolet-visible wavelength range (i.e., ∼310 to 750 nm) in the presence of a photoactive molecule. Upon photon absorption, photosensitive molecules can generate excited species that initiate photopolymerization through different reaction mechanisms. However, this process could promote undesired side reactions depending on the target zone or treatment type (e.g., oxidative stress and modification of biomolecules such as proteins and lipids). This review explores the basic concepts behind the photopolymerization process of ex situ and in situ biomaterials. Particular emphasis was put on the photosensitization initiated by the most employed photosensitizers and the photoreactions that they mediate in aqueous media. Finally, the undesired oxidation reactions at the bio-interface and potential solutions are presented.

Mimicking the Physicochemical Properties of the Cornea: A Low-Cost Approximation Using Highly Available Biopolymers.

Corneal diseases represent a significant global health challenge, often resulting in blindness, for which penetrating keratoplasty is the clinical gold standard. However, in cases involving compromised ocular surfaces or graft failure, osteo-odonto keratoprosthesis (OOKP) emerges as a vital yet costly and complex alternative. Thus, there is an urgent need to introduce soft biomaterials that mimic the corneal tissue, considering its translation's physicochemical, biological, and economic costs. This study introduces a cross-linked mixture of economically viable biomaterials, including gelatin, chitosan, and poly-D-lysine, that mimic corneal properties. The physicochemical evaluation of certain mixtures, specifically gelatin, chitosan, and poly-D-lysine cross-linked with 0.10% glutaraldehyde, demonstrates that properties such as swelling, optical transmittance, and thermal degradation are comparable to those of native corneas. Additionally, constructs fabricated with poly-D-lysine exhibit good cytocompatibility with fibroblasts at 72 h. These findings suggest that low-cost biopolymers, particularly those incorporating poly-D-lysine, mimic specific corneal characteristics and have the potential to foster fibroblast survival. While further studies are required to reach a final corneal-mimicking solution, this study contributes to positioning low-cost reagents as possible alternatives to develop biomaterials with physicochemical properties like those of the human cornea.

Oxidative stress promotes cytotoxicity in human cancer cell lines exposed to Escallonia spp. extracts.

BACKGROUND: Standard cancer treatments show a lack of selectivity that has led to the search for new strategies against cancer. The selective elimination of cancer cells modulating the redox environment, known as "selective oxycution", has emerged as a viable alternative. This research focuses on characterizing the unexplored Escallonia genus plant extracts and evaluating their potential effects on cancer's redox balance, cytotoxicity, and activation of death pathways. METHODS: 36 plant extracts were obtained from 4 different species of the Escallonia genus (E. illinita C. Presl, E. rubra (Ruiz & Pav.) Pers., E. revoluta (Ruiz & Pav.) Pers., and E. pulverulenta (Ruiz & Pav.) Pers.), which were posteriorly analyzed by their phytoconstituents, antioxidant capacity, and GC-MS. Further, redox balance assays (antioxidant enzymes, oxidative damage, and transcription factors) and cytotoxic effects (SRB, ∆Ψmt, and caspases actives) of those plant extracts were analyzed on four cell lines (HEK-293T, MCF-7, HT-29, and PC-3). RESULTS: 36 plant extracts were obtained, and their phytoconstituents and antioxidant capacity were established. Further, only six extracts had EC50 values < 10 µg*mL- 1, indicating high toxicity against the tested cells. From those, two plant extracts were selective against different cancer cell lines: the hexane extract of E. pulverulenta´s stem was selective for HT-29, and the ethyl acetate extract of E. rubra´s stem was selective for PC-3. Both extracts showed unbalanced redox effects and promoted selective cell death. CONCLUSIONS: This is the first study proving "selective oxycution" induced by Chilean native plant extracts.

Stability of ACE2 Peptide Mimetics and Their Implications on the Application for SARS-CoV2 Detection.

The SARS-CoV-2 worldwide outbreak prompted the development of several tools to detect and treat the disease. Among the new detection proposals, the use of peptides mimetics has surged as an alternative to avoid the use of antibodies, of which there has been a shortage during the COVID-19 pandemic. However, the use of peptides in detection systems still presents some questions to be answered, mainly referring to their stability under different environmental conditions. In this work, we synthesized an ACE2 peptide mimic and evaluated its stability in different pH, salinity, polarity, and temperature conditions. Further, the same conditions were assessed when using the ability of the peptide mimic to detect the recombinant SARS-CoV-2 spike protein in a biotin-streptavidin-enzyme-linked assay. Finally, we also tested the capacity of the peptide to detect SARS-CoV-2 from patients' samples. The results indicate that the peptide is structurally sensitive to the medium conditions, with relevance to the pH, where basic pH favored its performance when used as a SARS-CoV-2 detector. Further, the proposed peptide mimic was able to detect SARS-CoV-2 comparably to RT-qPCR results. Therefore, the present study promotes knowledge advancement, particularly in terms of stability considerations, in the application of peptide mimics as a replacement for antibodies in detection systems.

Poly(ethylene imine)-chitosan carbon dots: study of its physical-chemical properties and biological in vitro performance.

Carbon dots (CDs) have been quickly extended for nanomedicine uses because of their multiple applications, such as bioimaging, sensors, and drug delivery. However, the interest in increasing their photoluminescence properties is not always accompanied by cytocompatibility. Thus, a knowledge gap exists regarding their interactions with biological systems linked to the selected formulations and synthesis methods. In this work, we have developed carbon dots (CDs) based on poly (ethylene imine) (PEI) and chitosan (CS) by using microwave irradiation, hydrothermal synthesis, and a combination of both, and further characterized them by physicochemical and biological means. Our results indicate that synthesized CDs have sizes between 1 and 5 nm, a high presence of amine groups on the surface, and increased positive ζ potential values. Further, it is established that the choice and use of different synthesis procedures can contribute to a different answer to the CDs regarding their optical and biological properties. In this regard, PEI-only CDs showed the longest photoluminescent emission lifetime, non-hemolytic activity, and high toxicity against fibroblast. On the other hand, CS-only CDs have higher PL emission, non-cytotoxicity associated with fibroblast, and high hemolytic activity. Interestingly, their combination using the proposed methodologies allow a synergic effect in their CDs properties. Therefore, this work contributes to developing and characterizing CD formulations based on PEI and CS and better understanding the CD's properties and biological interaction.

Cytocompatible drug delivery hydrogels based on carboxymethylagarose/chitosan pH-responsive polyelectrolyte complexes.

Several drugs are chemically unstable in the gastric environment and have low bioavailability restricted by intestinal absorption, which motivates the development of alternative routes for drug release, such as transdermal drug carriers for drug delivery to specific areas of the skin. Herein, novel polyelectrolyte complexes (PEC) consisting of carboxymethylagarose (CMA) and chitosan (CS) were prepared. pH-responsive CMA/CS hydrogels were obtained by mixing CMA and CS at various weight ratios. Swelling ratio was modulated by varying the CMA and CS weight ratio, and the highest swelling values were achieved for 2:1 wt% hydrogels at 25 °C and pH 6.0. PEC films were characterized by ATR-FTIR spectroscopy, TGA, DSC, and SEM. Results indicated that CMA and CS were successfully crosslinked by ionic complexation. As a model drug, diclofenac sodium (DS) was loaded in CMA/CS PECs. Association efficiency and loading capacity were ca. 69% and 79%, respectively, exhibiting 67% cumulative release after 72 h at 37 °C and pH 6.0 through Fickian diffusion mechanism. Viability assay of immortalized human keratinocyte (HaCat) cells showed ca. 100% survival in the presence of hydrogels and DS. Therefore, this work suggests that CMA/CS PECs can be applied as pH-responsive carriers for dermal drug delivery.

Ag@Au bimetallic nanoparticles: an easy and highly reproducible synthetic approach for photocatalysis.

An Ag@Au bimetallic nanoparticle (BNP) formulation was developed in this work. The proposed formulation was developed using photochemical and chemical methods and non-toxic reagents, showing high reproducibility and homogeneity. The synthesized BNPs have an average size of 7 nm, a core-shell-like structure (silver core and gold shell), high colloidal and long-term stability, and superior catalytic activity under darkness and white light irradiation conditions when evaluating the reduction of 4-nitrophenol to 4-aminophenolate, with respect to the monometallic Ag and Au counterparts. Furthermore, BNP concentrations as low as 2 nM were required to reach 100% conversions in less than 30 minutes. Therefore, considering future applications, the high surface-to-volume ratio of the prepared BNPs coupled with their well-defined optical properties makes them a great candidate for developing heterogeneous catalyzer materials to be applicable under sunlight as an environmentally friendly catalytic system.

Genetic Regulation of Alginate Production in Azotobacter vinelandii a Bacterium of Biotechnological Interest: A Mini-Review.

Alginates are a family of polymers composed of guluronate and mannuronate monomers joined by ß (1-4) links. The different types of alginates have variations in their monomer content and molecular weight, which determine the rheological properties and their applications. In industry, alginates are commonly used as additives capable of viscosifying, stabilizing, emulsifying, and gelling aqueous solutions. Recently, additional specialized biomedical uses have been reported for this polymer. Currently, the production of alginates is based on the harvesting of seaweeds; however, the composition and structure of the extracts are highly variable. The production of alginates for specialized applications requires a precise composition of monomers and molecular weight, which could be achieved using bacterial production systems such as those based on Azotobacter vinelandii, a free-living, non-pathogenic bacterium. In this mini-review, we analyze the latest advances in the regulation of alginate synthesis in this model.

Corrigendum: Genetic Regulation of Alginate Production in Azotobacter vinelandii a Bacterium of Biotechnological Interest: A Mini-Review.

[This corrects the article DOI: 10.3389/fmicb.2022.845473.].

Multifunctional Nano and Collagen-Based Therapeutic Materials for Skin Repair.

A novel strategy is needed for treating nonhealing wounds, which is able to simultaneously eradicate pathogenic bacteria and promote tissue regeneration. This would improve patient outcome and reduce the number of lower limb amputations. In this work, we present a multifunctional therapeutic approach able to control bacterial infections, provide a protective barrier to a full-thickness wound, and improve wound healing in a clinically relevant animal model. Our approach uses a nanoengineered antimicrobial nanoparticle for creating a sprayable layer onto the wound bed that prevents bacterial proliferation and also eradicates preformed biofilms. As a protective barrier for the wound, we developed a thermoresponsive collagen-based matrix that has prohealing properties and is able to fill wounds independent of their geometries. Our results indicate that using a combination of the matrix with full-thickness microscopic skin tissue columns synergistically contributed to faster and superior skin regeneration in a nonhealing wound model in diabetic mice.

Molecular weight and viscosifying power of alginates produced by mutant strains of Azotobacter vinelandii under microaerophilic conditions.

Alginates are polysaccharides that are of interest in various industrial applications. This is due to the viscosifying properties of alginates, which depends on the weight-average molecular weight. The aim of the present study was to evaluate the changes in alginate quality, in terms of the viscosifying power and weight-average molecular weight of the polymer produced by Azotobacter vinelandii mutant strains in shake flasks under microaerophilic conditions. In cultures developed at oxygen transfer rate (OTR) values close to 5 mmol L-1 h-1, the highest viscosifying power (1.75 L g-1) and weight-average molecular weight (3112 ±â€¯150 kDa) were achieved in cultures performed with the AT9 strain. These values were higher than those obtained for the alginates produced by the parental strain ATCC 9046 grown under similar OTR conditions. In contrast, the alginate produced by the GG9 and OPAlgU + exhibited a very low weight-average molecular weight and therefore a poor viscosifying power. Our results have shown that by the cultivation of AT9 strain under microaerophilic conditions it is possible to obtain a polymer having a high weight-average molecular weight and excellent viscosifying capacity. Therefore, it could be a viable strategy for producing alginates for industrial applications.

Electroconductive materials as biomimetic platforms for tissue regeneration.

In many diseases, tissue regeneration is compromised and/or insufficient to restore tissue/organ function. Therefore, novel regenerative therapies are being developed to enhance resident and transplanted cell proliferation and functional differentiation. Numerous biomaterials engineered to include nanocomponents have emerged as promising candidates to fulfil the need of mimicking the properties of the healthy extracellular matrix. This is particularly important for tissues that require electroconductive support to achieve optimal cellular function, such as muscles and neurons. In this review, we summarize and discuss the current state-of-the-art for electroconductive materials in tissue regeneration, with particular emphasis on materials containing nanocomponents.

Protein capped nanosilver free radical oxidation: role of biomolecule capping on nanoparticle colloidal stability and protein oxidation.

We studied the effect of human serum albumin protein capped spherical nanosilver on the nanoparticle stability upon peroxyl radical oxidation. The nanoparticle-protein composite is less prone to oxidation compared to the individual components. However, higher concentrations of hydrogen peroxide were formed in the nanoparticle-protein system.