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BACKGROUND: Lupanine is a plant toxin contained in the wastewater of lupine bean processing industries, which could be used for semi-synthesis of various novel high added-value compounds. This paper introduces an environmental friendly process for microbial production of enantiopure lupanine. RESULTS: Previously isolated P. putida LPK411, R. rhodochrous LPK211 and Rhodococcus sp. LPK311, holding the capacity to utilize lupanine as single carbon source, were employed as biocatalysts for resolution of racemic lupanine. All strains achieved high enantiomeric excess (ee) of L-(-)-lupanine (> 95%), while with the use of LPK411 53% of the initial racemate content was not removed. LPK411 fed with lupanine enantiomers as single substrates achieved 92% of D-(+)-lupanine biodegradation, whereas L-(-)-lupanine was not metabolized. Monitoring the transcriptional kinetics of the luh gene in cultures supplemented with the racemate as well as each of the enantiomers supported the enantioselectivity of LPK411 for D-(+)-lupanine biotransformation, while (trans)-6-oxooctahydro-1H-quinolizine-3-carboxylic acid was detected as final biodegradation product from D-(+)-lupanine use. Ecotoxicological assessment demonstrated that lupanine enantiomers were less toxic to A. fischeri compared to the racemate exhibiting synergistic interaction. CONCLUSIONS: The biological chiral separation process of lupanine presented here constitutes an eco-friendly and low-cost alternative to widely used chemical methods for chiral separation.
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Biotransformação , Pseudomonas putida/metabolismo , Rhodococcus/metabolismo , Esparteína/análogos & derivados , Águas Residuárias/microbiologia , Indústria Alimentícia , Lupinus/química , Esparteína/metabolismo , Estereoisomerismo , Águas Residuárias/químicaRESUMO
INTRODUCTION AND OBJECTIVES: Non-alcoholic fatty liver disease (NAFLD) is the most common liver disorder in western countries. It is often related to metabolic syndrome, presenting an increased risk of advanced liver disease and cardiovascular-related death. In some etiologies of chronic liver disease, thrombocytopenia has been associated not only with advanced stages of fibrosis but also with autoimmune disease. In NAFLD, however, its prevalence and related factors are still unknown. The aim of this study is to evaluate the prevalence of thrombocytopenia in NAFLD patients without cirrhosis and to investigate its related risk factors. PATIENTS AND METHODS: This was a retrospective study carried out in two tertiary hospitals in the South and Southeast regions of Brazil. Patients diagnosed with NAFLD by liver biopsy were included. Those with other causes of liver disease and/or cirrhosis were excluded. For analysis, patients were divided into two groups, with and without thrombocytopenia. Data was analyzed using a significance level of 5%. RESULTS: 441 non-cirrhotic patients with NAFLD (evaluated by liver biopsy) were included in the study. The prevalence of thrombocytopenia was 3.2% (14/441 patients). In the comparative analysis between groups, thrombocytopenia was associated with male sex (p=0.007) and level of hemoglobin (p=0.023). CONCLUSION: Thrombocytopenia is an infrequent event in NAFLD patients without cirrhosis and is related with male sex and higher hemoglobin levels.
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Hepatopatia Gordurosa não Alcoólica/epidemiologia , Trombocitopenia/epidemiologia , Adulto , Idoso , Brasil/epidemiologia , Feminino , Hemoglobinas/metabolismo , Humanos , Fígado/patologia , Masculino , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/sangue , Hepatopatia Gordurosa não Alcoólica/patologia , Prevalência , Estudos Retrospectivos , Fatores Sexuais , Trombocitopenia/sangueRESUMO
INTRODUCTION AND AIM: The gold-standard for fibrosis diagnosis in non-alcoholic fatty liver disease (NAFLD) is liver biopsy, despite its invasive approach, sampling limitations and variability among observers. The objective was to validate the performance of non-invasive methods (Fibroscan™; APRI, FIB4 and NAFLD score) comparing with liver biopsy in the evaluation of liver fibrosis in patients with NAFLD. MATERIAL AND METHODS: NAFLD patients ≥18 years of age who were submitted to liver biopsy were included and evaluated at two reference tertiary hospitals in Brazil with transient hepatic elastography (THE) assessment through Fibroscan™, APRI, FIB4 and NAFLD scores were determined. Sensitivity, specificity, positive (PPV) and negative (NPV) predictive values for the diagnosis of advanced fibrosis were calculated to evaluate the performance of these non-invasive methods in NAFLD patients, adopting liver biopsy as the gold standard. RESULTS: A total of 104 patients were studied. At three different cutoff values (7.9, 8.7 and 9.6kPa) THE presented the highest sensitivity values (95%, 90% and 85% respectively), and the highest NPV (98%, 96.4% and 95.1% respectively) for the diagnosis of advanced fibrosis. It also presented the highest AUROC (0.87; CI 95% 0.78-0.97). CONCLUSION: When compared to the gold standard, transient hepatic elastography presented the best performance for the diagnosis and exclusion of advanced fibrosis in patients with NAFLD, overcoming APRI, FIB4 and NAFLD score.
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Biópsia Guiada por Imagem/métodos , Cirrose Hepática/diagnóstico por imagem , Cirrose Hepática/epidemiologia , Hepatopatia Gordurosa não Alcoólica/diagnóstico por imagem , Hepatopatia Gordurosa não Alcoólica/epidemiologia , Adulto , Idoso , Área Sob a Curva , Brasil , Comorbidade , Estudos Transversais , Técnicas de Imagem por Elasticidade/métodos , Feminino , Humanos , Cirrose Hepática/patologia , Masculino , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/patologia , Curva ROC , Sensibilidade e Especificidade , Índice de Gravidade de Doença , Centros de Atenção Terciária , Ultrassonografia/métodosRESUMO
Substantial research on complete Achilles tendon ruptures is available, but guidance on partial ruptures is comparatively sparse. Conservative management is considered acceptable in partial tendon ruptures affecting less than 50% of the tendon's width, but supporting experimental evidence is currently lacking. Using a previously validated finite element model of the Achilles tendon, this study aimed to assess whether loading conditions simulating an early functional rehabilitation protocol could elicit progression to a complete rupture in partial ruptures of varying severity. In silico tendon rupture simulations were performed to locate the most likely rupture site for least, moderate, and extreme subtendon twist configurations. These three models were split at the corresponding rupture site and two sets of partial ruptures were created for each, starting from the medial and lateral sides, and ranging from 10% to 50% loss of continuity. Simulations were conducted with material parameters from healthy and tendinopathic tendons. Partial ruptures were considered to progress if the volume of elements showing a maximum principal strain above 10% exceeded 3 mm3. To assess whether the tendinopathic tendons typical geometric characteristics could compensate for the inferior material properties found in tendinopathy, an additional model with increased cross-sectional area in the free tendon region was developed. Progression to complete ruptures occurred even with less than a 50% loss of continuity, regardless of subtendon twisting, and material parameters. The tendinopathic tendon model with increased cross-sectional area showed similar results. These findings suggest the current criteria for surgical treatment of partial ruptures should be reconsidered. Statement of clinical significance: The clinical significance and most appropriate treatment of partial ruptures of the Achilles tendon is unclear. Despite the widespread use of the "50% rule" in treatment decisions of partial tendon ruptures, experimental evidence supporting it is missing. The present study provides new data, from a validated aponeurotic and free Achilles tendon finite element model, showing that partial ruptures may progress to complete ruptures under loading conditions elicited from functional rehabilitation protocols, even for partial ruptures affecting less than 50% of the tendon's width. Under these novel findings, the current criteria for surgical treatment of partial ruptures should be reconsidered.
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Tendão do Calcâneo , Análise de Elementos Finitos , Tendão do Calcâneo/lesões , Tendão do Calcâneo/fisiopatologia , Humanos , Ruptura/reabilitação , Traumatismos dos Tendões/reabilitação , Traumatismos dos Tendões/fisiopatologia , Simulação por Computador , Progressão da DoençaRESUMO
Cultivated meat, also known as cultured or cell-based meat, is meat produced directly from cultured animal cells rather than from a whole animal. Cultivated meat and seafood have been proposed as a means of mitigating the substantial harms associated with current production methods, including damage to the environment, antibiotic resistance, food security challenges, poor animal welfare, and-in the case of seafood-overfishing and ecological damage associated with fishing and aquaculture. Because biomedical tissue engineering research, from which cultivated meat draws a great deal of inspiration, has thus far been conducted almost exclusively in mammals, cultivated seafood suffers from a lack of established protocols for producing complex tissues in vitro. At the same time, fish such as the zebrafish Danio rerio have been widely used as model organisms in developmental biology. Therefore, many of the mechanisms and signaling pathways involved in the formation of muscle, fat, and other relevant tissue are relatively well understood for this species. The same processes are understood to a lesser degree in aquatic invertebrates. This review discusses the differentiation and maturation of meat-relevant cell types in aquatic species and makes recommendations for future research aimed at recapitulating these processes to produce cultivated fish and shellfish.
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Conservação dos Recursos Naturais , Peixe-Zebra , Animais , Pesqueiros , Alimentos Marinhos/análise , Músculos , Adipócitos , Diferenciação Celular , Biologia do Desenvolvimento , MamíferosRESUMO
Over recent years, the field of cell and gene therapy has witnessed rapid growth due to the demonstrated benefits of using living cells as therapeutic agents in a broad range of clinical studies and trials. Bioprocess economic models (BEMs) are fundamental tools for guiding decision-making in bioprocess design, being capable of supporting process optimization and helping to reduce production costs. These tools are particularly important when it comes to guiding manufacturing decisions and increasing the likelihood of market acceptance of cell-based therapies, which are often cost-prohibitive because of high resource and quality control costs. Not only this, but the inherent biological variability of their underlying bioprocesses makes them particularly susceptible to unforeseen costs arising from failed or delayed production batches. The present work reviews important concepts concerning the development of bioprocesses for stem cell therapy products and highlights the valuable role which BEMs can play in this endeavor. Additionally, some theoretical concepts relevant to the building and structuring of BEMs are explored. Finally, a comprehensive review of the existent BEMs so far reported in the scientific literature for stem cell-related bioprocesses is provided to showcase their potential usefulness.
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Novel green materials not sourced from animals and with low environmental impact are becoming increasingly appealing for biomedical and cellular agriculture applications. Marine biomaterials are a rich source of structurally diverse compounds with various biological activities. Kappa-carrageenan (κ-c) is a potential candidate for tissue engineering applications due to its gelation properties, mechanical strength, and similar structural composition of glycosaminoglycans (GAGs), possessing several advantages when compared to other algae-based materials typically used in bioprinting such as alginate. For those reasons, this material was selected as the main polysaccharide component of the bioinks developed herein. In this work, pristine κ-carrageenan bioinks were successfully formulated for the first time and used to fabricate 3D scaffolds by bioprinting. Ink formulation and printing parameters were optimized, allowing for the manufacturing of complex 3D structures. Mechanical compression tests and dry weight determination revealed young's modulus between 24.26 and 99.90 kPa and water contents above 97%. Biocompatibility assays, using a mouse fibroblast cell line, showed high cell viability and attachment. The bioprinted cells were spread throughout the scaffolds with cells exhibiting a typical fibroblast-like morphology similar to controls. The 3D bio-/printed structures remained stable under cell culture conditions for up to 11 days, preserving high cell viability values. Overall, we established a strategy to manufacture 3D bio-/printed scaffolds through the formulation of novel bioinks with potential applications in tissue engineering and cellular agriculture.
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Conductive hydrogels are emerging as promising materials for bioelectronic applications as they minimize the mismatch between biological and electronic systems. We propose a strategy to bioprint biohybrid conductive bioinks based on decellularized extracellular matrix (dECM) and multiwalled carbon nanotubes. These inks contained conductive features and morphology of the dECM fibers. Electrical stimulation (ES) was applied to bioprinted structures containing human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). It was observed that in the absence of external ES, the conductive properties of the materials can improve the contractile behavior of the hPSC-CMs, and this effect is enhanced under the application of external ES. Genetic markers indicated a trend toward a more mature state of the cells with upregulated calcium handling proteins and downregulation of calcium channels involved in the generation of pacemaking currents. These results demonstrate the potential of our strategy to manufacture conductive hydrogels in complex geometries for actuating purposes.
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Microbially produced carboxylic acids (CAs) are considered key players in the implementation of more sustainable industrial processes due to their potential to replace a set of oil-derived commodity chemicals. Most CAs are intermediates of microbial central carbon metabolism, and therefore, a biochemical production pathway is described and can be transferred to a host of choice to enable/improve production at an industrial scale. However, for some CAs, the implementation of this approach is difficult, either because they do not occur naturally (as is the case for levulinic acid) or because the described production pathway cannot be easily ported (as it is the case for adipic, muconic or glucaric acids). Synthetic biology has been reshaping the range of molecules that can be produced by microbial cells by setting new-to-nature pathways that leverage on enzyme arrangements not observed in vivo, often in association with the use of substrates that are not enzymes' natural ones. In this review, we provide an overview of how the establishment of synthetic pathways, assisted by computational tools for metabolic retrobiosynthesis, has been applied to the field of CA production. The translation of these efforts in bridging the gap between the synthesis of CAs and of their more interesting derivatives, often themselves non-naturally occurring molecules, is also reviewed using as case studies the production of methacrylic, methylmethacrylic and poly-lactic acids.
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Levulinic acid is a versatile platform molecule with potential to be used as an intermediate in the synthesis of many value-added products used across different industries, from cosmetics to fuels. Thus far, microbial biosynthetic pathways having levulinic acid as a product or an intermediate are not known, which restrains the development and optimization of a microbe-based process envisaging the sustainable bioproduction of this chemical. One of the doors opened by synthetic biology in the design of microbial systems is the implementation of new-to-nature pathways, that is, the assembly of combinations of enzymes not observed in vivo, where the enzymes can use not only their native substrates but also non-native ones, creating synthetic steps that enable the production of novel compounds. Resorting to a combined approach involving complementary computational tools and extensive manual curation, in this work, we provide a thorough prospect of candidate biosynthetic pathways that can be assembled for the production of levulinic acid in Escherichia coli or Saccharomyces cerevisiae. Out of the hundreds of combinations screened, five pathways were selected as best candidates on the basis of the availability of substrates and of candidate enzymes to catalyze the synthetic steps (that is, those steps that involve conversions not previously described). Genome-scale metabolic modeling was used to assess the performance of these pathways in the two selected hosts and to anticipate possible bottlenecks. Not only does the herein described approach offer a platform for the future implementation of the microbial production of levulinic acid but also it provides an organized research strategy that can be used as a framework for the implementation of other new-to-nature biosynthetic pathways for the production of value-added chemicals, thus fostering the emerging field of synthetic industrial microbiotechnology.
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Ácidos Levulínicos/metabolismo , Vias Biossintéticas , Escherichia coli/metabolismo , Engenharia Metabólica/métodos , Saccharomyces cerevisiae/metabolismo , Biologia Sintética/métodosRESUMO
The induced pluripotent stem cells (iPSCs) are derived from somatic cells by using reprogramming factors such as Oct4, Sox2, Klf4, and c-Myc (OSKM) or Oct4, Sox2, Nanog and Lin28 (OSNL). They resemble embryonic stem cells (ESCs) and have the ability to differentiate into cell lineage of all three germ-layer, including cardiomyocytes (CMs). The CMs can be generated from iPSCs by inducing embryoid bodies (EBs) formation and treatment with activin A, bone morphogenic protein 4 (BMP4), and inhibitors of Wnt signaling. However, these iPSC-derived CMs are a heterogeneous population of cells and require purification and maturation to mimic the in vivo CMs. The matured CMs can be used for various therapeutic purposes in regenerative medicine by cardiomyoplasty or through the development of tissue-engineered cardiac patches. In recent years, significant advancements have been made in the isolation of iPSC and their differentiation, purification, and maturation into clinically usable CMs. Newer small molecules have also been identified to substitute the reprogramming factors for iPSC generation as well as for direct differentiation of somatic cells into CMs without an intermediary pluripotent state. This review provides a concise update on the generation of iPSC-derived CMs and their application in personalized cardiac regenerative medicine. It also discusses the current limitations and challenges in the application of iPSC-derived CMs. Graphical abstract.
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Células-Tronco Pluripotentes Induzidas , Diferenciação Celular/genética , Células-Tronco Embrionárias , Miócitos Cardíacos , Medicina RegenerativaRESUMO
The use of electrospun scaffolds for neural tissue engineering applications allows a closer mimicry of the native tissue extracellular matrix (ECM), important for the transplantation of cells in vivo. Moreover, the role of the electrospun fiber mat topography on neural stem cell (NSC) differentiation remains to be completely understood. In this work REN-VM cells (NSC model) were differentiated on polycaprolactone (PCL) nanofibers, obtained by wet/wet electrospinning, and on flat glass lamellas. The obtained differentiation profile of NSCs was evaluated using immunofluorescence and qPCR analysis. Glycosaminoglycan (GAG) analysis was successfully emplyed to evaluate changes in the GAG profile of differentiating cells through the use of the highly sensitive liquid chromatography-tandem mass/mass spectrometry (LC-MS/MS) method. Our results show that both culture platforms allow the differentiation of REN-VM cells into neural cells (neurons and astrocytes) similarly. Moreover, LC-MS/MS analysis shows changes in the production of GAGs present both in cell cultures and conditioned media samples. In the media, hyaluronic acid (HA) was detected and correlated with cellular activity and the production of a more plastic extracellular matrix. The cell samples evidence changes in chondroitin sulfate (CS4S, CS6S, CS4S6S) and heparan sulfate (HS6S, HS0S), similar to those previously described in vivo studies and possibly associated with the creation of complex structures, such as perineural networks. The GAG profile of differentiating REN-VM cells on electrospun scaffolds was analyzed for the first time. Our results highlight the advantage of using platforms obtain more reliable and robust neural tissue-engineered transplants.
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Diferenciação Celular , Sulfatos de Condroitina/biossíntese , Heparitina Sulfato/biossíntese , Células-Tronco Neurais/metabolismo , Alicerces Teciduais/química , Linhagem Celular Transformada , Humanos , Células-Tronco Neurais/citologiaRESUMO
In recent years, stem cell-based therapies shown to have promising effects on the clinical management of ischemic heart disease. Moreover, stem cells differentiation into cardiomyocytes (CMs) can overcome the cell source limitations. The current research involves the isolation and expansion of mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs), their differentiation into CMs and subsequent construction of tissue-engineered myocardium supported by random and aligned polycaprolactone (PCL) nanofibrous matrices (av. dia: 350-850 nm). Umbilical cord matrix (UCM)-derived MSCs were isolated successfully by routine enzymatic digestion and a nonenzymatic explant culture method and characterized by their morphology, differentiation into different lineages, and surface marker expression. Treatment of UCM-derived MSCs with 5-azacytidine (5 µM) induced their differentiation into putative cardiac cells, as revealed by the expression of cardiac-specific troponin T (cTnT), smooth muscles actin, myogenin (MYOG), smoothelin, cardiac α-actin genes and cTnT, α-actinin proteins by RT-PCR and immunocytochemistry, respectively. However, no beating cells were observed in differentiated MSCs. On the other hand, adult human foreskin-derived iPSCs cultured on Matrigel™-coated aligned PCL nanofibrous matrices showed anisotropic behavior along the PCL nanofibers and, upon differentiation, expressed cardiac-specific cTnT (23.34 vs. 32.55%) proteins and showed more synchronized beating than those differentiated on Matrigel™-coated tissue culture coated polystyrene surfaces. Moreover, aligned PCL nanofibers are able to promote cells orientation parallel to the fibers, thus providing an effective way to control anisotropic nature under in vitro condition.
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Prepúcio do Pênis/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Mesenquimais/citologia , Miócitos Cardíacos/citologia , Cordão Umbilical/citologia , Diferenciação Celular , Células Cultivadas , Humanos , Masculino , Engenharia Tecidual/métodos , Alicerces Teciduais/químicaRESUMO
Cell-derived extracellular matrices have emerged as promising scaffolds for tissue engineering (TE) strategies due to their ability to create a biomimetic microenvironment providing biochemical and physical cues to cells, without the limitations of availability and potential pathogen transmission associated with tissue-derived extracellular matrix (ECM) scaffolds. Glycosaminoglycans (GAGs) are important components of ECM with a crucial role in the maintenance of the mechanical properties of the tissue and as signaling regulators of several cellular processes, such as cell adhesion, growth and differentiation. However, despite their relevance to the field of TE, little information is available on the GAG composition of cell-derived ECM, mainly due to the lack of appropriate quantitative tools to determine different GAG and disaccharide subtypes in complex biological samples. In this chapter, we describe a highly sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to characterize decellularized cell-derived ECM generated in vitro in terms of their GAG and disaccharide composition.
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Dissacarídeos/metabolismo , Matriz Extracelular/metabolismo , Glicosaminoglicanos/metabolismo , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida , Matriz Extracelular/ultraestrutura , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/ultraestrutura , Humanos , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/ultraestruturaRESUMO
Multilayered skin substitutes comprising allogeneic cells have been tested for the treatment of nonhealing cutaneous ulcers. However, such nonnative skin grafts fail to permanently engraft because they lack dermal vascular networks important for integration with the host tissue. In this study, we describe the fabrication of an implantable multilayered vascularized bioengineered skin graft using 3D bioprinting. The graft is formed using one bioink containing human foreskin dermal fibroblasts (FBs), human endothelial cells (ECs) derived from cord blood human endothelial colony-forming cells (HECFCs), and human placental pericytes (PCs) suspended in rat tail type I collagen to form a dermis followed by printing with a second bioink containing human foreskin keratinocytes (KCs) to form an epidermis. In vitro, KCs replicate and mature to form a multilayered barrier, while the ECs and PCs self-assemble into interconnected microvascular networks. The PCs in the dermal bioink associate with EC-lined vascular structures and appear to improve KC maturation. When these 3D printed grafts are implanted on the dorsum of immunodeficient mice, the human EC-lined structures inosculate with mouse microvessels arising from the wound bed and become perfused within 4 weeks after implantation. The presence of PCs in the printed dermis enhances the invasion of the graft by host microvessels and the formation of an epidermal rete. Impact Statement Three Dimensional printing can be used to generate multilayered vascularized human skin grafts that can potentially overcome the limitations of graft survival observed in current avascular skin substitutes. Inclusion of human pericytes in the dermal bioink appears to improve both dermal and epidermal maturation.
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Bioimpressão/métodos , Células Endoteliais/citologia , Fibroblastos/citologia , Queratinócitos/citologia , Pericitos/citologia , Engenharia Tecidual/métodos , Animais , Células Cultivadas , Colágeno Tipo I/metabolismo , Células Endoteliais/metabolismo , Fibroblastos/metabolismo , Citometria de Fluxo , Humanos , Queratinócitos/metabolismo , Pericitos/metabolismo , Ratos , Medicina Regenerativa/métodosRESUMO
Neurodegenerative diseases compromise the quality of life of increasing numbers of the world's aging population. While diagnosis is possible no effective treatments are available. Strong efforts are needed to develop new therapeutic approaches, namely in the areas of tissue engineering and deep brain stimulation (DBS). Conductive polymers are the ideal material for these applications due to the positive effect of conducting electricity on neural cell's differentiation profile. This novel study assessed the biocompatibility of polybenzimidazole (PBI), as electrospun fibers and after being doped with different acids. Firstly, doped films of PBI were used to characterize the materials' contact angle and electroconductivity. After this, fibers were electrospun and characterized by SEM, FTIR and TGA. Neural Stem Cell's (NSC) proliferation was assessed and their growth rate and morphology on different samples was determined. Differentiation of NSCs on PBI - CSA fibers was also investigated and gene expression (SOX2, NES, GFAP, Tuj1) was assessed through Immunochemistry and qPCR. All the samples tested were able to support neural stem cell (NSC) proliferation without significant changes on the cell's typical morphology. Successfully differentiation of NSCs towards neural cells on PBI - CSA fibers was also achieved. This promising PBI fibrous scaffold material is envisioned to be used in neural cell engineering applications, including scaffolds, in vitro models for drug screening and electrodes.
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This work explores the potential for development of a lupanine valorization process evaluating different isolated microorganisms for their capacity to metabolize the alkaloid. Ecotoxicological assessment demonstrated that lupanine is toxic for Vibrio fischeri and Daphnia magna exhibiting EC50 values of 89 mg L-1 and 47 mg L-1 respectively, while acting both as growth inhibitor for a monocotyledonous and as promoter for a dicotyledonous plant. Among the eight aerobic and anaerobic strains isolated and identified Rhodococcus rhodochrous LPK211 achieved 81% removal for 1.5 g L-1 lupanine, while no end-products were detected by NMR constituting a promising microorganism for lupanine biodegradation. Moreover, Rhodococcus ruber LPK111 and Rhodococcus sp. LPK311 exhibited 66% and 71% of removal respectively, including potential formation of lupanine N-oxide. Pseudomonas putida LPK411 reached 80% of lupanine removal and generated three fermentation products potentially comprising 17-oxolupanine and lupanine derivatives with open ring structures enabling the development of alkaloid valorization processes.
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Alcaloides/metabolismo , Biodegradação Ambiental , Esparteína/análogos & derivados , Aliivibrio fischeri/metabolismo , Alcaloides/análise , Alcaloides/química , Animais , Daphnia/metabolismo , Magnoliopsida/metabolismo , Pseudomonas putida/metabolismo , Esparteína/análise , Esparteína/química , Esparteína/metabolismoRESUMO
Biomaterial properties and controlled architecture of scaffolds are essential features to provide an adequate biological and mechanical support for tissue regeneration, mimicking the ingrowth tissues. In this study, a bioextrusion system was used to produce 3D biodegradable scaffolds with controlled architecture, comprising three types of constructs: (i) poly(ε-caprolactone) (PCL) matrix as reference; (ii) PCL-based matrix reinforced with cellulose nanofibers (CNF); and (iii) PCL-based matrix reinforced with CNF and hydroxyapatite nanoparticles (HANP). The effect of the addition and/or combination of CNF and HANP into the polymeric matrix of PCL was investigated, with the effects of the biomaterial composition on the constructs (morphological, thermal, and mechanical performances) being analysed. Scaffolds were produced using a single lay-down pattern of 0/90°, with the same processing parameters among all constructs being assured. The performed morphological analyses showed a satisfactory distribution of CNF within the polymer matrix and high reliability was obtained among the produced scaffolds. Significant effects on surface wettability and thermal properties were observed, among scaffolds. Regarding the mechanical properties, higher scaffold stiffness in the reinforced scaffolds was obtained. Results from the cytotoxicity assay suggest that all the composite scaffolds presented good biocompatibility. The results of this first study on cellulose and hydroxyapatite reinforced constructs with controlled architecture clearly demonstrate the potential of these 3D composite constructs for cell cultivation with enhanced mechanical properties.
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Celulose/química , Durapatita/química , Nanofibras/química , Nanopartículas/química , Poliésteres/química , Alicerces Teciduais/química , Materiais Biocompatíveis/química , Varredura Diferencial de Calorimetria , Cristalização , Imageamento Tridimensional , Poliésteres/síntese química , Polímeros/química , Regeneração , Reprodutibilidade dos Testes , Estresse Mecânico , Propriedades de Superfície , Temperatura , Termogravimetria , MolhabilidadeRESUMO
A mathematical model is presented that describes the ionic transport across the cortical thick ascending limb (cTAL) of the Henle's loop, taking into account its tubular geometry. A comprehensive description of the cTAL is given for the first time in terms of potential, ion concentrations and ion fluxes along the tubule. For given ion concentrations at the entrance of the tubule, the model simulates steady-state profiles and allows the fitting of existing experimentally measured values at its exit. Moreover, the model expands the potentialities of experiments in situ and enables testing the effect of different perturbations induced by drugs or mutation-altering transport activity. One of the main insights given by this model is the increase of the lumenal electrical potential from the entrance to the exit of the tubule with a profile determined by the transepithelial electrical potential difference and by the chemical gradients along the lumen, both reflecting transepithelial salt transport. Furthermore, model and experimental results are consistent, showing that when the TAL is perfused at high rates with a diluted NaCl solution in relation to the bath, the transepithelial electrical potential difference increases from 6.7 to 23.0 mV and the potential difference across the basolateral barrier changes very little. The model predicts that the same static head is obtained independently of the NaCl concentration at the entrance of the tubule. A final important insight concerns the lowest reported NaCl concentrations (20-30 mM) at the exit of the tubule, which is controlled by a very tight epithelium, where the back-leak is substantially reduced.