Bone and cartilage regeneration can be improved by designing a functionalized biomaterial that includes bioactive drugs in a biocompatible and biodegradable scaffold. Based on our previous studies, we designed a vanadium-loaded collagen scaffold for osteochondral tissue engineering. Collagen-vanadium loaded scaffolds were characterized by SEM, FTIR, and permeability studies. Rat bone marrow progenitor cells were plated on collagen or vanadium-loaded membranes to evaluate differences in cell attachment, growth and osteogenic or chondrocytic differentiation. The potential cytotoxicity of the scaffolds was assessed by the MTT assay and by evaluation of morphological changes in cultured RAW 264.7 macrophages. Our results show that loading of VOAsc did not alter the grooved ordered structure of the collagen membrane although it increased membrane permeability, suggesting a more open structure. The VOAsc was released to the media, suggesting diffusion-controlled drug release. Vanadium-loaded membranes proved to be a better substratum than C0 for all evaluated aspects of BMPC biocompatibility (adhesion, growth, and osteoblastic and chondrocytic differentiation). In addition, there was no detectable effect of collagen or vanadium-loaded scaffolds on macrophage viability or cytotoxicity. Based on these findings, we have developed a new ordered collagen scaffold loaded with VOAsc that shows potential for osteochondral tissue engineering.
Background and Aims: Non-invasive biomarkers are urgently needed to identify patients with non-alcoholic fatty liver disease (NAFLD) especially those at risk of disease progression. This is particularly true in high prevalence areas such as Latin America. The gut microbiome and intestinal permeability may play a role in the risk of developing NAFLD and NASH, but the mechanism by which microbiota composition disruption (or dysbiosis) may affect NAFLD progression is still unknown. Targeted metabolomics is a powerful technology for discovering new associations between gut microbiome-derived metabolites and disease. Thus, we aimed to identify potential metabolomic biomarkers related to the NAFLD stage in Argentina, and to assess their relationship with clinical and host genetic factors. Materials and methods: Adult healthy volunteers (HV) and biopsy-proven simple steatosis (SS) or non-alcoholic steatohepatitis (NASH) patients were recruited. Demographic, clinical and food frequency consumption data, as well as plasma and stool samples were collected. SNP rs738409 (PNPLA3 gene) was determined in all volunteers. HPLC and flow injection analysis with MS/MS in tandem was applied for metabolomic studies using the MxP Quant 500 Kit (Biocrates Life Sciences AG, Austria). Significantly different metabolites among groups were identified with MetaboAnalyst v4.0. Bivariate and multivariate analyses were used to identify variables that were independently related to NAFLD stage. Forward stepwise logistic regression models were constructed to design the best feature combination that could distinguish between study groups. Receiver Operating Characteristic (ROC) curves were used to evaluate models′ accuracy. Results: A total of 53 volunteers were recruited: 19 HV, 12 SS and 22 NASH. Diet was similar between groups. The concentration of 33 out of 424 detected metabolites (25 in plasma and 8 in stool) was significantly different among study groups. Levels of triglycerides (TG) were higher among NAFLD patients, whereas levels of phosphatidylcholines (PC) and lysoPC were depleted relative to HV. The PNPLA3 risk genotype for NAFLD and NASH (GG) was related to higher plasma levels of eicosenoic acid FA(20:1) (p<0.001). Plasma metabolites showed a higher accuracy for diagnosis of NAFLD and NASH when compared to stool metabolites. Body mass index (BMI) and plasma levels of PC aa C24:0, FA(20:1) and TG(16:1_34:1) showed high accuracy for diagnosis of NAFLD; whereas the best AUROC for discriminating NASH from SS was that of plasma levels of PC aa C24:0 and PC ae C40:1. Conclusion: A panel of plasma and stool biomarkers could distinguish between NAFLD and NASH in a cohort of patients from Argentina. Plasma biomarkers may be diagnostic in these patients and could be used to assess disease progression. Further validation studies including a larger number of patients are needed.
Interactions between communities of the gut microbiome and with the host could affect the onset and progression of metabolic associated fatty liver disease (MAFLD), and can be useful as new diagnostic and prognostic biomarkers. In this study, we performed a multi‐omics approach to unravel gut microbiome signatures from 32 biopsy‐proven patients (10 simple steatosis ‐SS‐ and 22 steatohepatitis ‐SH‐) and 19 healthy volunteers (HV). Human and microbial transcripts were differentially identified between groups (MAFLD vs. HV/SH vs. SS), and analyzed for weighted correlation networks together with previously detected metabolites from the same set of samples. We observed that expression of Desulfobacteraceae bacterium, methanogenic archaea, Mushu phage, opportunistic pathogenic fungi Fusarium proliferatum and Candida sorbophila, protozoa Blastocystis spp. and Fonticula alba were upregulated in MAFLD and SH. Desulfobacteraceae bacterium and Mushu phage were hub species in the onset of MAFLD, whereas the activity of Fonticula alba, Faecalibacterium prausnitzii, and Mushu phage act as key regulators of the progression to SH. A combination of clinical, metabolomic, and transcriptomic parameters showed the highest predictive capacity for MAFLD and SH (AUC = 0.96). In conclusion, faecal microbiome markers from several community members contribute to the switch in signatures characteristic of MAFLD and its progression towards SH.
El desarrollo de los sistemas de expresión basados en plantas para la producción de proteínas recombinantes constituye un paradigma tanto en la producción de biológicos como de proteínas con diversas aplicaciones como son la investigación médica, la medicina regenerativa, como también las destinadas a la producción de biocombustibles, entre otros. El uso de esta tecnología se consolidó en distintos mercados gracias a algunas ventajas que permitían lidiar con las fallas de los sistemas tradicionales, como por ejemplo la rápida escalabilidad de la producción, la capacidad de síntesis de moléculas que no se sintetizan eficientemente en otros sistemas y la ausencia de endotoxinas y seguridad intrínseca. Las plantas representan además, un sistema de expresión sustentable y amigable con el medio ambiente. Este trabajo de tesis pretende contribuir al conocimiento de las plantas como fábricas de proteínas recombinantes y se basó en dos grandes aspectos. Por un lado, las condiciones de crecimiento de las plantas, teniendo en cuenta un factor clave que es el bajo costo tanto en la producción de biomasa, como de infraestructura y del proceso downstream. Y por otro lado, el estudio de estrategias que incrementen el rendimiento de producción, teniendo en cuenta que los mismos se encuentran limitados por procesos como el correcto plegado de proteínas y el transporte de las mismas a través de la vía secretoria hacia su destino final. En una primera sección, se analizó el efecto de la utilización de iluminación LED Full Spectrum o Rojo-Azul sobre el crecimiento de plantas Nicotiana benthamiana y sobre la producción de proteínas recombinantes en sistemas de expresión transitoria, con respecto a un sistema de iluminación tradicional como son los tubos fluorescentes blancos fríos. Para ello, en primer lugar se determinó la intensidad de luz emitida por cada una de las fuentes de luz estudiadas. Luego se evaluaron parámetros de crecimiento y fisiológicos de plantas Nicotiana benthamiana crecidas durante cuatro semanas bajo las distintas luces, como su altura, color, diámetro de hoja, y contenido relativo de agua (CRA). La mejor condición para el crecimiento de las plantas fue la de tubos fluorescentes o luces LED Full Spectrum para las cuales las plantas presentaron una coloración verde oscura, tamaños de planta y hojas adecuados para ensayos de agroinfiltración y valores de CRA superiores a 90%. Por otro lado, para evaluar el efecto de las luces sobre la producción de proteínas recombinantes, se analizaron los niveles de expresión de dos proteínas reporteras retenidas en el retículo endoplásmico: la proteína fluorescente verde (RE-GFP) y la enzima β glucuronidasa (RE-GUS). La mejor condición de luz para la producción de estas proteínas reporteras fue la de tubos fluorescentes, donde se alcanzaron los mayores niveles de RE-GFP y RE GUS y en donde se obtuvieron los niveles más bajos de la chaperona BiP, que es indicadora de estrés en el retículo endoplásmico. En la segunda sección de este trabajo, se exploró una estrategia de ingeniería de la vía secretoria a partir del estudio del efecto de la sobreexpresión de distintos genes candidatos sobre la producción de proteínas recombinantes. Los genes candidatos codifican para factores de transcripción del tipo bZIP, homólogos a aquellos que se expresan durante la diferenciación de células secretorias profesionales en otros organismos, o para chaperonas involucradas en el plegado proteico, que se expresan durante fase de acumulación de proteínas de reserva en semillas y también tienen un rol en los procesos vinculados a estrés, tal como la respuesta a proteínas mal plegadas o UPR. Los genes estudiados en este trabajo codifican para los factores AtbZIP60 (AT1G42990; NM_103458), AtbZIP28 (AT3G10800; NM_111917) y AtbZIP17 (AT2G40950; NM_129659), en sus formas completa o truncada (sin dominio C-terminal – ΔC) y para la chaperona Calnexina1 (CNX1) (AT5G61790). Se evaluó su efecto sobre la expresión de las proteínas reporteras sencillas RE GFP y RE-GUS y sobre la expresión de proteínas de interés más demandantes en su plegado como la versión simple cadena del anticuerpo 2G3 fusionado a la proteína fluorescente roja y un tag de histidinas (RFP-ScFv-KDEL) y el anticuerpo monoclonal tetramérico 14D9. Los resultados obtenidos muestran que la expresión de los genes codificantes para la forma activa del factor AtbZIP28ΔC o la chaperona Calnexina1, provoca un incremento en los niveles de GUS y GFP, como también de moléculas más demandantes para el plegado como los anticuerpos simple cadena 2G3 (RE-RFP-2G3) y multimérico 14D9. La co-expresión de la forma activa del factor bZIP60ΔC, también produjo incrementos en los niveles de acumulación de las reporteras GUS y GFP pero fue menos efectiva que la obtenida con los genes del bZIP28ΔC o Calnexina1. Este trabajo demuestra que la expresión de los genes codificantes para la forma activa del factor AtbZIP28ΔC o la chaperona Calnexina1, representa una estrategia válida para incrementar los niveles de proteínas reporteras con distintos requerimientos de plegado en plantas Nicotiana benthamiana por ensayos de expresión transitoria.
Plants are economical and sustainable factories for the production of recombinant proteins. Currently, numerous proteins produced using different plant-based systems with applications as cosmetic and tissue culture ingredients, research and diagnostic reagents, and industrial enzymes are marketed worldwide. In this study, we aimed to demonstrate the usefulness of a plant-based system to synthesize a single-chain antibody (scFv)-elastin-like polypeptide (ELP) fusion to be applied as an affinity precipitation reagent of the difficult to produce recombinant proteins. We used the human tissue transglutaminase (TG2), the main celiac disease autoantigen, as a proof of concept. We cloned a TG2-specific scFv and fused it to a short hydrophobic ELP tag. The anti-TG2-scFv-ELP was produced in Nicotiana benthamiana and was efficiently recovered by an inverse transition cycling procedure improved by coaggregation with bacteria-made free ELP. Finally, the scFv-ELP was used to purify both plant-synthesized human TG2 and also Caco-2-TG2. In conclusion, this study showed for the first time the usefulness of a plant-based expression system to produce an antibody-ELP fusion designed for the purification of low-yield proteins. K E Y W O R D S affinity precipitation, downstream processing, elastin like polypeptides, molecular farming, single-chain variable antibody fragments 1 | INTRODUCTION Many recombinant proteins produced in plants are currently in the market, such as tissue culture or cosmetic ingredients, research or diagnostic reagents, and industrial enzymes. Less successful has been the production of biopharmaceuticals, with only one approved product and several vaccines in advanced stages of clinical trials (Fischer & Buyel, 2020). Plant-based platforms can compete across different markets due to specific advantages like inexpensive and massively scalable transgenic plant-based systems, or the rapid scaleup of transient leaf expression systems (Fischer & Buyel, 2020). The bottlenecks of molecular farming include the low product yield, the
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