22,487 research outputs found

    Corncob as carbon source in the production of xanthan gum in different strains Xanthomonas sp.

    Get PDF
    Xanthan gum (XG) is a biopolymer obtained in fermentation and used as a rheology control agent in aqueous systems and in stabilizing emulsions and suspensions. XG, together with other polysaccharides, can form soft, cohesive composite gels. The carbon source in the fermentative process is responsible for one-third of the production costs, and the search for less expensive and sustainable alternatives is ongoing. The use of agricultural residues such as the corncob is highly suggestive due to their abundance. This study aims to evaluate the use of derived hemicellulose fractions from the alkaline extraction of corncob as a carbon source in the production of XG in trials using four strains of Xanthomonas sp. (629, 1078, 254, and S6). The results indicate that strain 629 provides the higher yield (8.37 ± 5.75 g L−1) while using a fermentation medium containing a carbon source of saccharose (1.25%), hemicellulose fractions (3.75%), and salts. In this same medium, the strain 629 produces gum in 3% aqueous solution, showing the higher apparent viscosity (9298 ± 31 mPa s−1) at a shear rate of 10 s−1 at 25 °C. In conclusion, corncob is proven to be a promising sustainable alternative carbon source in the obtention of XG, improving the economic viability of the process within a biorefinery context. Saccharose must, however, also be included in the fermentation medium.0F1A-358B-43B5 | Fernando Jorge Ribeiro da MataN/

    Formalin-free fixation and xylene-free tissue processing preserves cell-hydrogel interactions for histological evaluation of 3D calcium alginate tissue engineered constructs

    Get PDF
    Histological evaluation of tissue-engineered products, including hydrogels for cellular encapsulation, is a critical and invaluable tool for assessing the product across multiple stages of its lifecycle from manufacture to implantation. However, many tissue-engineered products are comprised of polymers and hydrogels which are not optimized for use with conventional methods of tissue fixation and histological processing. Routine histology utilizes a combination of chemical fixatives, such as formaldehyde, and solvents such as xylene which have been optimized for use with native biological tissues due to their high protein and lipid content. Previous work has highlighted the challenges associated with processing hydrogels for routine histology due to their high water content and lack of diverse chemical moieties amenable for tissue fixation with traditional fixatives. Thus, hydrogel-based tissue engineering products are prone to histological artifacts during their validation which can lead to challenges in correctly interpreting results. In addition, chemicals used in conventional histological approaches are associated with significant health and environmental concerns due to their toxicity and there is thus an urgent need to identify suitable replacements. Here we use a multifactorial design of experiments approach to identify processing parameters capable of preserving cell-biomaterial interactions in a prototypical hydrogel system: ionically crosslinked calcium alginate. We identify a formalin free fixative which better retains cell-biomaterial interactions and calcium alginate hydrogel integrity as compared to the state-of-the-art formalin-based approaches. In addition, we demonstrate that this approach is compatible with a diversity of manufacturing techniques used to fabricate calcium alginate-based scaffolds for tissue engineering and cell therapy, including histological evaluation of cellular encapsulation in 3D tubes and thin tissue engineering scaffolds (∼50 μm). Furthermore, we show that formalin-free fixation can be used to retain cell-biomaterial interactions and hydrogel architecture in hybrid alginate-gelatin based scaffolds for use with histology and scanning electron microscopy. Taken together, these findings are a significant step forward towards improving histological evaluation of ionically crosslinked calcium alginate hydrogels and help make their validation less toxic, thus more environmentally friendly and sustainable

    Desarrollo de papeles biocativos por injerto de moléculas específicas en celulosa

    Get PDF
    Tesis (DCI)--FCEFN-UNC, 2019En la presente tesis se presenta el desarrollo de papeles bioactivos con potencial aplicación en el envasado activo de alimentos. Para tal fin, se propuso el injerto de eugenol, un compuesto de origen natural con propiedades antimicrobiana, antioxidante y repelente de insectos, en celulosa, utilizando ácido policarboxílico como agente ligante. Con el objetivo de evaluar la escalabilidad del proceso propuesto, se estudiaron distintas tecnologías de curado, tales como calentamiento por convección, infrarrojo, microondas y conducción. En todos los casos, se analizaron la influencia de las variables operativas sobre el avance de la reacción y propiedades finales del papel preparado, utilizando un diseño de experimentos Doehlert para elegir las experiencias a realizar, y analizando los resultados mediante metodología de superficie de respuesta y análisis estadístico ANOVA. Se pudo comprobar que la reacción de injerto de eugenol en papel comercial se produjo con éxito en todas las tecnologías estudiadas. Asimismo, se encontraron las condiciones óptimas de reacción para cada una de las tecnologías, para lo cual se buscó un compromiso entre el avance de la reacción y las propiedades finales del material (mecánicas y color). A partir de estas condiciones, se prepararon papeles y se realizó una caracterización más específica para su aplicación como envase de alimentos comparando los papeles modificados con el papel virgen. Se analizaron las propiedades mecánicas por ensayo de tracción, rasgado y punzonado y se midió la absorción de agua y la capacidad de degradación. Por otro lado, las propiedades bioactivas analizadas fueron la actividad antioxidante, antimicrobiana, repelente e insecticida de gorgojos (T. castaneum y R. dominica). Una vez probado que el papel modificado presenta buenas características físicas y bioactivas para su posible aplicación en el envasado de alimentos, se realizaron prototipos de envasado para harina, como alimento representativo de alimentos derivados de cereales, susceptibles al ataque de plagas. En este estudio se analizó la migración de reactivos, propiedades organolépticas y conservación del alimento, arrojando resultados promisorios para la industria de envases de alimentos. Finalmente, se realizó una comparación de las tecnologías de curado ensayadas, analizando diferentes aspectos como avance de reacción, propiedades finales, apariencia, tiempo de reacción, consumo de energía, entre otros, como así también disponibilidad y uso de estas tecnologías a escala industrial, seleccionando la tecnología de conducción como la más adecuada para una propuesta de escalado industrial.Fil: Muratore, Florencia. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales; Argentina.Fil: Muratore, Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigación y Desarrollo en Ingeniería de Procesos y Química Aplicada; Argentina

    Targeting Fusion Proteins of HIV-1 and SARS-CoV-2

    Get PDF
    Viruses are disease-causing pathogenic agents that require host cells to replicate. Fusion of host and viral membranes is critical for the lifecycle of enveloped viruses. Studying viral fusion proteins can allow us to better understand how they shape immune responses and inform the design of therapeutics such as drugs, monoclonal antibodies, and vaccines. This thesis discusses two approaches to targeting two fusion proteins: Env from HIV-1 and S from SARS-CoV-2. The first chapter of this thesis is an introduction to viruses with a specific focus on HIV-1 CD4 mimetic drugs and antibodies against SARS-CoV-2. It discusses the architecture of these viruses and fusion proteins and how small molecules, peptides, and antibodies can target these proteins successfully to treat and prevent disease. In addition, a brief overview is included of the techniques involved in structural biology and how it has informed the study of viruses. For the interested reader, chapter 2 contains a review article that serves as a more in-depth introduction for both viruses as well as how the use of structural biology has informed the study of viral surface proteins and neutralizing antibody responses to them. The subsequent chapters provide a body of work divided into two parts. The first part in chapter 3 involves a study on conformational changes induced in the HIV-1 Env protein by CD4-mimemtic drugs using single particle cryo-EM. The second part encompassing chapters 4 and 5 includes two studies on antibodies isolated from convalescent COVID-19 donors. The former involves classification of antibody responses to the SARS-CoV-2 S receptor-binding domain (RBD). The latter discusses an anti-RBD antibody class that binds to a conserved epitope on the RBD and shows cross-binding and cross-neutralization to other coronaviruses in the sarbecovirus subgenus.</p

    Towards personalized immunotherapy : development of in vitro models for imaging natural killer cell behavior in the tumor microenvironment

    Get PDF
    Tremendous advances in the tumor immunology field have transformed immunotherapy from a promising approach to a standard clinical practice. However, a subset of cancer patients is non-responsive to immunotherapy. More research is therefore needed to understand the mechanisms underlying tumor resistance to immunotherapeutic treatments. The aim of this doctoral work was to develop new tools to study the mechanisms of cancer immunosurveillance and to test immunotherapeutic treatments in vitro. In this thesis, I describe the methods developed, and I discuss the main biological findings obtained by using these methods. The thesis is organized as follows. A short historical background of immunotherapy is provided in Chapter 1. Chapter 2 describes the principles of NK cell-mediated cancer immunosurveillance, and provides an overview on rare cancers, mainly focusing on sarcoma. The research aims are listed in Chapter 3. In Chapter 4, I describe the cell culture methods and cell analysis techniques relevant for my doctoral work. In Chapter 5, I describe the methods we developed to culture tumor spheroids in vitro using ultrasonic standing waves in microwell chips, focusing on the theory, design, and applications. Chapter 6 and Chapter 7 focus on the biological findings obtained using our platform in combination with traditional immunological methods, followed by future implementations discussed in Chapter 8. The constituent papers are provided at the end of the thesis. In Paper I, we combined the use of the microwell chip, ultrasonic standing waves and a protein-repellent polymer coating to enable the production of spheroids from multiple cell types. In absence of cell adhesion to the chip, spheroids could be collected and further analyzed by off-the-chip techniques. In Paper II, we designed a novel multichambered microwell chip to perform multiplexed fluorescence screening of two- or three-dimensional cell cultures. The platform allows the direct assessment of drug or immune cell cytotoxic efficacy, making it a promising tool for individualized cytotoxicity tests for personalized medicine. In Paper III, we investigate the function of PVR receptors in NK cells interacting with renal carcinoma spheroids, and the impact of PVR in NK cell-based cellular immunotherapy. We demonstrated that variations in PVR expression are primarily recognized by the inhibitory receptor TIGIT, while DNAM-1 strongly contributes to NK cell activation mainly through PVR-independent mechanisms. We performed NK cell-based cytotoxicity assays against renal carcinoma spheroids in the microwell chip. Anti-TIGIT treatment was effective only for TIGIThigh NK cells both when used as monotherapy or in combination with other drugs, suggesting that only a fraction of patients might respond to anti-TIGIT therapy. In Paper IV, a similar approach was used with primary sarcomas. We cultured patient-derived sarcoma spheroids and tested NK cell-based immunotherapy in the microwell chip, either alone or in combination with antibody therapy, and we identified promising treatment combinations. In Paper V, we applied the use of expansion microscopy to visualize NK cells infiltrating renal carcinoma spheroids. In conclusion, our multi-disciplinary work shows the development of new imaging-based platform and its use to study the mechanisms of NK cell-mediated tumor surveillance and for personalized therapy

    RNA pull-down-confocal nanoscanning (RP-CONA), a novel method for studying RNA/protein interactions in cell extracts that detected potential drugs for Parkinson’s disease targeting RNA/HuR complexes

    Get PDF
    MicroRNAs (miRNAs, miRs) are a class of small non-coding RNAs that regulate gene expression through specific base-pair targeting. The functional mature miRNAs usually undergo a two-step cleavage from primary miRNAs (pri-miRs), then precursor miRNAs (pre-miRs). The biogenesis of miRNAs is tightly controlled by different RNA-binding proteins (RBPs). The dysregulation of miRNAs is closely related to a plethora of diseases. Targeting miRNA biogenesis is becoming a promising therapeutic strategy. HuR and MSI2 are both RBPs. MiR-7 is post-transcriptionally inhibited by the HuR/MSI2 complex, through a direct interaction between HuR and the conserved terminal loop (CTL) of pri-miR-7-1. Small molecules dissociating pri-miR-7/HuR interaction may induce miR-7 production. Importantly, the miR-7 levels are negatively correlated with Parkinson’s disease (PD). PD is a common, incurable neurodegenerative disease causing serious motor deficits. A hallmark of PD is the presence of Lewy bodies in the human brain, which are inclusion bodies mainly composed of an aberrantly aggregated protein named α-synuclein (α-syn). Decreasing α-syn levels or preventing α-syn aggregation are under investigation as PD treatments. Notably, α-syn is negatively regulated by several miRNAs, including miR-7, miR-153, miR-133b and others. One hypothesis is that elevating these miRNA levels can inhibit α-syn expression and ameliorate PD pathologies. In this project, we identified miR-7 as the most effective α-syn inhibitor, among the miRNAs that are downregulated in PD, and with α-syn targeting potentials. We also observed potential post-transcriptional inhibition on miR-153 biogenesis in neuroblastoma, which may help to uncover novel therapeutic targets towards PD. To identify miR-7 inducers that benefit PD treatment by repressing α-syn expression, we developed a novel technique RNA Pull-down Confocal Nanoscaning (RP-CONA) to monitor the binding events between pri-miR-7 and HuR. By attaching FITC-pri-miR-7-1-CTL-biotin to streptavidin-coated agarose beads and incubating them in human cultured cell lysates containing overexpressed mCherry-HuR, the bound RNA and protein can be visualised as quantifiable fluorescent rings in corresponding channels in a confocal high-content image system. A pri-miR-7/HuR inhibitor can decrease the relative mCherry/FITC intensity ratio in RP-CONA. With this technique, we performed several small-scale screenings and identified that a bioflavonoid, quercetin can largely dissociate the pri-miR-7/HuR interaction. Further studies proved that quercetin was an effective miR-7 inducer as well as α-syn inhibitor in HeLa cells. To understand the mechanism of quercetin mediated α-syn inhibition, we tested the effects of quercetin treatment with miR-7-1 and HuR knockout HeLa cells. We found that HuR was essential in this pathway, while miR-7 hardly contributed to the α-syn inhibition. HuR can directly bind an AU-rich element (ARE) at the 3’ untranslated region (3’-UTR) of α-syn mRNA and promote translation. We believe quercetin mainly disrupts the ARE/HuR interaction and disables the HuR-induced α-syn expression. In conclusion, we developed and optimised RP-CONA, an on-bead, lysate-based technique detecting RNA/protein interactions, as well as identifying RNA/protein modulators. With RP-CONA, we found quercetin inducing miR-7 biogenesis, and inhibiting α-syn expression. With these beneficial effects, quercetin has great potential to be applied in the clinic of PD treatment. Finally, RP-CONA can be used in many other RNA/protein interactions studies

    The Role of Silane Sol-Gel Coatings on the Corrosion Protection of Magnesium Alloys

    Get PDF
    Magnesium alloys, as the lightest structural metallic material with promising physical, mechanical, and biodegradable properties, have become very attractive for different technical applications, especially for industrial and biomedical fields. However, rapid corrosion is the most critical obstacle that limits its use to play a major role in large-scale applications. The simplest way to control the corrosion rate is to prevent a direct contact of the magnesium substrate with the environment by using surface modification technologies. Silica sol-gel coatings are considered a promising solution to enhance the corrosion resistance of magnesium alloys because sol-gel-based coating systems form very stable chemical bonds with the metallic surface. In this chapter, an insight about the advances in silica sol-gel coatings as an alternative method to control the corrosion of Mg and its alloys will be exposed. A wide overview of the most relevant aspects and their current applications, specifically for aerospace, automobile, and biomedical applications will be described. The modification of silica sol-gel matrix by the incorporation of different types of inhibitors to achieve an active barrier property on Mg alloys has been also considered. Finally, the future perspective based on the development of new silica sol-gel coatings on Mg alloy will be presented

    Substrate-specificity of the DNA-protein crosslink repair protease SPRTN

    Get PDF
    • …
    corecore