Papers by Andrea Villarino
Frontiers in Cellular and Infection Microbiology, Jun 22, 2023
Characteristics of Mycobacterium tuberculosis PtpA interaction and activity on the alpha subunit ... more Characteristics of Mycobacterium tuberculosis PtpA interaction and activity on the alpha subunit of human mitochondrial trifunctional protein, a key enzyme of lipid metabolism.
Biotechnology and Applied Biochemistry, May 29, 2009
Addition of tags [such as His (histidine) tags] is extremely helpful for the affinity purificatio... more Addition of tags [such as His (histidine) tags] is extremely helpful for the affinity purification of recombinant proteins. In several cases, these tags must be removed before performing functional and structural studies. The enzyme most frequently used to cleave tags of recombinant proteins is the TEV-protease (tobacco-etch-virus NIa protease). The continuous production of this enzyme in soluble form is quite an expensive process and not easily accessible to many laboratories. Thus an interesting alternative is the use of TEV-protease in an immobilized form, which may be reutilized several times. The main objective of the present study was to obtain a TEV-protease in an immobilized form, by covalent immobilization on to solid supports through selective use of different amino acid residues, lysine or cysteine. High protein immobilization yields (75-97 %) were obtained with both strategies. The TEV-protease immobilized through its exposed cysteine thiol groups maintained its ability for cleaving a 20 kDa substrate. While the activity of the immobilized TEV-protease maintained only 30 % of the activity of the enzyme in soluble form, its stability at 4 • C was improved three times. Moreover, this enzyme could be reutilized in at least five cycles of cleavage without loss of performance. The present results indicate that the use of a TEV-protease in an immobilized form is a potentially useful tool for the cleavage of His tags of recombinant proteins and may be useful for reducing the cost of the total process of cleavage.
Acta Crystallographica Section A, Aug 23, 2005
Src homology 2 domain-containing protein tyrosine phosphatase [SHP] substrate 1 (SHPS-1), a recep... more Src homology 2 domain-containing protein tyrosine phosphatase [SHP] substrate 1 (SHPS-1), a receptor-type transmembrane glycoprotein whose cytoplasmic region binds and activates the protein tyrosine phosphatases SHP-1 and SHP-2, and thereby modulates multiple cellular functions. Its extracellular region regulates intercellular communication in the neural and immune systems through its association with CD47 on adjacent cells. Interactions between CD47 and SHPS-1 are implicated in multiple cellular processes, including cell motility [1], neutrophil transmigration, phagocytosis of red blood cells by splenic macrophages [2], and T cell activation. Although the roles of the CD47-SHPS-1 system has been presented, little is known about the cell surface organization of these ligand/receptor complexes and the structural basis for signal transduction. To gain new insights into the physiological and biological roles of the CD47-SHPS-1 system, we determined the crystal structure of the SHPS-1 extracellular domain. The domain adopts a classical immunoglobulin (Ig) fold that was observed to form an antiparallel dimer. A dimeric form of SHPS-1 was observed in vivo, and our structural and biophysical data shows that the extracellular domain of SHPS-1 is dimeric in solution, compatible with the view of SHPS-1 acting as a cis-dimeric adhesion receptor. Previous investigations showed native CD47 formed cis-dimers. These features suggest that both CD47 and SHPS-1 trans-interact each other by the formation of cis-dimers and offer perceptions into interactions of related Ig superfamily receptors.
Frontiers in Microbiology, Oct 22, 2020
Fish & Shellfish Immunology
OENO One
Issatchenkia terricola 0621 is a non-Saccharomyces yeast strain isolated from Tannat grapes from ... more Issatchenkia terricola 0621 is a non-Saccharomyces yeast strain isolated from Tannat grapes from Uruguayan vineyards; it stands out for its ability to produce high levels of β-glucosidase activity, which contributes to the aromatic complexity of wines. To delve into the potential oenological applications of this strain, its high-quality genome was obtained and explored, allowing the main central carbon and nitrogen metabolic pathways to be reconstructed. I. terricola is able to utilise glycerol as the sole carbon source in a way that has not previously been described for yeasts. The genes of the fermentome and those involved in stress resistance during winemaking were also identified, and differences were found when compared to S. cerevisiae, which may explain why I. terricola is unable to complete fermentation. The pathways responsible for natural aroma synthesis were also reconstructed, and the production of aromatic acids, alcohols, esters, acetates and lactones was verified expe...
Fish & Shellfish Immunology, 2021
Sturgeons are chondrostean fish critically endangered due to anthropogenic loss and degradation o... more Sturgeons are chondrostean fish critically endangered due to anthropogenic loss and degradation of natural habitat and overfishing for meat and caviar production. Consequently, sturgeon aquaculture has extensively developed lately, being Russian sturgeon (Acipenser gueldenstaedtii) the second most important species reared for caviar production. However, Russian sturgeon aquaculture in subtropical countries, such as Uruguay, confronts difficulties because fish have to endure excessive summertime warm temperatures, which weaken their innate defences facilitating opportunistic infections. To address this problem, we look for identifying putative acute phase proteins (APPs), which might be robust serum biomarkers of both infection and chronic thermal stress, applied to monitoring Russian sturgeon health status in farms. We focused on the C-Reactive Protein/Serum Amyloid P (CRP/SAP) pentraxin since the pentraxin family includes well-known APPs, better characterised in mammals than fish. We identified A. gueldenstaedtii CRP/SAP (AgCRP/SAP), as a member of the universal CRP/SAP pentraxin sub-family, and studied AgCRP/SAP involvement in sturgeon response to bacterial challenge and chronic thermal stress, in comparison with A. gueldenstaedtii Serum Amyloid A (AgSAA), a previously described positive APP. Results showed that AgCRP/SAP is a constitutive serum component that remained constant upon Aeromonas hydrophila challenge and chronic thermal stress. Contrastingly, serum AgSAA was subjected to regulation by bacterial and thermal stress challenges, showing a 50-fold increase and 3-fold decline in serum levels, respectively. Overall, results highlight the potential value of AgSAA, but not of AgCRP/SAP, as a biomarker of bacterial infection and the need to continue searching for robust chronic thermal stress biomarkers in sturgeons.
Scientific Reports, 2020
The immune system of sturgeons, one of the most ancient and economically valuable fish worldwide,... more The immune system of sturgeons, one of the most ancient and economically valuable fish worldwide, is poorly understood. The lack of molecular tools and data about infection biomarkers hinders the possibility to monitor sturgeon health during farming and detect infection outbreaks. To tackle this issue, we mined publicly available transcriptomic datasets and identified putative positive acute-phase proteins (APPs) of Russian sturgeons that could be induced by a bacterial infection and monitored using non-invasive methods. Teleost literature compelled us to focus on five promising candidates: hepcidin, a warm acclimation associated hemopexin, intelectin, serum amyloid A protein (SAA) and serotransferrin. Among them, SAA was the most upregulated protein at the mRNA level in the liver of sturgeons challenged with heat-inactivated or liveAeromonas hydrophila. To assess whether this upregulation yielded increasing SAA levels in circulation, we developed an in-house ELISA to quantify SAA l...
Frontiers in Microbiology, 2020
We describe our experience with the Muithu sports notational analysis system, a novel digital inf... more We describe our experience with the Muithu sports notational analysis system, a novel digital information system in the popular sports domain. The system integrates real-time coach notations with related video sequences, and is configured with small, off-the shelf and cheap components. Muithu requires little or no human post-processing, which is in strong contrast to stateof-the art resource-intensive competing systems. Muithu also provides a novel social network for athletes and their coaches for information management and interactive e-learning experiences based on video footage. This next generation digital information system is already in operational use by a Norwegian elite soccer club, both for training and game events.
Fish & Shellfish Immunology, 2019
of Epinephelus coioides in metamorphosis stages revealed that dissimilarity in distribution of th... more of Epinephelus coioides in metamorphosis stages revealed that dissimilarity in distribution of the individuals in 12dph, 18dph and 50dph. Analysis of immune gene (TLR-3, IL-1b, IFN-a, IFN-g, NF-kB and Mx gene) showed the fish were in pathogen-free environment. The Proteobacteria in intestine microbiome is most abundant microbes at each development stages. In the other hand, NNV-, GIV-, and Vibrio-infected fish exhibit different phylum composition of microbes. The relationship of pathogen and microbiome in grouper will be investigated in the future, which might be the solution for the grouper disease control.
Journal of Molecular Biology, 2017
Fish & shellfish immunology, Jan 22, 2017
Russian sturgeon (Acipenser gueldenstaedtii) has been successfully farmed in Uruguay for the past... more Russian sturgeon (Acipenser gueldenstaedtii) has been successfully farmed in Uruguay for the past ten years. However, during the Uruguayan summer fish endure high water temperatures and increased bacterial infections that threaten aquaculture. Our understanding of sturgeon's immune system and its interplay with environmental factors like temperature is almost unknown. This study analysed the way in which seasonal variations affect enzymatic blood components of Russian sturgeon's innate defences, including the serum alternative complement pathway (ACP), ceruloplasmin (Cp) and lysozyme activities. Results showed that summertime conditions in the farm altered these defences in different ways, inducing a significant decrease in ACP and Cp, and an increase in lysozyme. In addition, serum levels of total protein and cortisol decreased in summer, suggesting a chronic stress response was induced in parallel. Subsequently, we analysed whether the increase in water river temperature d...
Fish & Shellfish Immunology, 2016
Sturgeons (family Acipenseridae) are fish species of high ecological and economic value. Unfortun... more Sturgeons (family Acipenseridae) are fish species of high ecological and economic value. Unfortunately, sturgeon species are highly endangered; overfishing and pollution have led to a drastic decline of natural reserves. Aquaculture is of decisive importance for the conservation and restoration of sturgeon populations, representing also an activity with valuable socioeconomic impact. Acipenser gueldensteadtii (Russian sturgeon) is one of the most cultured sturgeon specie worldwide. In Uruguay, sturgeon farming has been successful, but a high susceptibility to bacterial infection has been observed during summer. We studied the health status of Russian sturgeons, cultured during winter and summer seasons in an Uruguayan farm, by analyzing the activity of some innate immune components, including serum ceruloplasmin (Cp), the alternative complement pathway (ACP) and lysozyme. Adult females exhibited a low serum level of Cp and ACP, and a slight increase of lysozyme level during summer. Since Cp and ACP activities depend strongly on liver synthesis, we explored whether their alterations were linked to a chronic stress response. Serum protein levels were significantly lower in adult sturgeons during summer, but no significant differences were observed in cortisol. Therefore, a chronic stress response might have altered liver metabolism, interfering with the synthesis of constitutive proteins, including Cp and ACP components. Examination of the contribution of temperature changes to the observed effects was carried out by analyzing the same indicators in young sturgeons cultured at 18⁰C or 24⁰C during 37 days. In addition, the effect of oral administration of a nutritional supplement containing Se, Zn and a yeast-derived immunostimulator (Biopack, Alltech) was analyzed. Exposure to 24⁰C caused a high mortality and a significant decrease in fish growth and serum protein levels, but not changes in cortisol. Cp and ACP activities were significantly diminished at 24⁰C. Results resemble those obtained for sturgeon reared in summer. Biopack administration enhanced fish growth, Cp, and ACP activities in fishes cultured at 18⁰C, although failed to reverse the detrimental effects observed at 24⁰C. In contrast, Biopack seems to revert the modest increase in lysozyme activity found at 24⁰C. Overall, the higher susceptibility of sturgeons to bacterial infections may be caused by the usual temperatures reported during the Uruguayan summer, which would lead to decrease innate defenses as Cp and ACP. Biopack supplementation may benefit sturgeon health status, but further studies are needed to determine if it could prevent defense deficiencies provoked by high temperatures.
Biotechnol Technique, 1998
European Journal of Biochemistry, 2003
Cells that have lost the ability to grow in culture could be defined operationally as either aliv... more Cells that have lost the ability to grow in culture could be defined operationally as either alive or dead depending on the method used to determine cell viability. As a consequence, the interpretation of the state of ‘nonculturable’ cells is often ambiguous. Escherichia coli K12 cells inactivated by UV‐irradiation with a low (UV1) and a high (UV2) dose were used as a model of nonculturable cells. Cells inactivated by the UV1 dose lost ‘culturability’ but they were not lysed and maintained the capacity to respond to nutrient addition by protein synthesis and cell wall synthesis. The cells also retained both a high level of glucose transport and the capacity for metabolizing glucose. Moreover, during glucose incorporation, UV1‐treated cells showed the capacity to respond to aeration conditions modifying their metabolic flux through the Embden–Meyerhof and pentose‐phosphate pathways. However, nonculturable cells obtained by irradiation with the high UV2 dose showed several levels of m...
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Papers by Andrea Villarino