Assessment of Notch1 ligands production - key protein targets in breast cancer

Cell-to-cell communication is required for many biological processes in development and adult life. One of the most common systems utilized by a wide range of eukaryotes is the Notch signalling pathway. Four Notch receptors and five ligands have been identified in mammals that interact via their extracellular domains leading to transcription activation. Studies have shown that the Notch ligands expression is undetectable in normal breast tissues, but moderate to high expression has been detected in breast cancer. Thus, any of the Notch1 ligands can be studied as possible therapeutic targets for breast cancer. To study Notch pathway proteins there is the need to obtain stable protein solutions. E. coli is the host of excellence for recombinant proteins for the ease of use, fast growth and high cell densities. However, the expression of mammalian proteins in such systems may overwhelm the bacterial cellular machinery, which does not possess the ability for post-translational modifications, or dedicated compartments for protein synthesis. Mammalian cells are therefore preferred, despite their technical and financial increased demands. We aim to determine the best expression and purification conditions for the different ligand protein constructs, to develop specific function-blocking antibodies using the Phage Display technology. Moreover, we propose to crystallize the Notch1 ligands alone and in complex with the phage display selected antibodies, unveiling molecular details. hJag2DE3 and hDll1DE6 proteins were purified from refolded inclusion bodies or mammalian cell culture supernatants, respectively, and purity was confirmed by SDS-PAGE (>95%). Protein produced in mammalian cells showed to be more stable, apparently with the physiological disulfide pattern, contrary to what was observed in the refolded protein. Several nano-scale crystallization experiments were set up in 96-well plates, but no positive result was obtained. We will continue to pursue for the best expression for the Notch ligand constructs in both expression systems.

Learning Activities with Semantic. Hypermedia in Higher Education

The increasing use of information and communication technologies (ICT) in diverse professional and personal contexts calls for new knowledge, and a set of abilities, competences and attitudes, for an active and participative citizenship. In this context it is acknowledged that universities have an important role innovating in the educational use of digital media to promote an inclusive digital literacy. The educational potential of digital technologies and resources has been recognized by both researchers and practitioners. Multiple pedagogical models and research approaches have already contributed to put in evidence the importance of adapting instructional and learning practices and processes to concrete contexts and educational goals. Still, academic and scientific communities believe further investments in ICT research is needed in higher education. This study focuses on educational models that may contribute to support digital technology uses, where these can have cognitive and educational relevance when compared to analogical technologies. A teaching and learning model, centered in the active role of the students in the exploration, production, presentation and discussion of interactive multimedia materials, was developed and applied using the internet and exploring emergent semantic hypermedia formats. The research approach focused on the definition of design principles for developing class activities that were applied in three different iterations in undergraduate courses from two institutions, namely the University of Texas at Austin, USA and the University of Lisbon, Portugal. The analysis of this study made possible to evaluate the potential and efficacy of the model proposed and the authoring tool chosen in the support of metacognitive skills and attitudes related to information structuring and management, storytelling and communication, using computers and the internet.

Lagochilascaris minor: antibody production in experimentally infected mice

Lagochilascaris minor is the causative agent of lagochilascariosis, a disease that affects the neck region and causes festering abscesses, with eggs, adult parasites and L3/L4 larvae within the purulent exudates. Today, mice are considered to be intermediate hosts for the parasite. C57BL/6 mice produce immunoglobulin IgM, IgA and IgG against the crude extract of the parasite; on the other hand, antibodies produced against the secreted/excreted antigens of Lagochilascaris minor present lower levels of IgM, IgA and IgG. This is the first description of antibody detection against different antigens of Lagochilascaris minor.

Production of anti-Cryptosporidium polyclonal antibodies and standardization of direct immunofluorescence for detecting oocysts in water

INTRODUCTION: The production of anti-Cryptosporidium polyclonal antibodies and its use in direct immunofluorescence assays to determine the presence of Cryptosporidium in water are described in the present work. METHODS: Two rabbits were immunized with soluble and particulate antigens from purified Cryptosporidium oocysts. The sera produced were prepared for immunoglobulin G extraction, which were then purified and conjugated with fluorescein isothiocyanate (FITC). Slides containing known amounts of oocysts were prepared to determine the sensitivity of the technique. To test the specificity, slides containing Giardia duodenalis cysts were prepared. RESULTS: The conjugate was successfully used in water samples experimentally contaminated with Cryptosporidium oocysts, and it was possible to detect up to five oocysts/spot, corresponding to contamination of 250 oocysts/mL. CONCLUSIONS: The three immunizations performed in the rabbits were enough to produce antibodies against Cryptosporidium, the standard direct immunofluorescence assay permitted the detection of five oocysts in 20% of the samples, and no cross-reaction with Giardia duodenalis cysts occurred.

Different culture media containing methyldopa for melanin production by Cryptococcus species

INTRODUCTION: Melanin production by species of Cryptococcus is widely used to characterize C. neoformans complex in mycology laboratories. This study aims to test the efficacy of methyldopa from pharmaceutical tablet as a substrate for melanin production, to compare the production of melanin using different agar base added with methyldopa, and to compare the melanin produced in those media with that produced in Niger seed agar and sunflower seed agar by C. neoformans, C. laurentii, and C. albidus. Two isolates of each species, C. neoformans, C. laurentii, and C. albidus, and one of Candida albicans were used to experimentally detect conditions for melanin production. METHODS: The following media were tested: Mueller-Hinton agar (MHA), brain and heart infusion agar (BHIA), blood agar base (BAB), and minimal medium agar (MMA), all added with methyldopa, and the media Niger seed agar (NSA) and sunflower seed agar (SSA). RESULTS: All isolates grew in most of the culture media after 24h. Strains planted on media BAB and BHIA showed growth only after 48h. All isolates produced melanin in MMA, MHA, SSA, and NSA media. CONCLUSIONS: Methyldopa in the form pharmaceutical tablet can be used as a substrate for melanin production by Cryptococcus species; minimal medium plus methyldopa was more efficient than the BAB, MHA, and BHIA in the melanin production; and NSA and SSA, followed by MMA added with methyldopa, were more efficient than other media studied for melanin production by all strains studied.

Production of cytokine and chemokines by human mononuclear cells and whole blood cells after infection with Trypanosoma cruzi

INTRODUCTION: The innate immune response is the first mechanism of protection against Trypanosoma cruzi, and the interaction of inflammatory cells with parasite molecules may activate this response and modulate the adaptive immune system. This study aimed to analyze the levels of cytokines and chemokines synthesized by the whole blood cells (WBC) and peripheral blood mononuclear cells (PBMC) of individuals seronegative for Chagas disease after interaction with live T. cruzi trypomastigotes. METHODS: IL-12, IL-10, TNF-α, TGF-β, CCL-5, CCL-2, CCL-3, and CXCL-9 were measured by ELISA. Nitrite was determined by the Griess method. RESULTS: IL-10 was produced at high levels by WBC compared with PBMC, even after incubation with live trypomastigotes. Production of TNF-α by both PBMC and WBC was significantly higher after stimulation with trypomastigotes. Only PBMC produced significantly higher levels of IL-12 after parasite stimulation. Stimulation of cultures with trypomastigotes induced an increase of CXCL-9 levels produced by WBC. Nitrite levels produced by PBMC increased after the addition of parasites to the culture. CONCLUSIONS: Surface molecules of T. cruzi may induce the production of cytokines and chemokines by cells of the innate immune system through the activation of specific receptors not evaluated in this experiment. The ability to induce IL-12 and TNF-α contributes to shift the adaptive response towards a Th1 profile.

O contrato de Project Finance

Currently, Angola portrays a notorious economic growth and due to recent innovative legislations, it has become the major investment attracting pole, especially in Sub-Saharan Africa, having, thus, an extraordinary potentiality for a rapid and sustainable development, likely to place her in outstanding positions in the world economic ranking. Yet, such economic growth entails demanding levels of intensive investment in infrastructure, what has been reported of the Angolan Government to be unable to respond to, save if recurring to very high index of external debt, poisoning, in this way, the future budgeting of the country. Due to these infrastructure investment shortages, the cost of production remains highly onerous and the cost of life extremely unaffordable. On this account, the current study disserts about the contract of Project Finance; an alternative finance resource given as a viable solution for the private financing of infrastructure, aiming to demonstrate that such contractual figure, likewise the experience of several emerging economies and others, is a contract bid framework to take into account in today’s world. It refers to a financing technique – through which the Government may satisfy a common need (for example, the construction of a public domain or public servicing), without having to pay neither offer any collateral – based on a complex legal-financial engineering, arranged throughout a coalition of typical and atypical agreements, whereby it is mandatory to look back at the basic concepts of corporate law. More than just a simple financial study, the dissertation at stake analyses the nature and legal framework of Project Finance, which is a legally atypical and innominate contract, concluding that there is a relevant need for regulating and devoting a special legal regime in the Angolan jurisdiction for this promising legal form in the contemporary corporate finance world.

Establishment and evaluation of flexible insect cell lines for rapid production of recombinant proteins

Fundação para a Ciência e a Tecnologia (FCT) - (PTDC/EBB-EBI/102266/2008 and SFRH/BD/43830/2008, respectively) and by European Community’s FP7/2007-2013 (grant agreement nº 270089)

Production and diagnostic application of recombinant domain III of West Nile envelope protein in Brazil

INTRODUCTION: West Nile virus (WNV) is a flavivirus with a natural cycle involving mosquitoes and birds. Over the last 11 years, WNV has spread throughout the Americas with the imminent risk of its introduction in Brazil. METHODS: Envelope protein domain III of WNV (rDIII) was bacterially expressed and purified. An enzyme-linked immunosorbent assay with WNV rDIII antigen was standardized against mouse immune fluids (MIAFs) of different flavivirus. RESULTS: WNV rDIII reacted strongly with St. Louis encephalitis virus (SLEV) MIAF but not with other flaviviruses. CONCLUSIONS: This antigen may be a potentially useful tool for serologic diagnosis and may contribute in future epidemiological surveillance of WNV infections in Brazil.

Determination of germ tube, phospholipase, and proteinase production by bloodstream isolates of Candida albicans

Introduction Candida albicans is a commensal and opportunistic agent that causes infection in immunocompromised individuals. Several attributes contribute to the virulence and pathogenicity of this yeast, including the production of germ tubes (GTs) and extracellular hydrolytic enzymes, particularly phospholipase and proteinase. This study aimed to investigate GT production and phospholipase and proteinase activities in bloodstream isolates of C. albicans. Methods One hundred fifty-three C. albicans isolates were obtained from blood samples and analyzed for GT, phospholipase, and proteinase production. The assays were performed in duplicate in egg yolk medium containing bovine serum albumin and human serum. Results Detectable amounts of proteinase were produced by 97% of the isolates, and 78% of the isolates produced phospholipase. GTs were produced by 95% of the isolates. A majority of the isolates exhibited low levels of phospholipase production and high levels of proteinase production. Conclusions Bloodstream isolates of C. albicans produce virulence factors such as GT and hydrolytic enzymes that enable them to cause infection under favorable conditions.

Evolution of coauthorship networks: worldwide scientific production on leishmaniasis

Introduction Collaboration is one of the defining features of contemporary scientific research, and it is particularly important with regard to neglected diseases that primarily affect developing countries. Methods The present study has identified publications on leishmaniasis in the Medline database from 1945 to 2010, analyzing them according to bibliometric indicators and statistics from social network analysis. Examining aspects such as scientific production, diachronic evolution, and collaboration and configuration of the research groups in the field, we have considered the different types of Leishmania studied and the institutional affiliation and nationality of the authors. Results Seven-hundred and thirty-five authors participate in 154 prominent research clusters or groups. Although the most predominant and consolidated collaborations are characterized by members from the same country studying the same type of Leishmania, there are also notable links between authors from different countries or who study different clinical strains of the disease. Brazil took the lead in this research, with numerous Brazilian researchers heading different clusters in the center of the collaboration network. Investigators from the USA, India, and European countries, such as France, Spain, the United Kingdom, and Italy, also stand out within the network. Conclusions Research should be fostered in countries such as Bangladesh, Nepal, Sudan, and Ethiopia, where there is a high prevalence of different forms of the disease but limited research development with reference authors integrated into the collaboration networks.

Production of metallo-β-lactamase among Pseudomonas aeruginosa strains isolated in the State of Sergipe, Brazil

INTRODUCTION: Acquired production of metallo-β-lactamases is an important mechanism of resistance in Pseudomonas aeruginosa. The objective of this study was to investigate the production of metallo-β-lactamase and the genetic diversity among ceftazidime-resistant P. aeruginosa isolates from State of Sergipe, Brazil. METHODS: Metallo-β-lactamase was investigated using the disk approximation test and polymerase chain reaction (PCR). Genetic diversity was evaluated by pulsed-field gel electrophoresis (PFGE). RESULTS: A total of 48 (51.6%) isolates were resistant to ceftazidime. Six (12.2%) of these were positive for metallo-β-lactamase production. Only two (4.1%) of the ceftazidime-resistant isolates carried the bla SPM-1 gene. CONCLUSIONS: Production of metallo-β-lactamases was not the main mechanism of resistance to ceftazidime and carbapenems among P. aeruginosa strains in Sergipe, Brazil.

Impact of carbon/nitrogen feeding strategy on polyhydroxyalkanoates production using mixed microbial cultures

Polyhydroxyalkanoates (PHA) production using mixed microbial cultures (MMC) requires a multi-stage process involving the microbial selection of PHA-storing microorganisms, typically operated in sequencing batch reactors (SBR), and an accumulation reactor. Since low-cost renewable feedstocks used as process feedstock are often nitrogen-deficient, nutrient supply in the selection stage is required to allow for microbial growth. In this context, the possibility to uncouple nitrogen supply from carbon feeding within the SBR cycle has been investigated in this study. Moreover, three different COD:N ratios (100:3.79, 100:3.03 and 100:2.43) were tested in three different runs which also allowed the study of COD:N ratio on the SBR performance. For each run, a synthetic mixture of acetic and propionic acids at an overall organic load rate of 8.5 gCOD L-1 d-1 was used as carbon feedstock, whereas ammonium sulfate was the nitrogen source in a lab-scale sequence batch reactor (SBR) with 1 L of working volume. Besides, a sludge retention time (SRT) of 1 d was used as well as a 6 h cycle length. The uncoupled feeding strategy significantly enhanced the selective pressure towards PHA-storing microorganisms, resulting in a two-fold increase in the PHA production (up to about 1.3 gCOD L-1). A high storage response was observed for the two runs with the COD:N ratios (gCOD:gN) of 100:3.79 and 100:3.03, whereas the lowest investigated nitrogen load resulted in very poor performance in terms of polymer production. In fact, strong nitrogen limitation caused fungi to grow and a very poor storage ability by microorganisms that thrived in those conditions. The COD:N ratio also affected the polymer composition, indeed the produced poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) showed a variable HV content (1-20 %, w/w) among the three runs, lessening as the COD:N increased. This clearly suggests the possibility to use the COD:N ratio as a tool for tuning polymer properties regardless the composition of the feedstock.