Contemporary approaches to site-selective protein modification

Proteins constitute the majority of nature’s worker biomolecules. Designed for specific functions, complex tertiary structures make proteins ideal candidates for analyzing natural systems and creating novel biological tools. Due to both large size and the need for proper folding, de novo synthesis of proteins has been quite a challenge, leading scientists to focus on modifying protein templates already provided by nature. Recently developed methods for protein modification fall into two broad categories: those that can modify the natural protein template directly and those that require genetic manipulation of the amino acid sequence prior to modification. The goal of this review is to provide not only a window through which to view the many opportunities created by novel protein modification techniques, but also to act as an initial guide to help scientists find direction and form ideas in an ever-growing field. In addition to the highlighting methods reported in the past five years, we aim to provide a broader sense of the goals and outcomes of protein modification and bioconjugation in general. While the main body of the paper comprises reactions directly involving proteins as a starting material, some further functionalization strategies as well as biological applications are also acknowledged. The discussion concludes by speculating what trends and discoveries will most likely come next in the field

Occurrence of deformed wing virus variants in the stingless Melipona subnitida and honey Apis mellifera bee populations in North Eastern Brazil

Deformed wing virus (DWV) is now a global insect pathogen. Brazilian stingless bees are a diverse group often managed in close proximity to honey bees. We investigated the prevalence and load of DWV in 33 stingless bees (Melipona subnitida) and 12 honey bees (Apis mellifera) colonies from NE Brazil. DWV was detected in all colonies with the A and C-variants dominating M. subnitida and A-variant in A. mellifera. Viral loads were 8.83E+07 and 7.19E+07 in M. subnitida and A. mellifera, respectively. On Fernando de Noronha island DWV is low (<1E+03) in honey bees, but we detected high loads (1.6E+08) in nine island M. subnitida colonies, indicating no viral spill-over of DWV has occurred during the past 34 years. Furthermore, the ubiquitous presence of the DWV-C variant in M. subnitida colonies, and rarity in A. mellifera, may suggest limited viral exchange between these two species

Caking kinetics of freeze-dried papaya determined by optica microscopy

O objetivo do presente trabalho foi o de estudar a cinética-de-caking em mamão (Carica papaya L.) liofilizado tomando por base seu condicionamento em diversos ambientes de umidade relativa (31, 52 e 89%) e temperatura (10 e 25 °C). A cinética-de-caking é estabelecida em fotomicrografias obtidas em microscópio ótico, a diversos intervalos de tempo. Os resultados mostra-ram a influência da temperatura e umidade relativa no grau de caking e a possibilidade da utilização de um método simples para sua análise em pós de alimentos liofilizados.The caking kinetics of freeze-dried papaya (Carica papaya L.) was estimated based on the environmental conditions of relative humidity (31, 52 and 89%) and of temperature (10 and 25 °C). The caking kinetics was established from photomicrographics obtained by an optical microscope used at different time intervals. The results showed the influence of temperature and relative humidity on the degree of caking and the possibility of utilizing a simple method for analysis of caking on freeze-dried fruit powders

Stoichiometric and irreversible cysteine-selective protein modification using carbonylacrylic reagents

Maleimides remain the reagents of choice for the preparation of therapeutic and imaging protein conjugates despite the known instability of the resulting products that undergo thiol-exchange reactions $\textit{in vivo}$. Here we present the rational design of carbonylacrylic reagents for chemoselective cysteine bioconjugation. These reagents undergo rapid thiol Michael-addition under biocompatible conditions in stoichiometric amounts. When using carbonylacrylic reagents equipped with PEG or fluorophore moieties, this method enables access to protein and antibody conjugates precisely modified at pre-determined sites. Importantly, the conjugates formed are resistant to degradation in plasma and are biologically functional, as demonstrated by the selective imaging and detection of apoptotic and HER2+ cells, respectively. The straightforward preparation, stoichiometric use and exquisite cysteine selectivity of the carbonylacrylic reagents combined with the stability of the products and the availability of biologically relevant cysteine-tagged proteins make this method suitable for the routine preparation of chemically defined conjugates for $\textit{in vivo}$ applications.FAPESP (Grant IDs: 2012/22274-2; BEPE 2015/07509-1, 2013/25504-1), Xunta de Galicia, FCT Portugal (FCT Investigator, SFRH/BPD/103172/2014 Postdoctoral fellowship, SFRH/BD/111556/2015 PhD Studentship), European Union (Marie-Sklodowska Curie ITN Protein Conjugates), Engineering and Physical Sciences Research Council, MECD (‘Salvador Madariaga’ mobility grant PRX15/00638), MINECO (CTQ2015-70524-R, RYC-2013-14706 ), Royal Society, European Research Council Starting Grant (TagIt

Discrete cutaneous lesions in a critically ill patient treated only for AIDS and miliary tuberculosis: a case report of disseminated histoplasmosis

Histoplasmosis is a systemic mycosis caused by the dimorphic fungus Histoplasma capsulatum, with disseminated histoplasmosis (HD) being one of its clinical forms. As a consequence of the HIV-AIDS pandemic, HD has become prevalent not only in regions that are recognized as endemic but also in areas not considered endemic, such as Europe and Asia. Its clinical manifestations are varied and mimic several infectious diseases, mainly tuberculosis. In endemic areas, it is the first manifestation of AIDS in 50 to 70% of patients. The diagnosis of histoplasmosis is difficult and HD can lead to death if not diagnosed early and if proper treatment is not instituted. The present report presents a patient with a recent diagnosis of HIV-AIDS, in treatment for miliary tuberculosis, who was diagnosed with disseminated histoplasmosis because of his dermatological manifestations

Combination with tomatidine improves the potency of posaconazole against Trypanosoma cruzi

Azoles such as posaconazole (Posa) are highly potent against Trypanosoma cruzi. However, when tested in chronic Chagas disease patients, a high rate of relapse after Posa treatment was observed. It appears that inhibition of T. cruzi cytochrome CYP51, the target of azoles, does not deliver sterile cure in monotherapy. Looking for suitable combination partners of azoles, we have selected a set of inhibitors of sterol and sphingolipid biosynthetic enzymes. A small-scale phenotypic screening was conducted in vitro against the proliferative forms of T. cruzi, extracellular epimastigotes and intracellular amastigotes. Against the intracellular, clinically relevant forms, four out of 15 tested compounds presented higher or equal activity as benznidazole (Bz), with EC50 values </=2.2 muM. Ro48-8071, an inhibitor of lanosterol synthase (ERG7), and the steroidal alkaloid tomatidine (TH), an inhibitor of C-24 sterol methyltransferase (ERG6), exhibited the highest potency and selectivity indices (SI = 12 and 115, respectively). Both were directed to combinatory assays using fixed-ratio protocols with Posa, Bz, and fexinidazole. The combination of TH with Posa displayed a synergistic profile against amastigotes, with a mean SigmaFICI value of 0.2. In vivo assays using an acute mouse model of T. cruzi infection demonstrated lack of antiparasitic activity of TH alone in doses ranging from 0.5 to 5 mg/kg. As observed in vitro, the best combo proportion in vivo was the ratio 3 TH:1 Posa. The combination of Posa at 1.25 mpk plus TH at 3.75 mpk displayed suppression of peak parasitemia of 80% and a survival rate of 60% in the acute infection model, as compared to 20% survival for Posa at 1.25 mpk alone and 40% for Posa at 10 mpk alone. These initial results indicate a potential for the combination of posaconazole with tomatidine against T. cruzi

Conversação e deliberação sobre questões sensíveis: um estudo sobre o uso das razões que circulam nos media

A discussão informal tem despertado crescente interesse em estudos sobre deliberação. Contudo, são raros os estudos que examinam o engajamento das pessoas em contextos conflitantes. Este artigo investiga o modo pelo qual sujeitos de grupos sociais em conflito fazem uso de razões concorrentes que circulam nos media para negociar suas opiniões sobre uma “questão sensível”: a redução da maioridade penal. Para tanto, foram realizados cinco grupos de discussão em Belém (PA) e Belo Horizonte (MG), com policiais militares e moradores e adolescentes de periferia. A partir da adaptação do Discourse Quality Index, este estudo analisa: as justificativas expressas pelos participantes; o respeito entre os grupos; o papel das histórias pessoais na discussão. Investiga-se, ainda, o fluxo de argumentos antes e após a apresentação do material dos media. Os resultados contribuem para compreender a dinâmica através da qual os sujeitos se situam num “espaço de razões” em debates plurais