Site-specific C-terminal and internal loop labeling of proteins using sortase-mediated reactions

Methods for site-specific modification of proteins should be quantitative and versatile with respect to the nature and size of the biological or chemical targets involved. They should require minimal modification of the target, and the underlying reactions should be completed in a reasonable amount of time under physiological conditions. Sortase-mediated transpeptidation reactions meet these criteria and are compatible with other labeling methods. Here we describe the expression and purification conditions for two sortase A enzymes that have different recognition sequences. We also provide a protocol that allows the functionalization of any given protein at its C terminus, or, for select proteins, at an internal site. The target protein is engineered with a sortase-recognition motif (LPXTG) at the place where modification is desired. Upon recognition, sortase cleaves the protein between the threonine and glycine residues, facilitating the attachment of an exogenously added oligoglycine peptide modified with the functional group of choice (e.g., fluorophore, biotin, protein or lipid). Expression and purification of sortase takes ∼3 d, and sortase-mediated reactions take only a few minutes, but reaction times can be extended to increase yields.National Institutes of Health (U.S.) (Grant RO1 AI08787

Site-Specific Chemoenzymatic Labeling of Aerolysin Enables the Identification of New Aerolysin Receptors

Aerolysin is a secreted bacterial toxin that perforates the plasma membrane of a target cell with lethal consequences. Previously explored native and epitope-tagged forms of the toxin do not allow site-specific modification of the mature toxin with a probe of choice. We explore sortase-mediated transpeptidation reactions (sortagging) to install fluorophores and biotin at three distinct sites in aerolysin, without impairing binding of the toxin to the cell membrane and with minimal impact on toxicity. Using a version of aerolysin labeled with different fluorophores at two distinct sites we followed the fate of the C-terminal peptide independently from the N-terminal part of the toxin, and show its loss in the course of intoxication. Making use of the biotinylated version of aerolysin, we identify mesothelin, urokinase plasminogen activator surface receptor (uPAR, CD87), glypican-1, and CD59 glycoprotein as aerolysin receptors, all predicted or known to be modified with a glycosylphosphatidylinositol anchor. The sortase-mediated reactions reported here can be readily extended to other pore forming proteins.National Institutes of Health (U.S.) (grant R01 AI087879

Fatores sociodemográficos e clínicos associados ao tempo de decisão para a procura de atendimento no infarto agudo do miocárdio

OBJETIVO: analizar la interacción del género en la asociación entre el tiempo de decisión para la búsqueda de servicio de salud y las variables sociodemográficas y clínicas. MÉTODO: estudio exploratorio, transversal, con 100 individuos entrevistados en hospitales de Salvador-BA. En el análisis se empleó el test Chi-cuadrado o Exacto de Fisher y el modelo de regresión linear robusto. La significancia estadística adoptada fue de 5%. RESULTADOS: hombres y mujeres presentaron tiempos de decisión elevados. Hubo menor tiempo de decisión para tabaquistas, con dolor constante y de fuerte intensidad. Hubo interacción entre género y tabaquismo y entre género y dolor irradiado para el cuello o mandíbula para el desenlace del tiempo de decisión. CONCLUSIÓN: los tiempos de decisión fueron elevados y sufrieron influencia de variables clínicas y de género. El estudio ofrece subsidios para prácticas de cuidar en enfermería enfocadas en la especificidad de esos factores y de los géneros objetivando obtener éxito en la reducción del tiempo de decisión.OBJETIVO: analisar a interação do sexo na associação entre o tempo de decisão para a procura de serviço de saúde e as variáveis sociodemográficas e clínicas. MÉTODO: estudo exploratório, transversal, com 100 indivíduos entrevistados em hospitais de Salvador, BA. Na análise, empregou-se o teste qui-quadrado ou exato de Fisher e o modelo de regressão linear robusto. A significância estatística adotada foi de 5%. RESULTADOS: Resultados: homens e mulheres apresentaram tempos de decisão elevados. Houve menor tempo de decisão para tabagistas, com dor constante e de forte intensidade. Houve interação entre sexo e tabagismo e entre sexo e dor irradiada para o pescoço ou a mandíbula para o desfecho tempo de decisão. CONCLUSÃO: os tempos de decisão foram elevados e sofreram influência de variáveis clínicas e do sexo. O estudo oferece subsídios para práticas de cuidar em enfermagem, focalizadas na especificidade desses fatores e dos gêneros, visando-se obter êxito na redução do tempo de decisão.OBJECTIVE: this study aimed to analyze the interaction of gender in the association between decision time for seeking healthcare services and the sociodemographic and clinical variables. METHOD: this exploratory, cross-sectional study was performed with 100 individuals interviewed in hospitals in Salvador, Bahia. The chi-square test or Fisher's exact test and the robust linear regression model were used in the analysis. A statistical significance of 5% was adopted. RESULTS: men and women presented long decision times. The decision time was less for smokers and those with constant and/or severe pain. There was an interaction between gender and smoking and between gender and pain radiating to the neck or jaw for the decision time outcome. CONCLUSION: decision times were long and were influenced by clinical and gender variables. The study provides support for nursing care practices focused on the specificity of these factors and of the genders aiming to reduce the decision time

Neoadjuvant multikinase inhibitor in patients with locally advanced unresectable thyroid carcinoma

Background: Papillary thyroid carcinoma (PTC) is the most common and less aggressive thyroid cancer, but some patients may display locally advanced disease. Therapeutic options are limited in these cases, particularly for those patients with unresectable tumors. Neoadjuvant therapy is not part of the recommended work up. Methods: Report a case of an unresectable grossly locally invasive PTC successfully managed with neoadjuvant therapy and provide a systematic review (SR) using the terms “Neoadjuvant therapy” AND “Thyroid carcinoma.” Results: A 32-year-old man with a 7.8 cm (in the largest dimension) PTC was referred to total thyroidectomy, but tumor resection was not feasible due to extensive local invasion (trachea, esophagus, and adjacent structures). Sorafenib, a multikinase inhibitor (MKI), was initiated; a 70% tumor reduction was observed after 6 months, allowing new surgical intervention and complete resection. Radioactive iodine (RAI) was administered as adjuvant therapy, and whole body scan (WBS) shows uptake on thyroid bed. One-year post-surgery the patient is asymptomatic with a status of disease defined as an incomplete biochemical response. The SR retrieved 123 studies on neoadjuvant therapy use in thyroid carcinoma; of them, 6 were extracted: 4 case reports and 2 observational studies. MKIs were used as neoadjuvant therapy in three clinical cases with 70–84% of tumor reduction allowing surgery. Conclusion: Our findings, along with other reports, suggest that MKIs is an effective neoadjuvant therapy and should be considered as a therapeutic strategy for unresectable grossly locally invasive thyroid carcinomas

Fish Oil Supplementation Reduces Heart Levels of Interleukin-6 in Rats with Chronic Inflammation due to Epilepsy

Sudden unexpected death in epilepsy (SUDEP) is a major cause of premature death related to epilepsy. The causes of SUDEP remain unknown, but cardiac arrhythmias and asphyxia have been suggested as a major mechanism of this event. Inflammation has been implicated in the pathogenesis of both epilepsy and ventricular arrhythmia, with interleukin-6 (IL-6) being recognized as a crucial orchestrator of inflammatory states. Our group previously reported that levels of IL-6 were increased in the hearts of epileptic rats. In this scenario, anti-inflammatory actions are among the beneficial effects of fish oil dietary supplementation. This investigation revealed that elevated levels of IL-6 in the heart were markedly reduced in epileptic rats that were treated in the long-term with fish oil, suggesting protective anti-inflammatory actions against dangerously high levels of IL-6. Based on these findings, our results suggest beneficial effects of long-term intake of fish oil in reducing the inflammation associated with chronic epilepsy.FAPESPCAPESCNPqFAPESP/CNPq/MCT-Instituto Nacional de Neurociencia TranslacionalFAPEMIGCEPID/FAPESPUniv Fed Sao Paulo, Escola Paulista Med, Disciplina Neurociencia, UNIFESP, Sao Paulo, BrazilUniv Fed Sao Paulo, Escola Paulista Med, Dept Fisiol, UNIFESP, Sao Paulo, BrazilUniv Fed Sao Joao Del Rei UFSJ, Dept Engn Biossistemas, Sao Joao Del Rei, BrazilUniv Presbiteriana Mackenzie, Programa Posgrad Disturbios Desenvolvimento, Ctr Ciencias Biol & Saude, Sao Paulo, BrazilUniv Fed Sao Paulo, Escola Paulista Med, Disciplina Neurociencia, UNIFESP, Sao Paulo, BrazilUniv Fed Sao Paulo, Escola Paulista Med, Dept Fisiol, UNIFESP, Sao Paulo, BrazilFAPESPCAPESCNPqFAPESP/CNPq/MCT-Instituto Nacional de Neurociencia TranslacionalFAPEMIGCEPID/FAPESPWeb of Scienc

Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation

While the catalog of mammalian transcripts and their expression levels in different cell types and disease states is rapidly expanding, our understanding of transcript function lags behind. We present a robust technology enabling systematic investigation of the cellular consequences of repressing or inducing individual transcripts. We identify rules for specific targeting of transcriptional repressors (CRISPRi), typically achieving 90%–99% knockdown with minimal off-target effects, and activators (CRISPRa) to endogenous genes via endonuclease-deficient Cas9. Together they enable modulation of gene expression over a ∼1,000-fold range. Using these rules, we construct genome-scale CRISPRi and CRISPRa libraries, each of which we validate with two pooled screens. Growth-based screens identify essential genes, tumor suppressors, and regulators of differentiation. Screens for sensitivity to a cholera-diphtheria toxin provide broad insights into the mechanisms of pathogen entry, retrotranslocation and toxicity. Our results establish CRISPRi and CRISPRa as powerful tools that provide rich and complementary information for mapping complex pathways

A Survey on Information Visualization for Network and Service Management

Network and service management encompasses a set of activities, methods, procedures, and tools whose ultimate goal is to guarantee the proper functioning of a networked system. Computational tools are essential to help network administrators in their daily tasks, and information visualization techniques are of great value in such context. In essence, information visualization techniques associated to visual analytics aim at facilitating the tasks of network administrators in the process of monitoring and maintaining the network health. This paper surveys the use of information visualization techniques as a tool to support the network and service management process. Through a Systematic Literature Review (SLR), we provide a historical overview and discuss the current state of the art in the field. We present a classification of 285 articles and papers from 1985 to 2013, according to an information visualization taxonomy as well as a network and service management taxonomy. Finally, we point out future research directions and opportunities regarding the use of information visualization in network and service management

Site-specific protein modification using immobilized sortase in batch and continuous-flow systems

Transpeptidation catalyzed by ​sortase A allows the preparation of proteins that are site-specifically and homogeneously modified with a wide variety of functional groups, such as fluorophores, PEG moieties, lipids, glycans, bio-orthogonal reactive groups and affinity handles. This protocol describes immobilization of ​sortase A on a solid support (Sepharose beads). Immobilization of ​sortase A simplifies downstream purification of a protein of interest after labeling of its N or C terminus. Smaller batch and larger-scale continuous-flow reactions require only a limited amount of enzyme. The immobilized enzyme can be reused for multiple cycles of protein modification reactions. The described protocol also works with a Ca²⁺-independent variant of ​sortase A with increased catalytic activity. This heptamutant variant of ​sortase A (7M) was generated by combining previously published mutations, and this immobilized enzyme can be used for the modification of calcium-senstive substrates or in instances in which low temperatures are needed. Preparation of immobilized ​sortase A takes 1–2 d. Batch reactions take 3–12 h and flow reactions proceed at 0.5 ml h⁻¹, depending on the geometry of the reactor used.United States. National Institutes of Health (RO1 AI087879