Optimal concentration of organic solvents to be used in the broth microdilution method to determine the antimicrobial activity of natural products against Paenibacillus larvae

American Foulbrood (AFB) is a bacterial disease, caused by Paenibacillus larvae, that affects honeybees (Apis mellifera). Alternative strategies to control AFB are based on the treatment of the beehives with antimicrobial natural substances such as extracts, essential oils and/or pure compounds from plants, honey by-products, bacteria and moulds. The broth microdilution method is currently one of the most widely used methods to determine the minimum inhibitory concentration (MIC) of a substance. In this regard, the fact that most natural products, due to their lipophilic nature, must be dissolved in organic solvents or their aqueous mixtures is an issue of major concern because the organic solvent becomes part of the dilution in the incubation medium, and therefore, can interfere with bacterial viability depending on its nature and concentration. A systematic study was carried out to determine by the broth microdilution method the MIC and the maximum non inhibitory concentration (MNIC) against P. larvae of the most common organic solvents used to extract or dissolve natural products, i.e. ethanol, methanol, acetonitrile, n-butanol, dimethylsulfoxide, and acidified hydromethanolic solutions. From the MIC and MNIC for each organic solvent, recommended maximum concentrations in contact with P. larvae were established: DMSO 5% (v/v), acetonitrile 7.5% (v/v), ethanol 7.5% (v/v), methanol 12% (v/v), n-butanol 1% (v/v), and methanol-water-acetic acid (1.25:98.71:0.04, v/v/v).Fil: Cugnata, Noelia Melina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Departamento de Biología; ArgentinaFil: Guaspari, Elisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Departamento de Biología; ArgentinaFil: Pellegrini, María Celeste. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Departamento de Biología; ArgentinaFil: Fuselli, Sandra Rosa. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Departamento de Biología; ArgentinaFil: Alonso Salces, Rosa Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Departamento de Biología; Argentin

Inhibitory action of essential oils against proteases activity of Paenibacillus larvae, the etiological agent of American Foulbrood disease

American foulbrood (AFB) is a disease affecting the larva of Apis mellifera. The etiological agent is Paenibacillus larvae, which releases metalloproteases involved in the degradation of larval tissues. Through quorum sensing (QS) mechanism, bacteria are able to activate specific genes such as virulence factors. The exoproteases regulation of P. larvae could be associated with QS. A promising mechanism of AFB control is to block QS mechanism with essential oils (EO). The aim of this study was to investigate the potential presence of QS signals in the regulation of P. larvae proteases and the effect of seven EOs on the exoproteases activity of P. larvae. From growth curves and evaluation of the presence of proteases by milk agar plates assay, it was observed protease activity during the late exponential phase of growth. Early production of protease activity (15 hours earlier than control) was observed when a low density culture was incubated with late exponential spent medium (SM) suggesting the presence of factor(s) inducing this activity. SM was obtained by the ultrafiltration of P. larvae cultures on late growth phase and was free of proteases. Proteolytic activity was quantified on P. larvae cultures in presence of sublethal concentration of EO by azocasein method. The EOs, except S. chilensis EO, reduced significantly protease activity (more than 50%). We report for the first time evidence on the possible role of QS on P. larvae and the antiproteolytic activity of EOs (except for S. chilensis) on exoproteases, an interesting therapeutic strategy to control AFB

Morfologia e hábitos alimentares de duas espécies de Engraulidae (Teleostei, Clupeiformes) na Baía de Sepetiba, Rio de Janeiro

Feeding habits of the engraulidid flsh - Anchoa januaria and Cetengraulis edentullus were studied in the Baía de Sepetiba, Rio de Janeiro, from July 1983 to July 1985. Dietary items and morphological structures "blted to feeding indicate that the surface structures of branchial rays of A. januaria and C. edentulus are more useful for food filtration than the distance between them. Those morphological structures provide a separation of the size of dietary items, thw resoltins in different forms of feeding behaviors between two species. A. januaria is a selective zooplanktivore, picking up food items, whereas C. edentulus is a filtering phytaplanktivore. Anchoa januaria is a diurnal feeder and exploits different layers in the water column during the warm and cold seasons.Foram estudados as hábitos alimentares dos peixes engraulídeos Anchoa januaria e Cetengraulis edentulus na Baía de Sepetiba, Rio de Janeiro, de julho de 1983 a julho de 1985. Os itens alimentares e as estruturas morfológicas relacionadas à alimentação indicaram que a área superficial dos rastros branquiais de A. januaria e C. edentulus é mais eficiente para a filtração dos alimentos, do que as distâncias entre os mesmos. Estas estruturas morfológicas possibilitam uma seletividade no tamanho dos itens alimentares, resultando em formas distintata de obtenção do alimento, entre as duas espécies. A. januaria seleciona o zooplâncton, por captura dos itens alimentares, enquanto C. edentuluss é um fitoplanctófago filtrador. Anchoa januaria tem hábitos alimentares diurnos e explgra diferentes estratos da coluna d'água durante os períodos frio e quente

Endogenous CCL2 neutralization restricts HIV-1 replication in primary human macrophages by inhibiting viral DNA accumulation

Macrophages are key targets of HIV-1 infection. We have previously described that the expressionof CC chemokine ligand 2 (CCL2) increases during monocyte differentiation to macrophages and it is furtherup-modulated by HIV-1 exposure. Moreover, CCL2 acts as an autocrine factor that promotes viral replication ininfected macrophages. In this study, we dissected the molecular mechanisms by which CCL2 neutralization inhibitsHIV-1 replication in monocyte-derived macrophages (MDM), and the potential involvement of the innate restrictionfactors protein sterile alpha motif (SAM) histidine/aspartic acid (HD) domain containing 1 (SAMHD1) and apolipoproteinB mRNA-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) family members.Results:CCL2 neutralization potently reduced the number of p24 Gag+cells during the course of either productive orsingle cycle infection with HIV-1. In contrast, CCL2 blocking did not modify entry of HIV-1 based Virus Like Particles, thusdemonstrating that the restriction involves post-entry steps of the viral life cycle. Notably, the accumulation of viralDNA, both total, integrated and 2-LTR circles, was strongly impaired by neutralization of CCL2. Looking for correlates ofHIV-1 DNA accumulation inhibition, we found that the antiviral effect of CCL2 neutralization was independent of themodulation of SAMHD1 expression or function. Conversely, a strong and selective induction of APOBEC3A expression,to levels comparable to those of freshly isolated monocytes, was associated with the inhibition of HIV-1 replicationmediated by CCL2 blocking. Interestingly, the CCL2 neutralization mediated increase of APOBEC3A expression was typeI IFN independent. Moreover, the transcriptome analysis of the effect of CCL2 blocking on global gene expressionrevealed that the neutralization of this chemokine resulted in the upmodulation of additional genes involved in thedefence response to viruses.Conclusions:Neutralization of endogenous CCL2 determines a profound restriction of HIV-1 replication in primaryMDM affecting post-entry steps of the viral life cycle with a mechanism independent of SAMHD1. In addition, CCL2blocking is associated with induction of APOBEC3A expression, thus unravelling a novel mechanism which mightcontribute to regulate the expression of innate intracellular viral antagonistsin vivo. Thus, our study may potentially leadto the development of new therapeutic strategies for enhancing innate cellular defences against HIV-1 and protecting macrophages from infection

The cyclin D1 carboxyl regulatory domain controls the division and differentiation of hematopoietic cells

International audienceAbstractBackgroundThe family of D cyclins has a fundamental role in cell cycle progression, but its members (D1, D2, D3) are believed to have redundant functions. However, there is some evidence that contradicts the notion of mutual redundancy and therefore this concept is still a matter of debate.ResultsOur data show that the cyclin D1 is indispensable for normal hematopoiesis. Indeed, in the absence of D1, either in genetic deficient mice, or after acute ablation by RNA interference, cyclins D2 and D3 are also not expressed preventing hematopoietic cell division and differentiation at its earliest stage. This role does not depend on the cyclin box, but on the carboxyl regulatory domain of D1 coded by exons 4–5, since hematopoietic differentiation is also blocked by the conditional ablation of this region.ConclusionThese results demonstrate that not all functions of individual D cyclins are redundant and highlight a master role of cyclin D1 in hematopoiesis

Guidelines on the treatment of primary immune thrombocytopenia in children and adolescents: Associacao Brasileira de Hematologia, Hemoterapia e Terapia Celular Guidelines Project: Associacao Medica Brasileira - 2012

Centro de Hematologia de São PauloUniversidade Federal de São Paulo (UNIFESP) Escola Paulista de MedicinaCentro Infantil BoldriniUniversidade de São Paulo Faculdade de MedicinaAssociacao Medica BrasileiraHospital Ana CostaCentro de Hematologia e Hemoterapia de Santa CatarinaUniversidade Federal de Santa CatarinaUNIFESP, EPMSciEL

Lipoproteins Are Critical TLR2 Activating Toxins in Group B Streptococcal Sepsis

Abstract Group B streptococcus (GBS) is the most important cause of neonatal sepsis, which is mediated in part by TLR2. However, GBS components that potently induce cytokines via TLR2 are largely unknown. We found that GBS strains of the same serotype differ in released factors that activate TLR2. Several lines of genetic and biochemical evidence indicated that lipoteichoic acid (LTA), the most widely studied TLR2 agonist in Gram-positive bacteria, was not essential for TLR2 activation. We thus examined the role of GBS lipoproteins in this process by inactivating two genes essential for bacterial lipoprotein (BLP) maturation: the prolipoprotein diacylglyceryl transferase gene (lgt) and the lipoprotein signal peptidase gene (lsp). We found that Lgt modification of the N-terminal sequence called lipobox was not critical for Lsp cleavage of BLPs. In the absence of lgt and lsp, lipoprotein signal peptides were processed by the type I signal peptidase. Importantly, both the Δlgt and the Δlsp mutant were impaired in TLR2 activation. In contrast to released factors, fixed Δlgt and Δlsp GBS cells exhibited normal inflammatory activity indicating that extracellular toxins and cell wall components activate phagocytes through independent pathways. In addition, the Δlgt mutant exhibited increased lethality in a model of neonatal GBS sepsis. Notably, LTA comprised little, if any, inflammatory potency when extracted from Δlgt GBS. In conclusion, mature BLPs, and not LTA, are the major TLR2 activating factors from GBS and significantly contribute to GBS sepsis

Necessidades de cuidados de crianças e adolescentes em cenários hospitalares: a ética do cuidado para a produção de vida

O estudo teve como objetivo identificar e analisar as necessidades de cuidado de crianças, adolescentes e seus cuidadores em um cenário hospitalar a partir das perspectivas da integralidade e da humanização. Foi realizado estudo retrospectivo, de caráter qualitativo, a partir de análise documental. Os resultados indicaram que as necessidades de cuidado estavam relacionadas ao impacto da situação de adoecimento, ao impacto da situação de hospitalização e ao impacto nas redes sociais, de serviço e de suporte. A análise transversal aponta para a exigência de considerar o sujeito protagonista de seu processo saúde-doença para uma leitura complexificada das necessidades de cuidado e articuladas com a história de vida do sujeito; indica que a própria instituição também produz necessidades, o que torna relevante a transformação das relações e da própria lógica da instituição; e assinala os desafios de compreender as necessidades de cuidado que se colocam para além das possibilidades da ação institucional. Conclui-se que é fundamental constituir modos mais potentes de escuta e leitura das necessidades de cuidado em cenários hospitalares para uma produção de saúde como produção de vida.The purpose of this study was to identify and analyze the care needs of children, adolescents and their caregivers in a hospital setting from the perspectives of integrality and humanization. A retrospective and qualitative study was conducted, based on documentary analysis. The results indicated that the care needs were related to the impact of the process of illness, the impact of the hospitalization and the impact on social, service and support networks. The analysis points to the need of considering the person as protagonist of his health-illness process, for a complexified understanding of care needs, articulated with the person's life history; indicates that sometimes the institution itself produces needs, which makes relevant the transformation of relations and of the logic of the institution; and points to the challenges of comprehend the care needs that go beyond the possibilities of institutional action. The study concluded that it is fundamental the constitution of more potent means of understanding the care needs in hospital settings, for a health production as a production of life