Preparação e caracterização de biovidros dopados com Sr e Zn

Mestrado em Ciência e Engenharia dos MateriaisOs vidros e vitro-cerâmicos do sistema ternário diopsite (CaO·MgO·2SiO2; posteriormente mencionado como Di) – fluorapatite (9CaO·3P2O5·CaF2; posteriormente mencionado como FA) e fosfato tricálcico (3CaO·P2O5; posteriormente mencionado como TCP) são potenciais candidatos para várias aplicações biomédicas incluindo estruturas de suporte para engenharia de tecido ósseo. O presente trabalho teve como objectivo investigar os vidros e os vitrocerâmicos do sistema Di-FA-TCP quanto à sua estrutura e capacidades de formação, sinterização e cristalização. Estudou-se também a influência da variação das razões SrO/CaO e ZnO/MgO na estrutura e em várias propriedades termo-físicas nos mesmos vidros, nos quais o CaO foi parcialmente substituído pelo SrO e o MgO pelo ZnO (0-10 mol.%). Todos os vidros estudados foram preparados pela técnica de fusão-arrefecimento rápido. A análise estrutural dos vidros foi feita por Ressonância Magnética Nuclear 29Si e 31P (NMR) e Espectroscopia de Infra-Vermelho (FTIR). Em todos os vidros a rede de silicato é coordenada predominantemente nas unidades Q2 (Si) enquanto que o fósforo tende a permanecer no ambiente do ortofosfato (Q0). O comportamento dos pós dos vidros na sinterização e cristalização foi estudado por Microscopia de Aquecimento (Hot Stage Microscopy - HSM) e por Análise Térmica Diferencial (DTA), respectivamente, enquanto a evolução das fases cristalinas e da microestrutura dos vitro-cerâmicos foi seguida por Difracção de Raios-X (XRD) e por Microscopia Electrónica de Varrimento (SEM). Na maioria das composições dos vidros investigados, a sinterização precedeu a cristalização tendo a DI e a FA cristalizado como fases primárias em todos os vitro-cerâmicos após sinterização a 850ºC durante 1 h.The glasses and glass-ceramics in ternary system of diopside (CaO·MgO·2SiO2; hereafter referred as Di) – fluorapatite [9CaO·3P2O5·CaF2; hereafter referred as FA] – tricalcium phosphate (3CaO·P2O5; hereafter referred as TCP) system are potential candidates for various biomedical applications including scaffolds for bone tissue engineering. The present work investigates the glass forming ability, structure, sintering ability and crystallization behaviour of some glasses along Di-FA-TCP join. Further, influence of varying SrO/CaO and ZnO/MgO ratio has been studied on structure and various thermo-physical properties of glasses in the above mentioned system where CaO has been partially substituted by SrO and MgO has been partially substituted by ZnO (0 – 10 mol.%). All the glasses have been prepared by melt-quenching technique. The structural analysis of glasses has been made by 29Si and 31P-nuclear magnetic resonance (NMR) and infrared spectroscopy (FTIR). The silicate network in all the investigated glasses is predominantly coordinated in Q2 (Si) units while phosphorus tends to remain in orthophosphate (Q0) environment. The sintering and crystallization behaviour of glass powders has been studied by hot stage microscopy (HSM) and by differential thermal analysis (DTA), respectively while crystalline phase evolution and microstructure of glass-ceramics has been followed by X-ray diffraction (XRD) and scanning electron microscopy (SEM). The sintering preceded crystallization in majority of the glass compositions and Di and FA crystallized as the primary crystalline phases in all the investigated glassceramics after sintering at 850ºC for 1 h

LNA probes substantially improve the detection of bacterial endosymbionts in whole mount of insects by fluorescent in-situ hybridization

BACKGROUND: Detection of unculturable bacteria and their localization in the host, by fluorescent in-situ hybridization (FISH), is a powerful technique in the study of host-bacteria interaction. FISH probes are designed to target the 16 s rRNA region of the bacteria to be detected. LNA probes have recently been used in FISH studies and proven to be more efficient. To date no report has employed LNA probes for FISH detection of bacterial endosymbiont in the whole mount tissues. Further, though speculated, bacteriocytes have not been reported from males of Bemisia tabaci. RESULTS: In this study, we compared the efficiency in detecting bacteria by fluorescent DNA oligonucleotides versus modified probes containing Locked Nucleic Acid (LNA) substitution in their structure. We used the insect Bemisia tabaci as the experimental material since it carried simultaneous infection by two bacteria: one a primary endosymbiont, Portiera (and present in more numbers) while the other a secondary endosymbiont Arsenophonus (and present in less numbers). Thus a variation in the abundance of bacteria was expected. While detecting both the bacteria, we found a significant increase in the signal whenever LNA probes were used. However, the difference was more pronounced in detecting the secondary endosymbiont, wherein DNA probes gave weak signals when compared to LNA probes. Also, signal to noise ratio for LNA probes was higher than DNA probes. We found that LNA considerably improved sensitivity of FISH, as compared to the commonly used DNA oligonucleotide probe. CONCLUSION: By employing LNA probes we could detect endosymbiotic bacteria in males, which have never been reported previously. We were able to detect bacteriocytes containing Portiera and Arsenophonus in the males of B. tabaci. Thus, employing LNA probes at optimized conditions will help to significantly improve detection of bacteria at the lowest concentration and may give a comprehensible depiction about their specific distribution within samples

Bacterial diversity analysis of larvae and adult midgut microflora using culture-dependent and culture-independent methods in lab-reared and field-collected Anopheles stephensi-an Asian malarial vector

<p>Abstract</p> <p>Background</p> <p>Mosquitoes are intermediate hosts for numerous disease causing organisms. Vector control is one of the most investigated strategy for the suppression of mosquito-borne diseases. <it>Anopheles stephensi </it>is one of the vectors of malaria parasite <it>Plasmodium vivax</it>. The parasite undergoes major developmental and maturation steps within the mosquito midgut and little is known about <it>Anopheles</it>-associated midgut microbiota. Identification and characterization of the mosquito midgut flora is likely to contribute towards better understanding of mosquito biology including longevity, reproduction and mosquito-pathogen interactions that are important to evolve strategies for vector control mechanisms.</p> <p>Results</p> <p>Lab-reared and field-collected <it>A. stephensi </it>male, female and larvae were screened by "culture-dependent and culture-independent" methods. Five 16S rRNA gene library were constructed form lab and field-caught <it>A. stephensi </it>mosquitoes and a total of 115 culturable isolates from both samples were analyzed further. Altogether, 68 genera were identified from midgut of adult and larval <it>A. stephensi</it>, 53 from field-caught and 15 from lab-reared mosquitoes. A total of 171 and 44 distinct phylotypes having 85 to 99% similarity with the closest database matches were detected among field and lab-reared <it>A. stephensi </it>midgut, respectively. These OTUs had a Shannon diversity index value of 1.74–2.14 for lab-reared and in the range of 2.75–3.49 for field-caught <it>A. stephensi </it>mosquitoes. The high species evenness values of 0.93 to 0.99 in field-collected adult and larvae midgut flora indicated the vastness of microbial diversity retrieved by these approaches. The dominant bacteria in field-caught adult male <it>A. stephensi </it>were uncultured <it>Paenibacillaceae </it>while in female and in larvae it was <it>Serratia marcescens</it>, on the other hand in lab-reared mosquitoes, <it>Serratia marcescens </it>and <it>Cryseobacterium meninqosepticum </it>bacteria were found to be abundant.</p> <p>Conclusion</p> <p>More than fifty percent of the phylotypes were related to uncultured class of bacteria. Interestingly, several of the bacteria identified are related to the known symbionts in other insects. Few of the isolates identified in our study are found to be novel species within the gammaproteobacteria which could not be phylogenetically placed within known classes. To the best of our knowledge, this is the first attempt to study the midgut microbiota of <it>A. stephensi </it>from lab-reared and field-collected adult and larvae using "culture-dependent and independent methods".</p

Knockdown of Aminopeptidase-N from Helicoverpa armigera Larvae and in Transfected Sf21 Cells by RNA Interference Reveals Its Functional Interaction with Bacillus thuringiensis Insecticidal Protein Cry1Ac

Aminopeptidase-N (APN) and cadherin proteins located at the midgut epithelium of Helicoverpa armigera have been implicated as receptors for the Cry1A subfamily of insecticidal proteins of Bacillus thuringiensis. Ligand blot analysis with heterologously expressed and purified H. armigera Bt receptor with three closely related Cry1A proteins tentatively identified HaAPN1 as an interacting ligand. However, to date there is no direct evidence of APN being a functional receptor to Cry1Ac in H. armigera. Sf21 insect cells expressing HaAPN1 displayed aberrant cell morphology upon overlaying with Cry1Ac protein. Down-regulating expression of HaAPN1 by RNA interference using double-stranded RNA correlated with a corresponding reduction in the sensitivity of HaAPN1-expressing cells to Cry1Ac protein. This clearly establishes that insect cells expressing the receptor recruit sensitivity to the insecticidal protein Cry1Ac, and their susceptibility is directly dependent on the amount of HaAPN1 protein expressed. Most importantly, silencing of HaAPN1 in H. armigera in vivo by RNA interference resulted in reduced transcript levels and a corresponding decrease in the susceptibility of larvae to Cry1Ac. BIAcore analysis of HaAPN1/Cry1Ac interaction further established HaAPN1 as a ligand for Cry1Ac. This is the first functional demonstration of insect aminopeptidase-N of H. armigera being a receptor of Cry1Ac protein of B. thuringiensis

Improved outcomes in the treatment of post-myocardial infarction ventricular septal defect with percutaneous TandemHeart left ventricular mechanical circulatory support

Background Post-myocardial infarction (MI) ventricular septal defect (VSD) is associated with 40% - 50% of peri-procedural mortalities; however, it is amenable to catheter-based therapies. We retrospectively investigated the impact of state-of-the-art bridging percutaneous left ventricular mechanical circulatory support (MCS) using the TandemHeart® (TH) ventricular assist device (VAD) on a patient with post-MI VSD. Results From July 2008 to March 2014, 23 patients were referred for treatment of post-MI VSD. Initially, 18/23 patients required MCS; 12 received an intra-aortic balloon pump (IABP), while 6 received initial TH support. Seven of the IABP patients later required TH support. Catheter-based device VSD closure was performed in 18 of the patients; however, three patients required conversion to conventional open cardiac surgical repair via VSD patch closure due to failure of the catheter-based approach. Five patients with TH underwent planned open cardiac surgical repair due to an anticipated lack of suitability for catheter-based treatment. Results revealed that delayed closure after MI correlated with improved survival. Overall, 30-day and 6-month survival rates were 83% (19/23) and 70% (16/23), respectively. Conclusions Further, Qp/Qs ratios of \u3c2.4 correlated with successful percutaneous VSD repair, and this assessment should be further explored as an assessment to inform clinical judgment in patients with post-MI VSD treatment

Aedes aegypti lachesin protein binds to the domain III of envelop protein of Dengue virus-2 and inhibits viral replication.

Dengue virus (DENV) comprises of four serotypes (DENV-1 to -4) and is medically one of the most important arboviruses (arthropod-borne virus). DENV infection is a major human health burden and is transmitted between humans by the insect vector, Aedes aegypti. Ae. aegypti ingests DENV while feeding on infected humans, which traverses through its gut, haemolymph and salivary glands of the mosquito before being injected into a healthy human. During this process of transmission, DENV must interact with many proteins of the insect vector, which are important for its successful transmission. Our study focused on the identification and characterisation of interacting protein partners in Ae. aegypti to DENV. Since domain III (DIII) of envelope protein (E) is exposed on the virion surface and is involved in virus entry into various cells, we performed phage display library screening against domain III of the envelope protein (EDIII) of DENV-2. A peptide sequence showing similarity to lachesin protein was found interacting with EDIII. The lachesin protein was cloned, heterologously expressed, purified and used for in vitro interaction studies. Lachesin protein interacted with EDIII and also with DENV. Further, lachesin protein was localised in neuronal cells of different organs of Ae. aegypti by confocal microscopy. Blocking of lachesin protein in Ae. aegypti with anti-lachesin antibody resulted in a significant reduction in DENV replication

Cellular Basis for Response Diversity in the Olfactory Periphery

An emerging idea in olfaction is that temporal coding of odor specificity can be intrinsic to the primary olfactory receptor neurons (ORNs). As a first step towards understanding whether lobster ORNs are capable of generating odor-specific temporal activity and what mechanisms underlie any such heterogeneity in discharge pattern, we characterized different patterns of activity in lobster ORNs individually and ensemble using patch-clamp recording and calcium imaging. We demonstrate that lobster ORNs show tonic excitation, tonic inhibition, phaso-tonic excitation, and bursting, and that these patterns are faithfully reflected in the calcium signal. We then demonstrate that the various dynamic patterns of response are inherent in the cells, and that this inherent heterogeneity is largely determined by heterogeneity in the underlying intrinsic conductances

Host Plant Induced Variation in Gut Bacteria of Helicoverpa armigera

Helicoverpa are important polyphagous agricultural insect pests and they have a worldwide distribution. In this study, we report the bacterial community structure in the midgut of fifth instar larvae of Helicoverpa armigera, a species prevalent in the India, China, South Asia, South East Asia, Southern & Eastern Africa and Australia. Using culturable techniques, we isolated and identified members of Bacillus firmus, Bacillus niabense, Paenibacillus jamilae, Cellulomonas variformis, Acinetobacter schindleri, Micrococcus yunnanesis, Enterobacter sp., and Enterococcus cassiliflavus in insect samples collected from host plants grown in different parts of India. Besides these the presence of Sphingomonas, Ralstonia, Delftia, Paracoccus and Bacteriodetes was determined by culture independent molecular analysis. We found that Enterobacter and Enterococcus were universally present in all our Helicoverpa samples collected from different crops and in different parts of India. The bacterial diversity varied greatly among insects that were from different host plants than those from the same host plant of different locations. This result suggested that the type of host plant greatly influences the midgut bacterial diversity of H. armigera, more than the location of the host plant. On further analyzing the leaf from which the larva was collected, it was found that the H. armigera midgut bacterial community was similar to that of the leaf phyllosphere. This finding indicates that the bacterial flora of the larval midgut is influenced by the leaf surface bacterial community of the crop on which it feeds. Additionally, we found that laboratory made media or the artificial diet is a poor bacterial source for these insects compared to a natural diet of crop plant

Challenge clusters facing LCA in environmental decision-making—what we can learn from biofuels

Purpose Bioenergy is increasingly used to help meet greenhouse gas (GHG) and renewable energy targets. However, bioenergy’s sustainability has been questioned, resulting in increasing use of life cycle assessment (LCA). Bioenergy systems are global and complex, and market forces can result in significant changes, relevant to LCA and policy. The goal of this paper is to illustrate the complexities associated with LCA, with particular focus on bioenergy and associated policy development, so that its use can more effectively inform policymakers. Methods The review is based on the results from a series of workshops focused on bioenergy life cycle assessment. Expert submissions were compiled and categorized within the first two workshops. Over 100 issues emerged. Accounting for redundancies and close similarities in the list, this reduced to around 60 challenges, many of which are deeply interrelated. Some of these issues were then explored further at a policyfacing workshop in London, UK. The authors applied a rigorous approach to categorize the challenges identified to be at the intersection of biofuels/bioenergy LCA and policy. Results and discussion The credibility of LCA is core to its use in policy. Even LCAs that comply with ISO standards and policy and regulatory instruments leave a great deal of scope for interpretation and flexibility. Within the bioenergy sector, this has led to frustration and at times a lack of obvious direction. This paper identifies the main challenge clusters: overarching issues, application and practice and value and ethical judgments. Many of these are reflective of the transition from application of LCA to assess individual products or systems to the wider approach that is becoming more common. Uncertainty in impact assessment strongly influences planning and compliance due to challenges in assigning accountability, and communicating the inherent complexity and uncertainty within bioenergy is becoming of greater importance. Conclusions The emergence of LCA in bioenergy governance is particularly significant because other sectors are likely to transition to similar governance models. LCA is being stretched to accommodate complex and broad policy-relevant questions, seeking to incorporate externalities that have major implications for long-term sustainability. As policy increasingly relies on LCA, the strains placed on the methodology are becoming both clearer and impedimentary. The implications for energy policy, and in particular bioenergy, are large

Persistencia de malezas gramíneas en cultivos de trigo del sudeste bonaerense

En la presente tesis se estudió la persistencia de especies poáceas en cultivos de trigo del sudeste de Buenos Aires. En dicha región, Avena fatua L. y Lolium multiflorum Lam. son las malezas poáceas más importantes, tanto por la dificultad de control como por sus efectos competitivos sobre el cultivo. A los efectos de cuantificar la persistencia de dichas especies, se estudió la composición de la comunidad de malezas en dos momentos del ciclo: preaplicación de herbicidas y precosecha. Individuos de ambas malezas fueron registrados en ambos momentos como consecuencia de “escapes” al control realizado con herbicidas, siendo A. fatua más constante que L. mutiflorum. Posteriormente, se estudiaron los procesos que definen la persistencia de ambas malezas. Los resultados obtenidos indican que el ajuste del momento de emergencia es jerárquicamente el factor más importante para explicar la persistencia de A. fatua. Se demostró que los modelos de germinación son diferentes según las semillas provengan de un lote agrícola o de una condición de no cultivo, siendo estas diferencias de naturaleza genética. Por otro lado, la variabilidad en la supervivencia a los herbicidas es el factor que mejor explica la persistencia de L. multiflorum, habiéndose documentado resistencia cruzada a los herbicidas inhibidores de la ALS, pyroxsulam, imazamox y flucarbazone, sin antecedentes previos en la región. Los índices de resistencia encontrados presentan variación con la temperatura ambiente en post-aplicación del herbicida, habiéndose registrado mayor resistencia con mayor temperatura. Además, se comprobó que los individuos resistentes presentan menor tiempo a floración que los susceptibles. Tal atributo puede significar una ventaja demográfica para dichas poblaciones. Queda así demostrada la persistencia de A. fatua y L. multiflorum durante el ciclo del cultivo más allá de las prácticas de control realizadas y la participación de dos procesos demográficos distintos (establecimiento y supervivencia) en dicha persistencia