Convergence of developmental mutants into a single tomato model system: 'Micro-Tom' as an effective toolkit for plant development research

Background: The tomato (Solanum lycopersicum L.) plant is both an economically important food crop and an ideal dicot model to investigate various physiological phenomena not possible in Arabidopsis thaliana. Due to the great diversity of tomato cultivars used by the research community, it is often difficult to reliably compare phenotypes. The lack of tomato developmental mutants in a single genetic background prevents the stacking of mutations to facilitate analysis of double and multiple mutants, often required for elucidating developmental pathways. Results: We took advantage of the small size and rapid life cycle of the tomato cultivar Micro-Tom (MT) to create near-isogenic lines (NILs) by introgressing a suite of hormonal and photomorphogenetic mutations (altered sensitivity or endogenous levels of auxin, ethylene, abscisic acid, gibberellin, brassinosteroid, and light response) into this genetic background. To demonstrate the usefulness of this collection, we compared developmental traits between the produced NILs. All expected mutant phenotypes were expressed in the NILs. We also created NILs harboring the wild type alleles for dwarf, self-pruning and uniform fruit, which are mutations characteristic of MT. This amplified both the applications of the mutant collection presented here and of MT as a genetic model system. Conclusions: The community resource presented here is a useful toolkit for plant research, particularly for future studies in plant development, which will require the simultaneous observation of the effect of various hormones, signaling pathways and crosstalk.

Behavior and viability of spontaneous oxidative stress-resistant Lactococcus lactis mutants in experimental fermented milk processing

Previously, we isolated two strains of spontaneous oxidative (SpOx2 and SpOx3) stress mutants of Lactococcus lactis subsp cremoris. Herein, we compared these mutants to a parental wild- type strain (J60011) and a commercial starter in experimental fermented milk production. Total solid contents of milk and fermentation temperature both affected the acidification profile of the spontaneous oxidative stress- resistant L. lactis mutants during fermented milk production. Fermentation times to pH 4.7 ranged from 6.40 h (J60011) to 9.36 h (SpOx2); V(max) values were inversely proportional to fermentation time. Bacterial counts increased to above 8.50 log(10) cfu/mL. The counts of viable SpOx3 mutants were higher than those of the parental wild strain in all treatments. All fermented milk products showed post-fermentation acidification after 24 h of storage at 4 degrees C; they remained stable after one week of storage.

Probing the hormonal activity of fractionated molecular humic components in tomato auxin mutants

We progressively reduced the complexity of humic matter by a mild sequential removal of unbound or free components, weakly, and strongly bound molecules. The auxin-like response of residues from each step was tested using tomato (cv. Micro-Tom) seedlings expressing DR5 auxin synthetic promoter fused to the beta-glucuronidase (GUS) reporter gene and the low auxin-sensitivity diageotropica (dgt) mutant. Both exogenous auxin and humic matter promoted lateral root emergence in the control, but failed to induce lateral roots in the dgt mutant. When strongly bound components were removed from humic matter by breaking the ester and ether bonds, the humic residues lost their ability to induce the DR5

Differential ultrastructural changes in tomato hormonal mutants exposed to cadmium

Cadmium (Cd) is a toxic heavy metal, which can cause severe damage to plant development. The aim of this work was to characterize ultrastructural changes induced by Cd in miniature tomato cultivar Micro-Tom (MT) mutants and their wild-type counterpart. Leaves of diageotropica (dgt) and Never ripe (Nr) tomato hormonal mutants and wild-type MT were analysed by light, scanning and transmission electron microscopy in order to characterize the structural changes caused by the exposure to 1 mM CdCl(2). The effect of Cd on leaf ultrastructure was observed most noticeably in the chloroplasts, which exhibited changes in organelle shape and internal organization, of the thylakoid membranes and stroma. Cd caused an increase in the intercellular spaces in Nr leaves, but a decrease in the intercellular spaces in dgt leaves, as well as a decrease in the size of mesophyll cells in the mutants. Roots of the tomato hormonal mutants, when analysed by light microscopy, exhibited alterations in root diameter and disintegration of the epidermis and the external layers of the cortex. A comparative analysis has allowed the identification of specific Cd-induced ultrastructural changes in wild-type tomato, the pattern of which was not always exhibited by the mutants. (C) 2009 Elsevier B.V. All rights reserved.

Study of the antimicrobial peptide indolicidin and mutants in eukaryotic modelled membrane by molecular dynamics simulations

In this work the interaction of the antimicrobial peptide indolicidin (IND) and its mutants CP10A and CP11 with a eukaryotic membrane model was examined by molecular dynamics simulations. The aim was to analyse the behaviour of these antimicrobial peptides when they interact with a eukaryotic modelled membrane, thereby obtaining atomic detailed observations that are not experimentally available. In the simulations, the widely studied dipalmitoylphosphatidylcholine hydrated bilayer was used as a eukaryotic membrane model. In agreement with experimental observations, the peptides IND, CP10A, and CP11 insert into the bilayer differently; the peptides that insert more deeply present the major hemolytic activities. The hydrophobic residues are responsible for the insertion, but some Trp residues of the peptides remain at the bilayer/water interface because they interact with the bilayer choline groups by cation-pi interactions that should be important for recognition of eukaryotic membrane by the three studied peptides.

Conservation of hepatitis C virus nonstructural protein 3 amino acid sequence in viral isolates during liver transplantation

As a consequence of selective pressure exerted by the immune response during hepatitis C virus (HCV) infection, a high rate of nucleotide mutations in the viral genome is observed which leads to the emergence of viral escape mutants. The aim of this study was to evaluate the evolution of the amino acid (aa) sequence of the HCV nonstructural protein 3 (NS3) in viral isolates after liver transplantation. Six patients with HCV-induced liver disease undergoing liver transplantation (LT) were followed up for sequence analysis. Hepatitis C recurrence was observed in all patients after LT. The rate of synonymous (dS) nucleotide substitutions was much higher than that of nonsynonymous (dN) ones in the NS3 encoding region. The high values of the dS/dN ratios suggest no sustained adaptive evolution selection pressure and, therefore, absence of specific NS3 viral populations. Clinical genotype assignments were supported by phylogenetic analysis. Serial samples from each patient showed lower mean nucleotide genetic distance when compared with samples of the same HCV genotype and subtype. The NS3 samples studied had an N-terminal aa sequence with several differences as compared with reference ones, mainly in genotype 1b-infected patients. After LT, as compared with the sequences before, a few reverted aa substitutions and several established aa substitutions were observed at the N-terminal of NS3. Sites described to be involved in important functions of NS3, notably those of the catalytic triad and zinc binding, remained unaltered in terms of aa sequence. Rare or frequent aa substitutions occurred indiscriminately in different positions. Several cytotoxic T lymphocyte epitopes described for HCV were present in our 1b samples. Nevertheless, the deduced secondary structure of the NS3 protease showed a few alterations in samples from genotype 3a patients, but none were seen in 1b cases. Our data, obtained from patients under important selective pressure during LT, show that the NS3 protease remains well conserved, mainly in HCV 3a patients. It reinforces its potential use as an antigenic candidate for further studies aiming at the development of a protective immune response.

Studies of aberrant phyllotaxy1 Mutants of Maize Indicate Complex Interactions between Auxin and Cytokinin Signaling in the Shoot Apical Meristem

One of the most fascinating aspects of plant morphology is the regular geometric arrangement of leaves and flowers, called phyllotaxy. The shoot apical meristem (SAM) determines these patterns, which vary depending on species and developmental stage. Auxin acts as an instructive signal in leaf initiation, and its transport has been implicated in phyllotaxy regulation in Arabidopsis (Arabidopsis thaliana). Altered phyllotactic patterns are observed in a maize (Zea mays) mutant, aberrant phyllotaxy1 (abph1, also known as abphyl1), and ABPH1 encodes a cytokinin-inducible type A response regulator, suggesting that cytokinin signals are also involved in the mechanism by which phyllotactic patterns are established. Therefore, we investigated the interaction between auxin and cytokinin signaling in phyllotaxy. Treatment of maize shoots with a polar auxin transport inhibitor, 1-naphthylphthalamic acid, strongly reduced ABPH1 expression, suggesting that auxin or its polar transport is required for ABPH1 expression. Immunolocalization of the PINFORMED1 (PIN1) polar auxin transporter revealed that PIN1 expression marks leaf primordia in maize, similarly to Arabidopsis. Interestingly, maize PIN1 expression at the incipient leaf primordium was greatly reduced in abph1 mutants. Consistently, auxin levels were reduced in abph1, and the maize PIN1 homolog was induced not only by auxin but also by cytokinin treatments. Our results indicate distinct roles for ABPH1 as a negative regulator of SAM size and a positive regulator of PIN1 expression. These studies highlight a complex interaction between auxin and cytokinin signaling in the specification of phyllotactic patterns and suggest an alternative model for the generation of altered phyllotactic patterns in abph1 mutants. We propose that reduced auxin levels and PIN1 expression in abph1 mutant SAMs delay leaf initiation, contributing to the enlarged SAM and altered phyllotaxy of these mutants.

Cys mutants in functional regions of Sticholysin I clarify the participation of these residues in pore formation

Experimental evidence shows that the mechanism of pore formation by actinoporins is a multistep process, involving binding of the water-soluble monomer to the membrane and subsequent oligomerization on the membrane surface, leading to the formation of a functional pore. However, as for other eukaryotic pore-forming toxins, the molecular details of the mechanism of membrane insertion and oligomerization are not clear. In order to obtain further insight with regard to the structure-function relationship in sticholysins, we designed and produced three cysteine mutants of recombinant sticholysin I (rStI) in relevant functional regions for membrane interaction: StI E2C and StI F15C (in the N-terminal region) and StI R52C (in the membrane binding site). The conformational characterization derived from fluorescence and CD spectroscopic studies of StI E2C, StI F15C and StI R52C suggests that replacement of these residues by Cys in rStI did not noticeably change the conformation of the protein. The substitution by Cys of Arg(52) in the phosphocholine-binding site, provoked noticeable changes in rStI permeabilizing activity; however, the substitutions in the N-terminal region (Glu(2), Phe(15)) did not modify the toxin`s permeabilizing ability. The presence of a dimerized population stabilized by a disulfide bond in the StI E2C mutant showed higher pore-forming activity than when the protein is in the monomeric state, suggesting that sticholysins pre-ensembled at the N-terminal region could facilitate pore formation. (C) 2011 Elsevier Ltd. All rights reserved.

Saccharomyces cerevisiae coq10 null mutants are responsive to antimycin A

Deletion of COQ10 in Saccharomyces cerevisiae elicits a respiratory defect characterized by the absence of cytochrome c reduction, which is correctable by the addition of exogenous diffusible coenzyme Q(2). Unlike other coq mutants with hampered coenzyme Q(6) (Q(6)) synthesis, coq10 mutants have near wild-type concentrations of Q(6). In the present study, we used Q-cycle inhibitors of the coenzyme QH(2)-cytochrome c reductase complex to assess the electron transfer properties of coq10 cells. Our results show that coq10 mutants respond to antimycin A, indicating an active Q-cycle in these mutants, even though they are unable to transport electrons through cytochrome c and are not responsive to myxothiazol. EPR spectroscopic analysis also suggests that wild-type and coq10 mitochondria accumulate similar amounts of Q(6) semiquinone, despite a lower steady-state level of coenzyme QH(2)-cytochrome c reductase complex in the coq10 cells. Confirming the reduced respiratory chain state in coq10 cells, we found that the expression of the Aspergillus fumigatus alternative oxidase in these cells leads to a decrease in antimycin-dependent H(2)O(2) release and improves their respiratory growth.

Termo de consentimento livre e esclarecido aos usuários de clínicas odontológicas brasileiras: aspectos éticos e legais

O consentimento do paciente antes do início de qualquer procedimento é uma condição a ser respeitada pelos profissionais da odontologia, sem nenhuma exceção. É necessário que o paciente esteja ciente de seu status de saúde, de suas necessidades específicas, do propósito de cada tratamento, conheça os planejamentos alternativos (incluindo o não tratamento), saiba do seu prognóstico, riscos, consequências, limitações e se conscientize das suas responsabilidades e as do seu cirurgião-dentista, proporcionando o sucesso do tratamento. O termo de consentimento livre e esclarecido (TCLE) visa fortalecer e esclarecer a posição do paciente, estabelecendo os direitos e deveres de ambas as partes paciente e profissional. O conhecimento integral do tratamento diminuirá a ansiedade do paciente e as complicações de tratamento, promoverá maior qualidade dos serviços odontológicos e maior satisfação do dentista e do paciente. Entretanto, no Brasil, poucos artigos são encontrados e existem alguns problemas éticos envolvendo as clínicas odontológicas, no que diz respeito a este documento de esclarecimento para o paciente. Diante disso, este trabalho tem por objetivo realizar uma revisão crítica sobre o tema abordado, demonstrando a importância do TCLE na clínica odontológica brasileira e na vida profissional dos cirurgiões-dentistas.

Comparison of periodontal parameters in individuals with syndromic craniosynostosis

Craniosynostosis syndromes are characterized by premature closure of one or more cranial sutures, associated with other malformations, the most frequent of which are the Crouzon and Apert syndromes. Few studies in the literature have addressed the oral health of these individuals. The purpose of this study was to compare the periodontal status of individuals with Apert, Crouzon, Pfeiffer and Saethre-Chotzen syndromes before toothbrushing and compare the efficiency of plaque removal before and after mechanical toothbrushing. The probing depth, plaque index (according to Löe and O'Leary), clinical attachment level, gingival index (according to Silness and Löe) and amount of keratinized mucosa were evaluated before toothbrushing, and the O'Leary plaque index was assessed before and immediately after toothbrushing, on the same day, in 27 individuals aged 11 to 36 years. There was statistically significant difference in the mean probing depth and clinical attachment level among regions (p=0.00; p=0.01, respectively). The gingival index did not reveal statistically significant differences. With regard to the plaque index, the left region exhibited higher plaque index values than the right and anterior regions. No significant results were found in the analysis of keratinized mucosa. Comparison of the O'Leary plaque index before and after toothbrushing revealed statistically significant difference for all syndromes except for the Pfeiffer syndrome (p<0.05). In conclusion, there was no difference in the periodontal status among individuals with syndromic craniosynostosis. The posterior region was more affected than the anterior region as to the presence of plaque, loss of insertion and probing depth. Individuals with Pfeiffer syndrome exhibited greater toothbrushing efficiency than individuals with the other craniosynostosis syndromes.

Indicações e uso da técnica "sonda-dedo"

A recomendação da Organização Mundial da Saúde é que todo recém nascido deva ser alimentado exclusivamente no seio materno até o sexto mês e, de forma complementar, até o segundo ano de vida. Assim, algumas técnicas são realizadas para facilitar a alimentação ao seio, dentre elas o uso do copo e, recentemente, a utilização da técnica "sonda-dedo". Tal prática é bastante controversa e há escassez de estudos na literatura sobre a descrição da técnica, sua indicação e uso. O objetivo do presente trabalho é relatar a experiência clínica para indicação e uso da técnica "sonda-dedo". A técnica "sonda-dedo" consiste no oferecimento do leite, de preferência humano, utilizando sonda gástrica conectada a uma seringa com êmbolo e fixada em dedo mínimo enluvado com fita adesiva. A sonda é posicionada na cavidade oral do recém nascido e deve servir como uma técnica de auxílio para adequação do padrão de sucção. Desta forma, sugere-se que sua indicação deve ser apenas nos casos em que seja caracterizada uma disfunção oral, seja em recém nascidos a termo ou pré-termo. Diante da avaliação específica, realizada pelo fonoaudiólogo, indica-se a técnica "sonda-dedo" com objetivo de adequar as alterações obtidas na avaliação da sucção não nutritiva ou em seio materno. Acredita-se que, para que a técnica "sonda-dedo" seja indicada como complemento do aleitamento materno, devam ser realizados novos estudos para esclarecer quais as repercussões da técnica "sonda-dedo" na prevalência do aleitamento materno e no desenvolvimento motor oral de recém nascidos.

A fala nas diferentes modalidades de reabilitação oral protética em idosos

TEMA: a produção da fala nas modalidades de reabilitação oral protética. OBJETIVO: verificar se o tipo de reabilitação oral interfere na produção da fala. MÉTODO: 36 idosos (média = 68 anos), divididos em 3 grupos, foram avaliados: 13 com dentes naturais (A), 13 com prótese total mucosossuportada superior e inferior (B) e 10 com prótese total mucosossuportada superior e implantossuportada inferior (C). A estabilidade das próteses foi avaliada por um dentista e amostras de fala foram analisadas por 5 fonoaudiólogos. Para determinar a freqüência de alteração dos sons da fala utilizou-se o cálculo da Porcentagem de Consoantes Corretas (PCC). RESULTADOS: observou-se poucos casos com alteração de fala, com maior freqüência no grupo C (23,08%), sendo a articulação travada presente em todos os grupos, a redução dos movimentos labiais em dois grupos (A e B) e a articulação exagerada e a falta de controle salivar em um dos grupos (C e B). Quanto à PCC, menor valor foi observado para os fones linguodentais nos grupos B e C (maior ocorrência de alteração), seguido dos fones alveolares, predominando casos sem alteração no grupo A, contrariamente aos demais grupos, sendo a projeção lingual e o ceceio as alterações mais encontradas. Não houve diferença entre os grupos e a maioria do grupo B estava com a prótese inferior insatisfatória, não havendo associação entre alteração de fala e prótese insatisfatória. CONCLUSÃO: apesar da amostra pequena, indivíduos reabilitados com prótese total apresentam alteração nos fones linguodentais e alveolares e o tipo de prótese, bem como a estabilidade desta parece não interferir na produção da fala.

Effects of light-curing time on the cytotoxicity of a restorative composite resin on odontoblast-like cells

This in vitro study evaluated the cytotoxicity of an experimental restorative composite resin subjected to different light-curing regimens. METHODS: Forty round-shaped specimens were prepared and randomly assigned to four experimental groups (n=10), as follows: in Group 1, no light-curing; in Groups 2, 3 and 4, the composite resin specimens were light-cured for 20, 40 or 60 s, respectively. In Group 5, filter paper discs soaked in 5 µL PBS were used as negative controls. The resin specimens and paper discs were placed in wells of 24-well plates in which the odontoblast-like cells MDPC-23 (30,000 cells/cm²) were plated and incubated in a humidified incubator with 5% CO2 and 95% air at 37ºC for 72 h. The cytotoxicity was evaluated by the cell metabolism (MTT assay) and cell morphology (SEM). The data were analyzed statistically by Kruskal-Wallis and Mann-Whitney tests (p<0.05). RESULTS: In G1, cell metabolism decreased by 86.2%, indicating a severe cytotoxicity of the non-light-cured composite resin. On the other hand, cell metabolism decreased by only 13.3% and 13.5% in G2 and G3, respectively. No cytotoxic effects were observed in G4 and G5. In G1, only a few round-shaped cells with short processes on their cytoplasmic membrane were observed. In the other experimental groups as well as in control group, a number of spindle-shaped cells with long cytoplasmic processes were found. CONCLUSION: Regardless of the photoactivation time used in the present investigation, the experimental composite resin presented mild to no toxic effects to the odontoblast-like MDPC-23 cells. However, intense cytotoxic effects occurred when no light-curing was performed.