Identification and Validation of EST-Derived Molecular Markers, TRAP and VNTRs, for Banana Research

The advent of high-throughput sequencing technology has generated abundant information on DNA sequences for the genomes of many plant species. Expressed Sequence Tags (ESTs), which are unique DNA sequences derived from a cDNA library and therefore representing genes transcribed in specific tissues or at some stage of development, are one type of DNA sequences highly available today for many important crop species. Molecular markers are used for bridging DNA sequence information with particular phenotypes and are useful tools for genotyping germplasm collections and also for tagging genes involved in desirable agronomic traits. In this sense, there is always a strong demand for suitable marker techniques to better utilise existing sequence information. A transcriptome database from banana (Musa spp.), DATAMusa, containing 42,724 ESTs from 11 different cDNA libraries and encompassing approximately 24 Mb of DNA sequence, was used in this study for the design of primers to PCR-amplify two types of EST-derived molecular markers, Variable Nucleotide Tandem Repeat (VNTR) and Target Region Amplification Polymorphism (TRAP). These primers were then validated against a panel of 14 diploid Musa genotypes and produced 32 (VNTR) and 119 (TRAP) alleles. Used separately or together, both types of markers were able to discriminate Musa genotypes from different genome background (A or B genomes). The TRAP alleles identified were derived from only one EST, while the VNTR alleles were derived from 12 unigenes. Based on the results of this study, EST-derived markers can be an important source of polymorphism to be used in genetic diversity and gene discovery studies in banan

Variable number of tandem repeat markers in the genome sequence of Mycosphaerella fijiensis, the causal agent of black leaf streak disease of banana (Musa spp)

ABSTRACT. We searched the genome of Mycosphaerella fijiensis for molecular markers that would allow population genetics analysis of this plant pathogen. M. fijiensis, the causal agent of banana leaf streak disease, also known as black Sigatoka, is the most devastating pathogen attacking bananas (Musa spp). Recently, the entire genome sequence of M. fijiensis became available. We screened this database for VNTR markers. Forty-two primer pairs were selected for validation, based on repeat type and length and the number of repeat units. Five VNTR markers showing multiple alleles were validated with a reference set of isolates from different parts of the world and a population from a banana plantation in Costa Rica. Polymorphism information content values varied from 0.6414 to 0.7544 for the reference set and from 0.0400 and 0.7373 for the population set. Eighty percent of the polymorphism information content values were above 0.60, indicating that the markers are highly informative. These markers allowed robust scoring of agarose gels and proved to be useful for variability and population genetics studies. In conclusion, the strategy we developed to identify and validate VNTR markers is an efficient means to incorporate markers that can be used for fungicide resistance management and to develop breeding strategies to control banana black leaf streak disease. This is the first report of VNTR-minisatellites from the M. fijiensis genome sequence. Key words: Molecular markers; VNTRs; Genetic diversity; Population genetics; Black Sigatok

Classes of exact wavefunctions for general time-dependent Dirac Hamiltonians in 1+1 dimensions

In this work we construct two classes of exact solutions for the most general time-dependent Dirac Hamiltonian in 1+1 dimensions. Some problems regarding to some formal solutions in the literature are discussed. Finally the existence of a generalized Lewis-Riesenfeld invariant connected with such solutions is discussed

Susceptibilidade antimicrobiana de cepas de estafilococcos coagulase-negativa isolados de ovinos de corte com mastite

Avaliou-se a sensibilidade antimicrobiana in vitro de 121 cepas de estafilococos coagulase-negativa isolada de leite de ovelhas Santa Inês, aos fármacos: penicilina, amoxicilina, ampicilina, estreptomicina, oxaciclina, neomicina, cefalotina, gentamicina e sulfonamida. A resistência à sulfonamida foi a mais frequente (27,3%), seguida pela estreptomicina (14,0%) e pela oxaciclina (14,0%), enquanto da gentamicina (1,6%) foi a menos frequente. Todas as cepas foram sensíveis a pelo menos um antimicrobiano, e 20,3% das cepas apresentaram resistência múltipla. Os resultados mostram a importância de Staphylococci coagulase-negativas como agentes causadores de mastite em ovinos, e o perfil de resistência múltipla indica a importância da determinação da resistência à oxaciclina como indicador da presença de ilhas de patogenicidade que contêm fatores de virulência e resistência a outros antimicrobianos que contribuem para a sobrevivência da bactéria ao tratamento.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Polymorphonuclear leukocytes CH138+ apoptosis evaluation in milk with high and low somatic cell count - preliminary data

A apoptose de leucócitos polimorfonucleares (PMN) é um evento central no processo de resolução da inflamação. Sendo a contagem de células somáticas (CCS) um indicador da situação imunológica da glândula mamária, o presente estudo buscou esclarecer a influência que esses fatores têm um sobre o outro e sobre a evolução do processo inflamatório. Marcaram-se as amostras de leite com anexina-V, iodeto de propídeo (PI), anticorpo anti-CH138A. Encontrou-se correlação negativa entre apoptose de PMN e CCS, além de diferença estatística entre um grupo de alta CCS e um grupo de baixa CCS quanto à taxa de PMN viáveis, em apoptose, em necrose e em necrose e/ou apoptose. De modo geral, o grupo de alta celularidade apresentou menos CH138+ em apoptose e mais células em necrose ou viáveis do que o grupo de baixa celularidade. Conclui-se que apoptose de PMN e CCS estão relacionados, e que em mamas com CCS elevada este evento está diminuído. Apesar de haver maior disponibilidade de fagócitos para a defesa nessa situação, os efeitos anti-inflamatórios da apoptose também estão diminuídos, enquanto os efeitos pró-inflamatórios da necrose estão aumentados, o que pode colaborar com a cronificação da inflamação

Cytotoxic Effects Of Zoledronic Acid On Human Epithelial Cells And Gingival Fibroblasts

Bisphosphonate-induced osteonecrosis has been related to the cytotoxicity of these drugs on oral mucosa cells. A previous study showed that 5 μM of zoledronic acid (ZA), a nitrogen-containing bisphosphonate, is the highest concentration of this drug found in the oral cavity of patients under treatment. Therefore, in order to simulate an osteonecrosis clinical condition, the aim of this study was to evaluate the highest concentration of ZA applied on human epithelial cells (HaCaT) and gingival fibroblasts. For this purpose, cells (3x104 cells/cm2) were seeded in wells for 48 h using complete culture medium (cDMEM). After 48 h incubation, the cDMEM was replaced by fresh serum-free culture medium (DMEM-FBS) in which the cells were maintained for additional 24 h. Then, 5 μM ZA were added to the DMEM-FBS and the cells incubated in contact with the drug for 48 h. After this period, the number of viable cells (trypan blue), cell viability (MTT assay), total protein (TP) production and cell morphology (SEM analysis) were assessed. Data were analyzed statistically by Mann-Whitney, ANOVA and Tukey's test (α=0.05). ZA caused a significant reduction in the number of viable cells and decreased the metabolic activity of both cell lines. However, decrease of TP production occurred only in the epithelial cell cultures. Morphological alterations were observed in both cell types treated with ZA. In conclusion, ZA (5 μM) was cytotoxic to human epithelial cells and gingival fibroblast cultures, which could be associated, clinically, with the development of bisphosphonateinduced osteonecrosis.246551558Civitelli, R., Napoli, N., Armamento-Villareal, R., Use of intravenous bisphosphonates in osteoporosis (2007) Curr Osteoporos Rep, 5, pp. 8-13Cohen, S.B., An update on bisphosphonates (2004) Curr Rheumatol Rep, 6, pp. 59-65Rogers, M.J., Watts, D.J., Russel, R.G., Overview of bisphosphonates (1997) Cancer, 80, pp. 1652-1660Rogers, M.J., Gordon, S., Benford, H.L., Coxon, F.P., Luckman, S.P., Monkkonen, J., Cellular and molecular mechanisms of action of bisphosphonates (2000) Cancer Supl, 88, pp. 2961-2978Lawson, M.A., Xia, Z., Barnett, B.L., Triffitt, J.T., Phipps, R.J., Dunford, J.E., Differences between bisphosphonates in binding affinities for hydroxyapatite (2010) J Biomed Mater Res Part B: Appl Biomater, 92, pp. 149-155Allen, M.R., Burr, D.B., The pathogenesis of bisphosphonate-related osteonecrosis of the jaw: So many hypotheses, so few data (2009) J Oral Maxillofac Surg, 67, pp. 61-70Otto, S., Pautke, C., Opelz, C., Wesphal, I., Drosse, I., Swager, J., Osteonecrosis of the jaw: Effects of bisphosphonate type, local concentration, and acidic milieu on the pathomechanism (2010) J Oral Maxillofac Surg, 68, pp. 2837-2845Reid, I.R., Booland, M.J., Is bisphosphonate-associated osteonecrosis of the jaw caused by soft tissue toxicity? (2007) Bone, 41, pp. 318-320Scheper, M.A., Badros, A., Chausparat, R., Cullen, K.J., Meiller, T.F., Effect of zoledronic acid on oral fibroblasts and epithelial cells: A potential mechanism of bisphosphonate-associated osteonecrosis (2009) Br J Haematol, 144, pp. 667-676Scheper, M.A., Badros, A., Salama, A.R., Wartburton, G., Cullen, K.J., Weikel, D.S., A novel bioassay model to determine clinically significant bisphosphonate levels (2009) Support Care Cancer, 17, pp. 1553-1557Ruggiero, S.L., Mehrotra, B., Rosenberg, T.J., Engroff, S.L., Osteonecrosis of the jaws associated with the use of bisphosphonates: A review of 63 cases (2004) J Oral Maxillofac Surg, 62, pp. 527-534Walter, C., Klein, M.O., Pabst, A., Al-Nawas, B., Duscher, H., Ziebart, T., Influence of bisphosphonates on endothelial cells, fibroblasts, and osteogenic cells (2010) Clin Oral Investig, 14, pp. 35-41Kumar, S.K.S., Gorur, A., Schaauddin, C., Shuler, C.F., Costerton, J.W., Sedghizadeh, P.P., The role of microbial biofilms in osteonecrosis of the jaw associated with bisphosphonate therapy (2010) Curr Osteoporos Rep, 8, pp. 40-48Aas, J.A., Paster, B.J., Stokes, L.N., Olsen, I., Dewhirst, F.E., Defining the normal bacterial flora of the oral cavity (2005) J Clin Microbiol, 43, pp. 5721-5732Basso, F.G., Pansani, T.N., Turrioni, A.P.S., Bagnato, V.S., Hebling, J., de Souza Costa, C.A., In vitro wound healing improvement by low-level laser therapy application in cultured gingival fibroblasts (2012) Int J Dent, , [Epub ahead of print. DOI: 10.1155/2012/719452]Wiegand, C., Hipler, U., Methods for the measurement of cell and tissue compatibility including tissue regeneration process (2008) GMS Krankenhhyg Interdiszip, 3, pp. 1863-5245Basso, F.G., Oliveira, C.F., Kurachi, C., Hebling, J., de Souza Costa, C.A., Biostimulatory effect of low-level laser therapy on keratinocytes in vitro (2013) Lasers Med Sci, 28, pp. 367-374De Souza Costa, C.A., Duarte, P.T., de Souza, P.P., Giro, E.M., Hebling, J., Cytotoxic effects and pulpal response caused by a mineral trioxide aggregate formulation and calcium hydroxide (2008) Am J Dent, 21, pp. 255-261Oliveira, C.F., Basso, F.G., Lins, E.C.C., Kurachi, C., Hebling, J., Bagnato, V.S., Increased viability of odontoblast-like cells subjected to low-level laser irradiation (2010) Laser Phys, 20, pp. 1659-1666Read, S.M., Northcote, D.H., Minimization of variation in the response to different proteins of the Coomassie blue G dye-binding assay for protein (1981) Anal Biochem, 116, pp. 53-64Oliveira, C.F., Basso, F.G., Lins, E.C., Kurachi, C., Hebling, J., Bagnato, V.S., In vitro effect of low-level laser on odontoblast-like cells (2011) Laser Phys Lett, 8, pp. 155-163Simon, M.J.K., Niehoff, P., Kimming, B., Wiltfang, J., Açil, Y., Expression profile and synthesis of different collagen types I, II III and V of human gingival fibroblasts, osteoblasts, ans SaOs-2 cells after bisphosphonate treatment (2010) Clin Oral Investig, 14, pp. 51-58Migliorati, C.A., Siegel, M.A., Elting, L.S., Bisphosphonate-associated osteonecrosis: A long-term complication of bisphosphonate treatment (2006) Lancet Oncol, 7, pp. 508-514Werner, S., Krieg, T., Smola, H., Keratinocyte-fibroblast interactions in wound healing (2007) J Investigative Dermatol, 127, pp. 998-1008Ravosa, M.J., Ning, J., Liu, Y., Stack, M.S., Bisphosphonate effects on the behavior of oral epithelial cells and oral fibroblasts (2011) Arch Oral Biol, 56, pp. 491-49

USE OF COMBINATION OF FLUORESCENT PROBES TO IDENTIFY SPERM SUBPOPULATIONS FOR QUALITY ASSESSMENT OF FRESH AND CRYOPRESERVED CANINE SEMEN. PRELIMINARY RESULTS

The use of fluorescent markers in the evaluation of sperm morphophysiology allows a better accuracy, compared to the subjective nature of some routine tests in semen qualification. In this study was used the combination of fluorescence probes: propionate iodide, Hoechst 33342 and FITC-PSA in fresh and thawed dog semen, to the identification of the following morphological subpopulations: II (intact plasma and acrosomal membranes), IL (intact plasma membrane and lesioned acrosomal membrane), LI (lesioned plasma membrane and intact acrosomal membrane) and LL (both membranes lesioned). When comparing the results obtained with the results of the tests used conventionally in semen evaluation (sperm motility and vigor, hypoosmotic test and morphological alterations), little correlation was observed. The II population declined from fresh semen to thawed, while LL population increased (p <0.05). The IL population was composed of extremely small numbers of cells but increased (p <0.05) from fresh semen to thawed semen. In the thawed semen the major defects had a positive correlation with the LL population (p <0.01). For the thawed semen, the results of the hypoosmotic test (number of cells that reacted to the medium) correlated positively with population II (p <0.025), that is, different from that observed in fresh semen. Although all tests were able to detect decrease in sperm quality post-thawing (p <0.05). The use of this fluorescent probe association allowed qualification and more accurately quantification of plasma membrane and acrosomal insults mediated by cryopreservation. El uso de marcadores fluorescentes en la evaluación de la morfofisiología espermática permite una mayor precisión, comparada con la naturaleza subjetiva de algunas pruebas de rutina en la valoración del semen. En este estudio se usó la combinación de pruebas fluorescentes: yoduro de propidio; Hoechst 33342 y FITC-PSA en semen fresco y descongelado de perro, para la identificación de las siguientes subpoblaciones morfológicas: II (membranas plasmática y acrosomal intactas), IL (membrana plasmática intacta y membrana acrosomal dañada), LI (membrana plasmática dañada y membrana acrosomal intacta) y LL (ambas membranas dañadas). Cuando se comparan los resultados obtenidos con los resultados de las pruebas usadas convencionalmente en la evaluación seminal (motilidad y vigor espermáticos, prueba hipoosmótica y alteraciones morfológicas), se observó poca correlación. La población II disminuyó desde el semen fresco al descongelado, mientras que la población LL se incrementó (p<0.05). la población IL estuvo compuesta por un número extremadamente pequeño de células, pero incremento (p<0.05) desde el semen fresco al descongelado. En el semen descongelado los defectos mayores tuvieron una correlación positiva con la población LL (p<0.01). En el semen descongelado, los resultados de la prueba hipoosmótica (número de células que reaccionan al medio) se correlacionaron positivamente con la población II (p<0.05). El uso de esta asociación de pruebas fluorescentes permitió la valoración y la cuantificación más precisa de los daños a la membrana plasmática y acrosomal mediados por la criopreservación.

In Vitro Effect Of Low-level Laser Therapy On Typical Oral Microbial Biofilms

The aim of this study was to evaluate the effect of specific parameters of low-level laser therapy (LLLT) on biofilms formed by Streptococcus mutans, Candida albicans or an association of both species. Single and dual-species biofilms - SSB and DSB - were exposed to laser doses of 5, 10 or 20 J/cm 2 from a near infrared InGaAsP diode laser prototype (LASERTable; 780 ± 3 nm, 0.04 W). After irradiation, the analysis of biobilm viability (MTT assay), biofilm growth (cfu/mL) and cell morphology (SEM) showed that LLLT reduced cell viability as well as the growth of biofilms. The response of S. mutans (SSB) to irradiation was similar for all laser doses and the biofilm growth was dose dependent. However, when associated with C. albicans (DSB), S. mutans was resistant to LLLT. For C. albicans, the association with S. mutans (DSB) caused a significant decrease in biofilm growth in a dose-dependent fashion. The morphology of the microorganisms in the SSB was not altered by LLLT, while the association of microbial species (DSB) promoted a reduction in the formation of C. albicans hyphae. LLLT had an inhibitory effect on the microorganisms, and this capacity can be altered according to the interactions between different microbial species.226502510Marques, M.M., Pereira, A.N., Fujihara, N.A., Nogueira, F.N., Eduardo, C.P., Effect of low-power laser irradiation on protein synthesis and ultrastructure of human gingival fibroblasts (2004) Lasers Surg Med, 34, pp. 260-265Damante, C.A., de Micheli, G., Miyagi, S.P.H., Feist, I.S., Marques, M.M., Effect of laser phototherapy on the release of fibroblast growth factors by human gingival fibroblasts (2009) Lasers Med Sci, 24, pp. 885-891Moritz, A., Schoop, U., Goharkhay, K., Schauer, P., Doertbudak, O., Wernisch, J., Treatment of periodontal pockets with a diode laser (1998) Lasers Surg Med, 22, pp. 302-311Nussbaum, E.L., Lilge, L., Mazzulli, T., Effects of 630-, 660-, 810-, and 905-nm laser irradiation delivering radiant exposure of 1-50 J/cm 2 on three species of bacteria in vitro (2002) J Clin Laser Med Surg, 20, pp. 325-333Nussbaum, E.L., Lilge, L., Mazzulli, T., Effects of low-level laser therapy (LLLT) of 810 nm upon in vitro growth of bacteria: Relevance of irradiance and radiant exposure (2003) J Clin Laser Med Surg, 21, pp. 283-290Lino, M.D.M.C., Carvalho, F.B., Oliveira, L.R., Magalhães, E.B., Pinheiro, A.L.B., Ramalho, L.M.P., Laser phototherapy as a treatment for radiotherapy-induced oral mucositis (2011) Braz Dent J, 22, pp. 162-165Maver-Biscanin, M., Mravak-Stipetic, M., Jerolimov, V., Biscanin, A., Fungicidal effect of diode laser irradiation in patients with denture stomatitis (2004) Lasers Surg Med, 35, pp. 259-262Dworkin, M., Endogenous photosensitization in a carotinoidless mutant of Rhodopseudomonas speroides (1958) J Gen Physiol, 43, pp. 1099-1112Rosenberg, B., Kemeny, G., Switzer, R.C., Hamilton, T.C., Quantitative evidence for protein denaturation as the cause of thermal death (1971) Nature, 232, pp. 471-473Krespi, Y.P., Kizhner, V., Nistico, L., Hall-Stoodley, L., Stoodley, P., Laser disruption and killing of methicillin-resistant Staphylococcus aureus biofilms (2011) Am J Otolaryngol, 32, pp. 198-202Shirtliff, M.E., Peters, B.M., Jabra-Rizk, M.A., Cross-kingdom interactions: Candida albicans and bacteria (2009) FEMS Microbiol Lett, 299, pp. 1-8Pereira-Cenci, T., Deng, D.M., Kraneveld, E.A., Manders, E.M.M., del Bel, C.A.A., ten Cate, J.M., The effect of Streptococcus mutans and Candida glabrata on Candida albicans biofilms formed on different surfaces (2008) Arch Oral Biol, 53, pp. 755-764Marsh, P.D., Microbial ecology of dental plaque and its significance in health and disease (1994) Adv Dent Res, 8, pp. 263-271Karkowska-Kuleta, J., Rapala-Kozik, M., Kozik, A., Fungi pathogenic to humans: Molecular bases of virulence of Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus (2009) Acta Biochim Pol, 56, pp. 211-224Thein, Z.M., Samaranayake, Y.H., Samaranayake, L.P., Dietary sugars, serum and the biocide chlorhexidine digluconate modify the population and structural dynamics of mixed Candida albicans and Escherichia coli biofilms (2007) APMIS, 115, pp. 1241-1251Kwieciński, J., Eick, S., Wójcik, K., Effects of tea tre (Melaleuca alternifolia) oil on Staphylococcus aureus in biofilms and stationary phase (2009) Int J Antimicrob Agents, 33, pp. 343-347Wang, Z.C., Fan, L.Y., Jiang, J.Q., Cai, W., Ding, Y., Study on the counting of Streptococcus mutans, Streptococcus sanguis, Haemophilus actinomycetemcomitans by methyl thiazolyl tetrazolium colorimetric method (2010) Hua Xi Kou Qiang Yi Xue Za Zhi, 28, pp. 306-310Nguyen, P.T.M., Abranches, J., Phan, T., Marquis, R.E., Repressed respiration of oral Streptococci grow in biofilms (2002) Curr Microbiol, 44, pp. 262-266Singleton, S., Treloar, R., Warren, P., Watson, G.K., Hodgson, R., Allison, C., Methods for microscopic characterization of oral biofilms: Analysis of colonization, microstructure, and molecular transport phenomena (1997) Adv Dent Res, 11, pp. 133-149Jarosz, L.M., Deng, D.M., van der Mei, H.C., Crielaard, W., Krom, B.P., Streptococcus mutans competence-stimulating peptide inhibits Candida albicans hypha formation (2009) Eukaryot Cell, 8, pp. 1658-1664Dortbudak, O., Haas, R., Bernhart, T., Mailath-Pokorny, G., Lethal photosensitization for decontamination of implant surface in the treatment of peri-implantitis (2001) Clin Oral Implant Res, 12, pp. 104-10

Highly Endemic, Waterborne Toxoplasmosis in North Rio de Janeiro State, Brazil

In Campos dos Goytacazes, northern Rio de Janeiro state, Brazil, reports of uveitis consistent with toxoplasmosis led to a survey of the prevalence and risk factors for Toxoplasma gondii infection in 1997–1999. The survey population was selected randomly from schools, randomly chosen communities, and an army battalion. Serum samples from 1,436 persons were tested. With results adjusted for age, 84% of the population in the lower socioeconomic group was seropositive, compared with 62% and 23% of the middle and upper socioeconomic groups, respectively (p<0.001). When multivariate analysis was performed, drinking unfiltered water was found to increase the risk of seropositivity for the lower socioeconomic (odds ratio [OR]: 3.0, 95% confidence interval [CI] 1.3 to 6.9) and middle socioeconomic (OR: 1.7, 95% CI 1.2 to 2.3) populations. We also found a high T. gondii seroprevalence in this Brazilian community. Drinking unfiltered water increased the risk of T. gondii seropositivity, indicating the potential importance of oocyst transmission in water in this region