Measurement of eroded dentine tubule patency and roughness following novel dab-on or brushing abrasion

Objectives To investigate the effect of dab-on or brushing of stannous-fluoride SnF2 or sodium-fluoride NaF dentifrice on eroded dentine tubule patency, surface and inter-tubular dentine roughness, using Confocal-Laser-Scanning-Microscopy (CLSM), Atomic-Force-Microscopy (AFM), Energy-Dispersive-X-ray-Spectroscopy (EDX), Scanning-Electron-Microscopy (SEM) and Contact-Profilometry (CP). Methods 75-polished human dentine samples were prepared and eroded in agitated 6% citric acid to expose patent tubules and ‘initiate’ DH. Samples were randomly allocated into 5 intervention groups; artificial saliva control (1); electric tooth-brushing with NaF (2) or SnF2 (3), and dab-on application of NaF (4) or SnF2 (5). Samples underwent three cycles of intervention followed by further acid challenge. Patent tubules, likely to cause DH clinically, were measured using validated biocomputational methods with CLSM images of dentine surfaces taken baseline and post-intervention. Randomised samples (n = 15, 20 %) were investigated using AFM, EDX and SEM to study surface and sub-surface tubular occlusion. Dentine surface and inter-tubular roughness were measured using CP and AFM respectively. Results At baseline, mean tubule patency in all samples was 216 (SD 58) with no significant inter-group differences. Post-intervention, the mean patency was 220 (40) and 208 (35) in groups 1 and 2 respectively (p ≥ 0.06), but decreased to 62 (41), 62 (21) and 63 (19) in groups 3, 4 and 5 respectively (p < 0.0001). Patency was confirmed using AFM, SEM and EDX. SnF2 interventions created greater sub-surface occlusion (p < 0.01), and increased CP surface roughness (p = 0.015). Significant negative correlation (-0.6) existed between CP surface roughness and tubule patency (p = 0.009). Conclusions Dab-on with NaF and SnF2 or brushing with SnF2 reduces DH in eroded dentine with ongoing acid challenges. Contacting surface roughness measures indicate risk of DH. Clinical significance Dab-on is a convenient supplementary method of dentifrice application to reduce DH; it beneficially avoids brushing post-erosion or overzealous brushing, enables re-establishment of an appropriate brushing regime post-DH and supports oral health. Significant modes of action of SnF2 in reducing DH are revealed. Finally, CP roughness measures provide indication of dentine lesions that may cause DH clinically

Contribution to unveiling the roles played by small non-coding RNAs in the biology and pathogenesis of Burkholderia cepacia complex bacteria

Tese de mestrado, Microbiologia Aplicada, Universidade de Lisboa, Faculdade de Ciências, 2017The development of high-throughput sequencing techniques and continued decrease of associated costs, together with advances in bioinformatics and increasing availability of powerful software for nucleotide and amino acid sequence analyses contributed to the exponential increase of available microbial genomes. These developments have unveiled several novel sequences presenting a size within the range of 50-500 nucleotides (nt) and encoding the now called small non-coding regulatory RNAs (sRNAs). These sRNAs are frequently encoded in intergenic regions, often partially overlapping with the 5’ or 3’ untranslated regions of the vicinal genes or annotated-opening reading frames (ORFs). The current known function for some sRNAs comprises the gene expression regulation through interference RNA mechanisms, mainly at the posttranscriptional level. Bacterial sRNAs may regulate their targets (messenger RNAs) through positive or negative regulation mechanisms. To carry out a negative regulation, the sRNA base pairs with its target in the region containing the initiation codon and/or the ribosome-binding site (RBS), precluding the ribosome binding and consequently preventing the mRNA translation. The Burkholderia cepacia (Bcc) complex comprises nowadays 20 validated and phylogenetically-related species of Gram-negative bacteria. These bacteria are phenotypically similar and genotypically distinct, being widely distributed in different ecological niches such as soil, water, plants, animals and in humans. Some Bcc bacteria have potential application as bioremediation and biocontrol/biopesticides agents due to their unusual metabolic abilities, which include xenobiotics metabolism, production of antifungal compounds and promotion of plant growth. However, Burkholderia species have emerged in 1980s as important opportunistic pathogens, especially to cystic fibrosis (CF) patients. Bcc bacteria have gained the attention of medical and scientific community because they can cause in CF patients a rapid evolving clinical state known as the cepacia syndrome, which can cause a fast and necrotizing pneumonia, septicemia and ultimately results in the patient early death. Bcc bacteria produce a wide variety of virulence factors, possessing intrinsic resistance mechanisms to different antibiotics which lead to a difficult eradication. Over the last years, some Bcc species have also been recognized as emerging nosocomial pathogenic agents in hospitalized non-CF patients, particularly in cancer patients. To identify potential genes involved in the Bcc bacteria virulence, researchers from iBB (Instituto de Bioengenharia e Biociências) prepared mutant libraries derived from B. contaminans IST408 and B. cenocepacia J2315 by random plasposon mutagenesis. This work aims to characterize a mutant derived from B. cenocepacia J2315, identified in an attenuated virulence screening using as model of infection the nematode Caenorhabditis elegans. This mutant, B. cenocepacia SJ2, contains the plasposon inserted in the intergenic region of B. cenocepacia J2315 chromosome 1, located between the coding gene of DNA gyrase subunit A (GyrA) enzyme and the coding gene of an outer membrane protein of the OmpA family. Bioinformatic and Southern blot analyses allowed the identification of the plasposon insertion in the mutant B. cenocepacia SJ2. The plasposon is located 8 nucleotides (nt) after the predicted 5’ UTR beginning of the B. cenocepacia J2315 OmpA-like protein. Therefore, the presence and functionality of the transcript ompA were evaluated by RT-PCR and Western blot, respectively. The results obtained indicated that the plasposon insertion did not affect ompA mRNA transcription or functionality. Bioinformatic analyses also led to the identification of a possible opening reading frame (ORF) in the complementary strand (named ORF3) and encoding a 93 amino acids protein. The presence of this ORF was supported by Northern blot assays previously performed. These assays allowed the identification of a transcript from the complementary strand. However, the amplification of complementary DNA ends (5’ and 3’ RACE) using specific primers for ORF3 region demonstrated that the transcript in study has a 5’ end with a lower size than the expected transcript of the potential protein (ORF3). A transcript containing approximately 179 base pairs and non-interrupted by the plasposon was identified. In the presence of these results, primers for the potential DNA sequence of ORF3 were designed and the results showed that this is the region interrupted by the plasposon. Due to the absence of initiation and termination codons in the region closer to the 179 base pairs transcript, it was hypothesized that the transcript corresponds to a sRNA (MavA). In this work, the presence of two genetic elements, a sRNA and a protein, in the intergenic region of B. cenocepacia J2315 chromosome 1 is described. The coding sequences of both the sRNA and protein are overlapped in, at least, 100 nucleotides. Bioinformatic analysis of the genetic elements showed that they are conserved among the Burkholderia genus. Moreover, the sequence of ORF3 protein also showed to be identical to the sequence of one protein encoded by an annotated ORF in the B. multivorans genome and to five proteins of B. pseudomallei. The assays performed with the strain containing the ORF3 interrupted by the plasposon revealed the involvement of this protein in the resistance of B. cenocepacia J2315 strain to heat-shock stress (50 ºC), susceptibility to the detergent sodium dodecyl sulphate (SDS), biofilms formation, cellular hydrophobicity and antibiotics resistance (imipenem, ceftazidime and tetracycline). In general, these results suggest that this protein is likely involved in the maintenance of the outer membrane integrity and virulence of B. cenocepacia J2315 towards the nematode C. elegans. MavA sRNA identified in the work herein presented was predicted to be a functional homologue of IstR-2 sRNA of Escherichia coli K-12 MG1655. Preliminary results of the MavA sRNA overexpression characterization indicate a possible direct or indirect role in the overexpression of ribosomal protein S12. Phenotypic analysis also showed the involvement in the swimming motility of B. cenocepacia J2315 strain and in resistance to thermal (50 ºC) stress. In addition, this sRNA is not involved in the virulence of B. cenocepacia J2315 towards the C. elegans nematode. Overall, the results presented in this study contribute to a better knowledge of the intergenic region under study and to the identification of a putative ORF and a sRNA, contributing to a better understanding of the biology of Bcc bacteria and the role of sRNAs in the regulation of the expression of putative virulence factors. The identification of MavA sRNA and ORF3 protein highlight the importance of these studies in identifying genetic elements that might be exploited as targets for the development of effective treatments to the B. cenocepacia J2315 strain infections.O recente desenvolvimento de técnicas de elevado rendimento para sequenciar genomas e a diminuição contínua do custo associado, conjuntamente com os avanços na bioinformática e a disponibilidade de número crescente de ferramentas bioinformáticas para análise de sequências nucleotídicas e aminoacídicas, tornou possível o aumento exponencial dos genomas microbianos disponíveis. Consequentemente, têm vindo a ser descobertos diversos pequenos transcritos com ação regulatória denominados de pequenos RNAs reguladores não codificantes (sRNAs), com tamanhos compreendidos entre 50-500 nucleótidos. Estes sRNAs têm sido identificados essencialmente em regiões intergénicas, junto a genes ou grelhas de leitura (ORFs) anotadas. A função conhecida de um grupo importante desses sRNAs consiste na regulação da expressão génica, principalmente ao nível pós-transcricional, por mecanismos de interferência de RNA. Os sRNAs bacterianos podem exercer nos seus alvos (RNAs mensageiros) uma regulação positiva ou negativa. No caso de ser exercida uma regulação negativa o sRNA estabelece com o seu alvo um emparelhamento na região que contém o codão de iniciação e/ou a região de ligação do ribossoma (RBS), tornando este local inacessível para a ligação do ribossoma e impedindo, assim, a tradução do mRNA. O complexo Burkholderia cepacia (Bcc) é atualmente constituído por 20 espécies de bactérias Gram-negativas validadas e filogeneticamente próximas. As bactérias que compõem este grupo são fenotipicamente semelhantes e genotipicamente distintas, podendo ser isoladas de várias fontes. São exemplo de fontes o solo, a água, a rizosfera de plantas, animais e Humanos. Devido às suas capacidades metabólicas invulgares, algumas estirpes do Bcc apresentam um elevado potencial de aplicação ao nível do biocontrolo, biorremediação e agricultura, pois são capazes de metabolizar xenobióticos, produzir compostos com atividade antifúngica e promover o crescimento de plantas. Contudo, na década de 80 as bactérias incluídas no género Burkholderia, incluindo as englobadas no complexo Bcc, emergiram como agentes patogénicos oportunistas em indivíduos com fibrose quística (FQ). O facto de as bactérias deste complexo produzirem vários fatores de virulência, apresentarem mecanismos de resistência a um largo espetro de antibióticos sendo difíceis de erradicar e de causarem em doentes com FQ infeções por vezes acompanhadas por um estado clínico de evolução rápida que inclui pneumonia necrotizante e septicémia, levando à morte do doente – síndrome cepacia -, faz com que tenham merecido uma elevada atenção da comunidade médica e científica. Acresce que algumas espécies do Bcc têm vindo a ser reconhecidas nos últimos anos como sendo agentes patogénicos nosocomiais emergentes em doentes hospitalizados, principalmente em doentes oncológicos. Com o objetivo de identificar possíveis genes envolvidos na virulência de bactérias do Bcc, investigadores do iBB (Instituto de Bioengenharia e Biociências) construíram bibliotecas de mutantes de B. contaminans IST408 e B. cenocepacia J2315 utilizando plasposões. As bibliotecas de mutantes foram rastreadas tendo como objetivo a identificação de mutantes que exibiam virulência atenuada, utilizando como modelo de infeção o nemátodo Caenorhabditis elegans. O trabalho aqui apresentado teve como objetivo a caracterização de um mutante identificado no rastreio acima mencionado, contendo um plasposão inserido na região intergénica do cromossoma 1 de B. cenocepacia J2315, localizada entre o gene codificante da subunidade A da enzima ADN girase (GyrA) e o gene codificante de uma proteína da membrana externa do tipo A (OmpA). Através de análises bioinformáticas e experimentais, foi possível localizar no mutante B. cenocepacia SJ2 a inserção do plasposão 8 nucleótidos (nt) após o início da região 5’ não traduzida (5’ UTR) do gene que codifica a proteína do tipo OmpA. Neste sentido, foram avaliados a presença e a funcionalidade do transcrito ompA por ensaios de RT-PCR e Western blot, respetivamente. Os resultados obtidos confirmaram que a inserção do plasposão não afetou a transcrição nem a funcionalidade do transcrito correspondente ao gene ompA. As análises bioinformáticas realizadas permitiram ainda a identificação de uma possível grelha de leitura aberta (ORF) na cadeia complementar da região intergénica (designada de ORF3), codificando para uma proteína de aproximadamente 93 aminoácidos. A confirmação da presença desta ORF foi suportada por ensaios de Northern blot realizados anteriormente e que permitiram a identificação de um transcrito a partir da cadeia complementar. No entanto, análises de amplificação em cadeia pela polimerase (PCR) das extremidades do ADN complementar (5’ e 3’ RACE) com sequências oligonucleotídicas iniciadoras específicas para a região da ORF3 demonstraram que o transcrito em causa possui uma extremidade 5’ menor que o esperado para o transcrito da possível proteína (ORF3), permitindo assim a identificação de um transcrito com cerca de 179 pares de bases que se verificou não ser interrompido pelo plasposão. Deste modo, tendo em conta os resultados aqui apresentados foram desenhadas sequências oligonucleotídicas iniciadoras específicas para a sequência de ADN da possível ORF3, tendo-se verificado que esta era a região interrompida pelo plasposão. Devido à ausência de codões de iniciação e de terminação na região próxima do transcrito de 179 pares de base, colocou-se a hipótese deste transcrito corresponder a outro elemento genético, nomeadamente um sRNA, que se denominou de MavA. Neste trabalho é descrita a existência de dois elementos genéticos na região intergénica do cromossoma 1 de B. cenocepacia J2315, um sRNA e um gene que codifica uma proteína, sendo que as sequências codificantes de ambos encontram-se parcialmente sobrepostas em, pelo menos, 100 nucleótidos. As análises bioinformáticas realizadas dos elementos genéticos permitiram a sua identificação como sendo ambos conservados no género Burkholderia. Além disso, demonstraram também que a proteína ORF3 é idêntica à sequência de uma proteína codificada por uma ORF anotada no genoma de B. multivorans e à de cinco proteínas de B. pseudomallei. Ensaios realizados com a estirpe contendo o plasposão a interromper a ORF3 demonstraram que esta proteína está envolvida na resistência da estirpe B. cenocepacia J2315 a choque térmico (50 ºC), suscetibilidade à presença do detergente dodecil sulfato de sódio (SDS), capacidade para formação de biofilmes e hidrofobicidade das células, bem como na resistência a antibióticos (imipeneme, ceftazidima e tetraciclina). De um modo geral, estes resultados apontam para que esta proteína esteja envolvida na manutenção da integridade da membrana externa e virulência da estirpe B. cenocepacia J2315 para o nemátodo C. elegans. O sRNA MavA identificado neste trabalho foi bioinformaticamente previsto como tendo como seu homólogo funcional o sRNA IstR-2 de Escherichia coli K-12 MG1655. Os resultados preliminares da caracterização do sRNA MavA através da sua sobrexpressão demonstram um possível envolvimento direto ou indireto na sobrexpressão da proteína ribossomal S12. As análises fenotípicas mostraram ainda o seu envolvimento na mobilidade da estirpe B. cenocepacia J2315 por swimming e na resistência ao choque térmico (50 ºC). Os resultados obtidos, baseados em ensaios de morte lenta do nemátodo C. elegans sugerem que este sRNA não está envolvido na virulência de B. cenocepacia J2315 neste modelo animal de infeção. Os resultados obtidos no presente trabalho contribuem para um conhecimento mais aprofundado da região intergénica estudada, tendo permitido identificar 2 elementos genéticos parcialmente sobrepostos que codificam uma proteína e um sRNA. Este trabalho constitui assim um contributo para o melhor conhecimento da biologia das bactérias do complexo Bcc e do papel dos sRNAs na regulação da expressão de fatores de virulência. A identificação do sRNA MavA e da proteína ORF3 realça a importância deste tipo de estudos na identificação de elementos genéticos que possam ser explorados como alvos para o desenvolvimento de estratégias terapêuticas mais eficazes com vista ao tratamento das infeções causadas pela estirpe B. cenocepacia J2315

De novo assembly of the rooibos genome

>Magister Scientiae - MScRooibos (Aspalathus linearis) is endemic to the Cederberg region of South Africa, and one of the few indigenous medicinal plants commercially cultivated in the country. International interest in rooibos is growing, and currently most of the rooibos harvest is exported overseas to more than 30 countries. Various problems hamper the growth of the rooibos industry, including insect pests, diseases, drought and a decreasing lifespan of the plants. The availability of whole-genome data for rooibos can contribute to the selection of genetically superior plants, facilitating not only the identification of important genes and metabolic pathways in rooibos, but also the establishment of breeding programs

Data Science techniques for predicting plant genes involved in secondary metabolites production

Masters of SciencePlant genome analysis is currently experiencing a boost due to reduced costs associated with the development of next generation sequencing technologies. Knowledge on genetic background can be applied to guide targeted plant selection and breeding, and to facilitate natural product discovery and biological engineering. In medicinal plants, secondary metabolites are of particular interest because they often represent the main active ingredients associated with health-promoting qualities. Plant polyphenols are a highly diverse family of aromatic secondary metabolites that act as antimicrobial agents, UV protectants, and insect or herbivore repellents. Most of the genome mining tools developed to understand genetic materials have very seldom addressed secondary metabolite genes and biosynthesis pathways. Little significant research has been conducted to study key enzyme factors that can predict a class of secondary metabolite genes from polyketide synthases. The objectives of this study were twofold: Primarily, it aimed to identify the biological properties of secondary metabolite genes and the selection of a specific gene, naringenin-chalcone synthase or chalcone synthase (CHS). The study hypothesized that data science approaches in mining biological data, particularly secondary metabolite genes, would enable the compulsory disclosure of some aspects of secondary metabolite (SM). Secondarily, the aim was to propose a proof of concept for classifying or predicting plant genes involved in polyphenol biosynthesis from data science techniques and convey these techniques in computational analysis through machine learning algorithms and mathematical and statistical approaches. Three specific challenges experienced while analysing secondary metabolite datasets were: 1) class imbalance, which refers to lack of proportionality among protein sequence classes; 2) high dimensionality, which alludes to a phenomenon feature space that arises when analysing bioinformatics datasets; and 3) the difference in protein sequences lengths, which alludes to a phenomenon that protein sequences have different lengths. Considering these inherent issues, developing precise classification models and statistical models proves a challenge. Therefore, the prerequisite for effective SM plant gene mining is dedicated data science techniques that can collect, prepare and analyse SM genes

Imaging, Tracking and Computational Analyses of Virus Entry and Egress with the Cytoskeleton

Viruses have a dual nature: particles are &ldquo;passive substances&rdquo; lacking chemical energy transformation, whereas infected cells are &ldquo;active substances&rdquo; turning-over energy. How passive viral substances convert to active substances, comprising viral replication and assembly compartments has been of intense interest to virologists, cell and molecular biologists and immunologists. Infection starts with virus entry into a susceptible cell and delivers the viral genome to the replication site. This is a multi-step process, and involves the cytoskeleton and associated motor proteins. Likewise, the egress of progeny virus particles from the replication site to the extracellular space is enhanced by the cytoskeleton and associated motor proteins. This overcomes the limitation of thermal diffusion, and transports virions and virion components, often in association with cellular organelles. This review explores how the analysis of viral trajectories informs about mechanisms of infection. We discuss the methodology enabling researchers to visualize single virions in cells by fluorescence imaging and tracking. Virus visualization and tracking are increasingly enhanced by computational analyses of virus trajectories as well as in silico modeling. Combined approaches reveal previously unrecognized features of virus-infected cells. Using select examples of complementary methodology, we highlight the role of actin filaments and microtubules, and their associated motors in virus infections. In-depth studies of single virion dynamics at high temporal and spatial resolutions thereby provide deep insight into virus infection processes, and are a basis for uncovering underlying mechanisms of how cells function

Acidic Pericellular Ph: Effects On Proteolysis And Gene Expression As Determined In 3d Models Of Breast Carcinoma

Among the non-cellular microenvironmental factors that contribute to malignancy of solid tumors is an acidic peritumoral pH. The first objective was to determine if an acidic extracellular pH observed in vivo (i.e., pHe 6.8) affects the activity of proteases, such as cathepsin B, that contribute to degradation of collagen IV by tumor cells when grown in biologically relevant three-dimensional cultures. At pHe 6.8 there were increases in pericellular active cysteine cathepsins and in degradation of DQ-collagen IV, which was partially blocked by a cathepsin B inhibitor. Imaging probes for active cysteine cathepsins localized to tumors in vivo. The amount of bound probe decreased in tumors in bicarbonate-treated mice, a treatment previously shown to increase peritumoral pHe and reduce local invasion of the tumors. Our results are consistent with the acid-mediated invasion hypothesis and with a role for cathepsin B in promoting degradation of a basement membrane protein substrate, i.e. type IV collagen, in an acidic peritumoral environment. The second objective was to use a microarray approach to identify significantly altered genes that may be of importance in the response to acidic pH in ductal carcinoma in situ (DCIS). We have found that STAT1-directed signaling is up-regulated in response to acidic pH extracellularly. Its role in altering glycolytic pathways suggests an important role for this transcription factor in the development of an acidic extracellular pH

J Mol Diagn

This report of the Whole Genome Analysis group of the Association for Molecular Pathology illuminates the opportunities and challenges associated with clinical diagnostic genome sequencing. With the reality of clinical application of next-generation sequencing, technical aspects of molecular testing can be accomplished at greater speed and with higher volume, while much information is obtained. Although this testing is a next logical step for molecular pathology laboratories, the potential impact on the diagnostic process and clinical correlations is extraordinary and clinical interpretation will be challenging. We review the rapidly evolving technologies; provide application examples; discuss aspects of clinical utility, ethics, and consent; and address the analytic, postanalytic, and professional implications.CC999999/Intramural CDC HHS/United States2019-05-07T00:00:00Z22918138PMC65041716242vault:3210