Cellular polarity in aging: role of redox regulation and nutrition

Cellular polarity concerns the spatial asymmetric organization of cellular components and structures. Such organization is important not only for biological behavior at the individual cell level, but also for the 3D organization of tissues and organs in living organisms. Processes like cell migration and motility, asymmetric inheritance, and spatial organization of daughter cells in tissues are all dependent of cell polarity. Many of these processes are compromised during aging and cellular senescence. For example, permeability epithelium barriers are leakier during aging; elderly people have impaired vascular function and increased frequency of cancer, and asymmetrical inheritance is compromised in senescent cells, including stem cells. Here, we review the cellular regulation of polarity, as well as the signaling mechanisms and respective redox regulation of the pathways involved in defining cellular polarity. Emphasis will be put on the role of cytoskeleton and the AMP-activated protein kinase pathway. We also discuss how nutrients can affect polarity-dependent processes, both by direct exposure of the gastrointestinal epithelium to nutrients and by indirect effects elicited by the metabolism of nutrients, such as activation of antioxidant response and phase-II detoxification enzymes through the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2). In summary, cellular polarity emerges as a key process whose redox deregulation is hypothesized to have a central role in aging and cellular senescence.

Translation termination and protein folding pathway genes are not correlated in gastric cancer

Background: The eukaryotic release factor 3 (eRF3) has been shown to affect both tubulin and actin cytoskeleton, suggesting a role in cytoskeleton assembly, mitotic spindle formation and chromosome segregation. Also, direct interactions between eRF3 and subunits of the cytosolic chaperonin CCT have been described. Moreover, both eRF3a and CCT subunits have been described to be up-regulated in cancer tissues. Our aim was to evaluate the hypothesis that eRF3 expression levels are correlated with the expression of genes encoding proteins involved in the tubulin folding pathways. Methods: Relative expression levels of eRF1, eRF3a/GSPT1, PFDN4, CCT2, CCT4, and TBCA genes in tumour samples relative to their adjacent normal tissues were investigated using real time-polymerase chain reaction in 20 gastric cancer patients. Results: The expression levels of eRF3a/GSPT1 were not correlated with the expression levels of the other genes studied. However, significant correlations were detected between the other genes, both within intestinal and diffuse type tumours. Conclusions: eRF3a/GSPT1 expression at the mRNA level is independent from both cell translation rates and from the expression of the genes involved in tubulin-folding pathways. The differences in the patterns of expression of the genes studied support the hypothesis of genetically independent pathways in the origin of intestinal and diffuse type gastric tumours.

Fat intake interacts with polymorphisms of Caspase9, FasLigand and PPARgamma apoptotic genes in modulating Crohn's disease activity

Background & aims: Crohn’s disease (CD) is a multifactorial disease where resistance to apoptosis is one major defect. Also, dietary fat intake has been shown to modulate disease activity. We aimed to explore the interaction between four single nucleotide polymorphisms (SNPs) in apoptotic genes and dietary fat intake in modulating disease activity in CD patients. Methods: Polymerase Chain Reaction (PCR) and Restriction Fragment Length Polymorphism (RFLP) techniques were used to analyze Caspase9þ93C/T, FasLigand-843C/T, Peroxisome Proliferator-Activated Receptor gammaþ161C/T and Peroxisome Proliferator-Activated Receptor gamma Pro12Ala SNPs in 99 patients with CD and 116 healthy controls. Interactions between SNPs and fat intake in modulating disease activity were analyzed using regression analysis. Results: None of the polymorphisms analyzed influenced disease susceptibility and/or activity, but a high intake of total, saturated and monounsaturated fats and a higher ratio of n-6/n-3 polyunsaturated fatty acids (PUFA), was associated with a more active phenotype (p < 0.05). We observed that the detrimental effect of a high intake of total and trans fat was more marked in wild type carriers of the Caspase9þ93C/T polymorphism [O.R (95%CI) 4.64 (1.27e16.89) and O.R (95%CI) 4.84 (1.34e17.50)]. In the Peroxisome Proliferator-Activated Receptor gamma Pro12Ala SNP, we also observed that a high intake of saturated and monounsaturated fat was associated to a more active disease in wild type carriers [OR (95%CI) 4.21 (1.33e13.26) and 4.37 (1.52e12.51)]. Finally, a high intake of n-6 PUFA was associated with a more active disease in wild type carriers for the FasLigand-843C/T polymorphism [O.R (95%CI) 5.15 (1.07e24.74)]. Conclusions: To our knowledge, this is the first study to disclose a synergism between fat intake and SNPs in apoptotic genes in modulating disease activity in CD patients.

Gene expression regulation and lineage evolution: the North and South tale of the hybrid polyploid Squalius alburnoides complex

The evolution of hybrid polyploid vertebrates, their viability and their perpetuation over evolutionary time have always been questions of great interest. However, little is known about the impact of hybridization and polyploidization on the regulatory networks that guarantee the appropriate quantitative and qualitative gene expression programme. The Squalius alburnoides complex of hybrid fish is an attractive system to address these questions, as it includes a wide variety of diploid and polyploid forms, and intricate systems of genetic exchange. Through the study of genome-specific allele expression of seven housekeeping and tissue-specific genes, we found that a gene copy silencing mechanism of dosage compensation exists throughout the distribution range of the complex. Here we show that the allele-specific patterns of silencing vary within the complex, according to the geographical origin and the type of genome involved in the hybridization process. In southern populations, triploids of S. alburnoides show an overall tendency for silencing the allele from the minority genome, while northern population polyploids exhibit preferential biallelic gene expression patterns, irrespective of genomic composition. The present findings further suggest that gene copy silencing and variable expression of specific allele combinations may be important processes in vertebrate polyploid evolution.

Modulation of translation factor's gene expression by histone deacetylase inhibitors in breast cancer cells

The histone deacetylase inhibitors sodium butyrate (NaBu) and trichostatin A (TSA) exhibit anti-proliferative activity by causing cell cycle arrest and apoptosis. The mechanisms by which NaBu and TSA cause apoptosis and cell cycle arrest are not yet completely clarified, although these agents are known to modulate the expression of several genes including cell-cycle- and apoptosis-related genes. The enzymes involved in the process of translation have important roles in controlling cell growth and apoptosis, and several of these translation factors have been described as having a causal role in the development of cancer. The expression patterns of the translation mechanism, namely of the elongation factors eEF1A1 and eEF1A2, and of the termination factors eRF1 and eRF3, were studied in the breast cancer cell line MCF-7 by real-time quantitative reverse transcription-polymerase chain reaction after a 24-h treatment with NaBu and TSA. NaBu induced inhibition of translation factors' transcription, whereas TSA caused an increase in mRNA levels. Thus, these two agents may modulate the expression of translation factors through different pathways. We propose that the inhibition caused by NaBu may, in part, be responsible for the cell cycle arrest and apoptosis induced by this agent in MCF-7 cells.

Analysis of a 17.9 kb region from Saccharomyces cerevisiae chromosome VII reveals the presence of eight open reading frames, including BRF1 (TFIIIB70) and GCN5 genes

We report the nucleotide sequence of a 17,893 bp DNA segment from the right arm of Saccharomyces cerevisiae chromosome VII. This fragment begins at 482 kb from the centromere. The sequence includes the BRF1 gene, encoding TFIIIB70, the 5' portion of the GCN5 gene, an open reading frame (ORF) previously identified as ORF MGA1, whose translation product shows similarity to heat-shock transcription factors and five new ORFs. Among these, YGR250 encodes a polypeptide that harbours a domain present in several polyA binding proteins. YGR245 is similar to a putative Schizosaccharomyces pombe gene, YGR248 shows significant similarity with three ORFs of S. cerevisiae situated on different chromosomes, while the remaining two ORFs, YGR247 and YGR251, do not show significant similarity to sequences present in databases.

Sequencing of a 17.6 kb segment on the right arm of yeast chromosome VII reveals 12 ORFs, including CCT, ADE3 and TR-I genes, homologues of the yeast PMT and EF1G genes, of the human and bacterial electron-transferring flavoproteins (beta-chain) and of the Escherichia coli phosphoserine phosphohydrolase, and five new ORFs.

A 17.6 kb DNA fragment from the right arm of chromosome VII of Saccharomyces cerevisiae has been sequenced and analysed. The sequence contains twelve open reading frames (ORFs) longer than 100 amino acids. Three genes had already been cloned and sequenced: CCT, ADE3 and TR-I. Two ORFs are similar to other yeast genes: G7722 with the YAL023 (PMT2) and PMT1 genes, encoding two integral membrane proteins, and G7727 with the first half of the genes encoding elongation factors 1gamma, TEF3 and TEF4. Two other ORFs, G7742 and G7744, are most probably yeast orthologues of the human and Paracoccus denitrificans electron-transferring flavoproteins (beta chain) and of the Escherichia coli phosphoserine phosphohydrolase. The five remaining identified ORFs do not show detectable homology with other protein sequences deposited in data banks. The sequence has been deposited in the EMBL data library under Accession Number Z49133.

Clusters of 5S rRNAs in the intergenic region of ubiquitin genes in Tetrahymena pyriformis

Here, we report the molecular analysis of two independent 5S rRNA clusters found in the intergenic region of two ubiquitin genomic clones isolated from Tetrahymena pyriformis. Each cluster contains two 120-bp-long coding regions organized in tandem with 142/145-bp-long spacers.

The Tetrahymena chaperonin subunit CCT eta gene is coexpressed with CCT gamma gene during cilia biogenesis and cell sexual reproduction

We report here the cloning and the characterization of the T. pyriformis CCT eta gene (TpCCT eta) and also a partial sequence of the corresponding T. thermophila gene (TtCCT eta). The TpCCt eta gene encodes a protein sharing a 60.3% identity with the mouse CCT eta. We have studied the expression of these genes in Tetrahymena exponentially growing cells, cells regenerating their cilia for different periods and during different stages of the cell sexual reproduction. These genes have similar patterns of expression to those of the previously identified TpCCt gamma gene. Indeed, the Tetrahymena CCT eta and CCT gamma genes are up-regulated at 60-120 min of cilia recovery, and in conjugation when vegetative growth was resumed and cell division took place. Our results seem to indicate that both CCT subunits play an important role in the biogenesis of the newly synthesized cilia of Tetrahymena and during its cell division.

Diversity and characterisation of coagulasenegative staphylococci isolated from healthy individuals in Portugal

In the past few years the interest in coagulase-negative staphylococci (CoNS) has significantly increased in human medicine. CoNS are common commensal colonisers of the human skin, although now also recognised as major nosocomial pathogens. Over the last decades, several studies have been carried out in order to understand the pathogenicity mechanisms of CoNS. The well known determinants in the pathogenesis of CoNS infections are their ability to form biofilms and an exceptional resistance to several antibiotics. Nevertheless, there is a lack of studies regarding the commensal lifestyle of these microorganisms. Additionally, it is now hypothesised that commensal bacteria might be a reservoir of pathogenic determinants. Therefore, the work described throughout this thesis was aimed to perform a phenotypic and genotypic characterisation of different CoNS species isolated from healthy Portuguese individuals. A total of 61 CoNS isolates, comprising 7 different species, were obtained and characterised at the level of biofilm formation and antibiotic susceptibility profiles. According to the results, biofilm formation ability and presence of biofilm-associated genes were commonly found features, highlighting their pivotal role in the colonising lifestyle of CoNS. This study also addressed the correlation between phenotypic and genotypic characteristics of biofilm formation, corroborating and raising questions about the importance of some genes in this process. Moreover, it was observed a great proportion of isolates with decreased susceptibility and multiple resistances to some important antibiotics. A significant association between antibiotic resistance and biofilm formation was also demonstrated, and some hypotheses about the nature of such association were provided. Lastly, the expression patterns of two biofilm-associated genes at two distinct biofilm developmental stages were determined, confirming their importance in the accumulative stage of biofilm formation. Overall, the results presented in this thesis indicate that staphylococcal skin flora might be an important reservoir of potentially pathogenic bacteria and, simultaneously, bring to light new perceptions about the molecular basis of staphylococcal biofilm formation, and the nature of the association between antibiotic resistance and biofilm formation.

Cholesterol 24S-Hydroxylase overexpression inhibits the liver X receptor (LXR) pathway by activating small guanosine triphosphate-binding proteins (sGTPases) in neuronal cells

The neuronal-specific cholesterol 24S-hydroxylase (CYP46A1) is important for brain cholesterol elimination. Cyp46a1 null mice exhibit severe deficiencies in learning and hippocampal long-term potentiation, suggested to be caused by a decrease in isoprenoid intermediates of the mevalonate pathway. Conversely, transgenic mice overexpressing CYP46A1 show an improved cognitive function. These results raised the question of whether CYP46A1 expression can modulate the activity of proteins that are crucial for neuronal function, namely of isoprenylated small guanosine triphosphate-binding proteins (sGTPases). Our results show that CYP46A1 overexpression in SH-SY5Y neuroblastoma cells and in primary cultures of rat cortical neurons leads to an increase in 3-hydroxy-3-methyl-glutaryl-CoA reductase activity and to an overall increase in membrane levels of RhoA, Rac1, Cdc42 and Rab8. This increase is accompanied by a specific increase in RhoA activation. Interestingly, treatment with lovastatin or a geranylgeranyltransferase-I inhibitor abolished the CYP46A1 effect. The CYP46A1-mediated increase in sGTPases membrane abundance was confirmed in vivo, in membrane fractions obtained from transgenic mice overexpressing this enzyme. Moreover, CYP46A1 overexpression leads to a decrease in the liver X receptor (LXR) transcriptional activity and in the mRNA levels of ATP-binding cassette transporter 1, sub-family A, member 1 and apolipoprotein E. This effect was abolished by inhibition of prenylation or by co-transfection of a RhoA dominant-negative mutant. Our results suggest a novel regulatory axis in neurons; under conditions of membrane cholesterol reduction by increased CYP46A1 expression, neurons increase isoprenoid synthesis and sGTPase prenylation. This leads to a reduction in LXR activity, and consequently to a decrease in the expression of LXR target genes.

Caracterização molecular da resistência a antimicrobianos em Escherichia coli produtoras de β-lactamases de espectro alargado isoladas em cães e estudo da partilha de clones bacterianos entre animais de companhia e coabitantes humanos

A pressão seletiva originada pelo uso excessivo de antimicrobianos na medicina humana e veterinária tem contribuído para a emergência de estirpes bacterianas multirresistentes, sendo os estudos mais escassos relativamente à sua presença nos animais de companhia. Porque os animais e os seus proprietários partilham o mesmo espaço habitacional, apresentando comportamentos de contacto próximo, existe uma hipótese elevada de transferência microbiana inter-espécie. Ante esta possibilidade é importante escrutinar o papel dos animais de companhia enquanto reservatórios de estirpes e de genes de resistência, bem como a sua envolvência na disseminação de estirpes bacterianas multirresistentes. Importa também, investigar o papel das superfícies e objetos domésticos partilhados por ambos, como potenciadores deste fenómeno. O objetivo deste trabalho foi, identificar o filogrupo e fazer a caracterização molecular dos genes que conferem resistência aos β-lactâmicos e às quinolonas, em quarenta isolados de Escherichia coli produtoras de β-lactamases de espectro alargado (ESBL), obtidas em zaragatoas fecais de cães consultados no Hospital Veterinário do ICBAS-UP. Complementarmente pretendeu-se inferir sobre a partilha de clones de Escherichia coli e Enterococcus spp. com elevadas resistências, em cinco agregados familiares (humanos e seus animais de companhia) assim como avaliar a potencial disseminação de estirpes multirresistentes no ambiente doméstico. Previamente foram recolhidas zaragatoas de fezes, pelo e mucosa oral dos animais e em alguns casos, dos seus proprietários, e ainda do ambiente doméstico. As zaragatoas foram processadas e as estirpes isoladas com base em meios seletivos. Foram realizados testes de suscetibilidade antimicrobiana de modo a estabelecer o fenótipo de resistência de cada isolado. O DNA foi extraído por varias metodologias e técnicas de PCR foram utilizadas para caracterização de filogrupos (Escherichia coli) e identificação da espécie (Enterococcus spp.). A avaliação da proximidade filogenética entre isolados foi efetuada por ERIC PCR e PFGE. No conjunto de quarenta isolados produtores de ESBL e/ou resistentes a quinolonas verificou-se que 47,5% pertenciam ao filogrupo A, havendo uma menor prevalência do filogrupo D (25,0%), B1 (17,5%), e B2 (10,0%).A frequência de resistência nestes isolados é factualmente elevada, sendo reveladora de uma elevada pressão seletiva. Com exceção de dois isolados, os fenótipos foram justificados pela presença de β-lactamases. A frequência da presença de genes foi: 47% blaTEM, 34% blaSHV, 24% blaOXA , 18% blaCTX-M-15, 8% blaCTX-M-2, 3% blaCTX-M-9. Nos isolados resistentes às quinolonas verificou-se maioritariamente a presença de mutações nos genes cromossomais gyrA e parC, e em alguns casos a presença de um determinante de resistência mediado por plasmídeo – qnrS. Nos cinco “agregados familiares” (humanos e animais) estudados foi observada uma partilha frequente de clones de E. coli e Enterococcus faecalis com múltiplas resistências, isolados em fezes e mucosa oral de cães e gatos e fezes e mãos dos respetivos proprietários, evidenciando-se assim uma possível transferência direta entre coabitantes (agregados A, C, D, E). Ficou também comprovado com percentagens de similaridade genotípica superiores a 94% que essa disseminação também ocorre para o ambiente doméstico, envolvendo objetos dos animais e de uso comum (agregados A, E). Os resultados obtidos reforçam a necessidade de um uso prudente dos antimicrobianos, pois elevados padrões de resistências terão um impacto não só na qualidade de vida dos animais mas também na saúde humana. Adicionalmente importa sensibilizar os proprietários para a necessidade de uma maior vigilância relativamente às formas de interação com os animais, bem como para a adoção de medidas higiénicas cautelares após essa mesma interação.

Fungal burden in waste industry: an occupational risk to be solved

High loads of fungi have been reported in different types of waste management plants. This study intends to assess fungal contamination in one waste-sorting plant before and after cleaning procedures in order to analyze their effectiveness. Air samples of 50 L were collected through an impaction method, while surface samples, taken at the same time, were collected by the swabbing method and subject to further macro- and microscopic observations. In addition, we collected air samples of 250 L using the impinger Coriolis μ air sampler (Bertin Technologies) at 300 L/min airflow rate in order to perform real-time quantitative PCR (qPCR) amplification of genes from specific fungal species, namely Aspergillus fumigatus and Aspergillus flavus complexes, as well as Stachybotrys chartarum species. Fungal quantification in the air ranged from 180 to 5,280 CFU m−3 before cleaning and from 220 to 2,460 CFU m−3 after cleaning procedures. Surfaces presented results that ranged from 29 × 104 to 109 × 104 CFU m−2 before cleaning and from 11 × 104 to 89 × 104 CFU m−2 after cleaning. Statistically significant differences regarding fungal load were not detected between before and after cleaning procedures. Toxigenic strains from A. flavus complex and S. chartarum were not detected by qPCR. Conversely, the A. fumigatus species was successfully detected by qPCR and interestingly it was amplified in two samples where no detection by conventional methods was observed. Overall, these results reveal the inefficacy of the cleaning procedures and that it is important to determine fungal burden in order to carry out risk assessment.

Task-based occupational exposure assessment and particle number concentration: two important data resources to perform risk assessment for occupational exposure to particles

Environment monitoring has an important role in occupational exposure assessment. However, due to several factors is done with insufficient frequency and normally don´t give the necessary information to choose the most adequate safety measures to avoid or control exposure. Identifying all the tasks developed in each workplace and conducting a task-based exposure assessment help to refine the exposure characterization and reduce assessment errors. A task-based assessment can provide also a better evaluation of exposure variability, instead of assessing personal exposures using continuous 8-hour time weighted average measurements. Health effects related with exposure to particles have mainly been investigated with mass-measuring instruments or gravimetric analysis. However, more recently, there are some studies that support that size distribution and particle number concentration may have advantages over particle mass concentration for assessing the health effects of airborne particles. Several exposure assessments were performed in different occupational settings (bakery, grill house, cork industry and horse stable) and were applied these two resources: task-based exposure assessment and particle number concentration by size. The results showed interesting results: task-based approach applied permitted to identify the tasks with higher exposure to the smaller particles (0.3 μm) in the different occupational settings. The data obtained allow more concrete and effective risk assessment and the identification of priorities for safety investments.

Bisphenol A at the reference level counteracts doxorubicin transcriptional effects on cancer related genes in HT29 cells

Human exposure to Bisphenol A (BPA) results mainly from ingestion of food and beverages. Information regarding BPA effects on colon cancer, one of the major causes of death in developed countries, is still scarce. Likewise, little is known about BPA drug interactions although its potential role in doxorubicin (DOX) chemoresistance has been suggested. This study aims to assess potential interactions between BPA and DOX on HT29 colon cancer cells. HT29 cell response was evaluated after exposure to BPA, DOX, or co-exposure to both chemicals. Transcriptional analysis of several cancer-associated genes (c-fos, AURKA, p21, bcl-xl and CLU) shows that BPA exposure induces slight up-regulation exclusively of bcl-xl without affecting cell viability. On the other hand, a sub-therapeutic DOX concentration (40nM) results in highly altered c-fos, bcl-xl, and CLU transcript levels, and this is not affected by co-exposure with BPA. Conversely, DOX at a therapeutic concentration (4μM) results in distinct and very severe transcriptional alterations of c-fos, AURKA, p21 and CLU that are counteracted by co-exposure with BPA resulting in transcript levels similar to those of control. Co-exposure with BPA slightly decreases apoptosis in relation to DOX 4μM alone without affecting DOX-induced loss of cell viability. These results suggest that BPA exposure can influence chemotherapy outcomes and therefore emphasize the necessity of a better understanding of BPA interactions with chemotherapeutic agents in the context of risk assessment.