Pedro Madureira - Academia.edu (original) (raw)
Papers by Pedro Madureira
Glyceraldehyde 3-phosphate dehydrogenases (GAPDH) are cytoplasmic glycolytic enzymes that, despit... more Glyceraldehyde 3-phosphate dehydrogenases (GAPDH) are cytoplasmic glycolytic enzymes that, despite lacking identifiable secretion signals, have been detected at the surface of several prokaryotic and eukaryotic organisms where they exhibit non-glycolytic functions including adhesion to host components. Group B Streptococcus (GBS) is a human commensal bacterium that has the capacity to cause life-threatening meningitis and septicemia in newborns. Electron microscopy and fluorescence-activated cell sorter (FACS) analysis demonstrated the surface localization of GAPDH in GBS. By addressing the question of GAPDH export to the cell surface of GBS strain NEM316 and isogenic mutant derivatives of our collection, we found that impaired GAPDH presence in the surface and supernatant of GBS was associated with a lower level of bacterial lysis. We also found that following GBS lysis, GAPDH can associate to the surface of many living bacteria. Finally, we provide
Carbohydrate Polymers, 2016
International Journal of Pharmaceutics, 2010
Journal of Bacteriology, 2003
The Journal of Immunology, 2007
Certain extracellular proteins produced by several pathogenic microorganisms interfere with the h... more Certain extracellular proteins produced by several pathogenic microorganisms interfere with the host immune system facilitating microbial colonization and were thus designated virulence-associated immunomodulatory proteins. In this study, a protein with B lymphocyte stimulatory activity was isolated from culture supernatants of Streptococcus agalactiae strain NEM316. This protein, with an apparent molecular mass of 45 kDa, was identified as GAPDH by N-terminal amino acid sequencing. The gapC gene was cloned and expressed in Escherichia coli for the production of a recombinant histidyl-tagged protein. The recombinant GAPDH (rGAPDH), purified in an enzymatically active form, induced in vitro an up-regulation of CD69 expression on B cells from normal and BCR transgenic mice. In addition, rGAPDH induced an increase in the numbers of total, but not of rGAPDH-specific, splenic Ig-secreting cells in C57BL/6 mice treated i.p. with this protein. These in vitro-and in vivo-elicited B cell responses suggest that the B cell stimulatory effect of rGAPDH is independent of BCR specificity. A S. agalactiae strain overexpressing GAPDH showed increased virulence as compared with the wild-type strain in C57BL/6 mice. This virulence was markedly reduced in IL-10deficient and anti-rGAPDH antiserum-treated mice. These results suggest that IL-10 production, which was detected at higher concentrations in the serum of rGAPDH-treated mice, is important in determining the successfulness of the host colonization by S. agalactiae and they highlight the direct role of GAPDH in this process. Taken together, our data demonstrate that S. agalactiae GAPDH is a virulence-associated immunomodulatory protein.
Oncotarget, Feb 20, 2018
Tyrosine kinase inhibition of the epidermal growth factor receptor (EGFR) is the standard in the ... more Tyrosine kinase inhibition of the epidermal growth factor receptor (EGFR) is the standard in the first line treatment of patients with advanced non-small-cell lung cancer (NSCLC) harbouring EGFR activating mutations. Here we aim to discern efficacy and toxicity measures through a meta-analysis of published studies that could aid treatment selection. We performed a meta-analysis of the main randomized clinical trials evaluating the currently approved EGFR-TKIs in first-line of treatment of EGFR-positive advanced NSCLC. Cochrane guidelines were used for statistical analysis. 3,179 patients were included. All EGFR TKIs showed improved outcomes with respect to ORR and PFS when compared to standard platinum-doublet chemotherapy. Comparative ORR for gefitinib, erlotinib and afatinib were 52.1%, 67.3% and 61.6% respectively. HRs for PFS were 0.62 (95% CI, 0.38-1.00) for gefitinib, 0.28 (95% CI, 0.17-0.45) for erlotinib and 0.40 (95% CI, 0.20-0.83) for afatinib. HRs for OS were not statisti...
Food Chemistry
An instant coffee fraction, rich in arabinogalactans, obtained by ultrafiltration, using 1 and 5k... more An instant coffee fraction, rich in arabinogalactans, obtained by ultrafiltration, using 1 and 5kDa membranes, has previously shown in vitro stimulatory activity on BALB/c mice spleen B lymphocytes. The compounds inducing lymphocytic activation were shown to elute, mainly near the void volume by size-exclusion chromatography, using Bio-Gel P6 (1-6kDa). Treatment of the compounds with chymotrypsin, a digestive protease, did not affect the induced B lymphocyte activation. On the contrary, hydrolysis with an α-l-arabinofuranosidase, removing single terminally-linked arabinose residues, decreased the extent of B cell activation. The immunostimulatory activity of these compounds was also observed by in vitro experiments, using bone marrow-derived macrophages and dendritic cells as responders. Altogether, these results show the relevance of single arabinose residues, present at the non-reducing end of polymeric compounds, to the coffee stimulatory activity in cells mediating innate and acquired immunity.
Biochemical Engineering Journal, 2016
Fluorescence in situ hybridization (FISH) is based on the use of fluorescent staining dyes, howev... more Fluorescence in situ hybridization (FISH) is based on the use of fluorescent staining dyes, however, the signal intensity of the images obtained by microscopy is seldom quantified with accuracy by the researcher. The development of innovative digital image processing programs and tools has been trying to overcome this problem, however, the determination of fluorescent intensity in microscopy images still has issues due to the lack of precision in the results and the complexity of existing software. This work presents FISHji, a set of new ImageJ methods for automated quantification of fluorescence in images obtained by epifluorescence microscopy. To validate the methods, results obtained by FISHji were compared with results obtained by flow cytometry. The mean correlation between FISHji and flow cytometry was high and significant, showing that the imaging methods are able to accurately assess the signal intensity of fluorescence images. FISHji are available for non-commercial use at http://paginas.fe. up.pt/∼nazevedo/.
Applied microbiology and biotechnology, Jan 12, 2016
Fluorescence in situ hybridization (FISH) employing nucleic acid mimics as probes is becoming an ... more Fluorescence in situ hybridization (FISH) employing nucleic acid mimics as probes is becoming an emerging molecular tool in the microbiology area for the detection and visualization of microorganisms. However, the impact that locked nucleic acid (LNA) and 2'-O-methyl (2'-OMe) RNA modifications have on the probe that is targeting microorganisms is unknown. In this study, the melting and hybridization efficiency properties of 18 different probes in regards to their use in FISH for the detection of the 16S rRNA of Helicobacter pylori were compared. For the same sequence and target, probe length and the type of nucleic acid mimics used as mixmers in LNA-based probes strongly influence the efficiency of detection. LNA probes with 10 to 15 mers showed the highest efficiency. Additionally, the combination of 2'-OMe RNA with LNA allowed an increase on the fluorescence intensities of the probes. Overall, these results have significant implications for the design and applications ...
Biomaterials, Jun 30, 2005
We have used self-assembled monolayers (SAMs) of alkanethiolates on gold to study the contributio... more We have used self-assembled monolayers (SAMs) of alkanethiolates on gold to study the contribution of methyl terminal functional groups in implant-triggered inflammation. The CH 3-coated biomaterials were inserted in an air-pouch cavity of the BALB/c mouse and the in situ inflammatory response was monitored 4, 24, 48 and 72 h later. Flow cytometry was applied to define surface expression of the adhesion receptor Mac-1 (CD11b/CD18), a marker of activated leukocytes, and also of CD3 and B220 antigens (T and B lymphocytes). The CH 3-coated surfaces caused a significant enhancement in the number of Mac-1 + cells in the implant. The only significant change in T and B lymphocytes was a transient increase in T cells detected 48 h after the implantation. Peak numbers of Mac-1 + phagocytes were observed 24 h after implantation. We conclude that if CH 3 is present at the surface of implants, this chemical group will trigger a significant enhancement of activated phagocytes involved in the inflammatory reaction, and this phenomenon may extend the local phlogistic event.
PloS one, 2015
Group B Streptococcus (GBS), a commensal organism, can turn into a life-threatening pathogen in n... more Group B Streptococcus (GBS), a commensal organism, can turn into a life-threatening pathogen in neonates and elderly, or in adults with severe underlying diseases such as diabetes. We developed a vaccine targeting the GBS glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme detected at the bacterial surface, which was proven to be effective in a neonatal mouse model of infection. Since this bacterium has emerged as an important pathogen in non-pregnant adults, here we investigated whether this vaccine also confers protection in an adult susceptible and in a diabetic mouse model of infection. For immunoprotection studies, sham or immunized adult mice were infected with GBS serotype Ia and V strains, the two most prevalent serotypes isolated in adults. Sham and vaccinated mice were also rendered diabetic and infected with a serotype V GBS strain. For toxicological (pre-clinical) studies, adult mice were vaccinated three times, with three concentrations of recombinant ...
Carbohydrate Polymers, 2015
Immunostimulatory polysaccharides are compounds capable of interacting with the immune system and... more Immunostimulatory polysaccharides are compounds capable of interacting with the immune system and enhance specific mechanisms of the host response. Glucans, mannans, pectic polysaccharides, arabinogalactans, fucoidans, galactans, hyaluronans, fructans, and xylans are polysaccharides with reported immunostimulatory activity. The structural features that have been related with such activity are the monosaccharide and glycosidic-linkage composition, conformation, molecular weight, functional groups, and branching characteristics. However, the establishment of structure-function relationships is possible only if purified and characterized polysaccharides are used and selective structural modifications performed. Aiming at contributing to the definition of the structure-function relationships necessary to design immunostimulatory polysaccharides with potential for preventive or therapeutical purposes or to be recognized as health-improving ingredients in functional foods, this review introduces basic immunological concepts required to understand the mechanisms that rule the potential claimed immunostimulatory activity of polysaccharides and critically presents a literature survey on the structural features of the polysaccharides and reported immunostimulatory activity.
Biochemical Engineering Journal, 2015
Multispecies biofilms are the dominant form of biofilms found in Nature. The application of fluor... more Multispecies biofilms are the dominant form of biofilms found in Nature. The application of fluorescence in situ hybridization (FISH)-based techniques to the discrimination of biofilm populations might contribute to the understanding of microorganism interactions in these structures, and might allow the development of efficient strategies to prevent or minimize biofilmassociated diseases. This work presents the first study that develops, optimizes and validates a multiplex FISH procedure using locked nucleic acid (LNA) and 2'-O-methyl RNA (2'OMe) oligonucleotides probes for the in vitro discrimination within mixed populations. As a case study, Escherichia coli, the major cause of urinary tract infections (UTIs), and three other atypical colonizers of urinary catheters (Delftia tsuruhatensis, Achromobacter xylosoxidans and Burkholderia fungorum) with unproven pathogenic potential, were selected. Specific probes for these species were designed and optimized for specific hybridization in multiplex experiments. Results showed that the LNA/2'OMe-FISH method performed well in multiplex experiments and presented a good correlation with total and cultivability counts, regardless of the cells physiological state. In fact, the method was also able to report variations of viable but noncultivable populations. Further analysis of mixed biofilm structures by confocal laser scanning microscopy provided a clear discrimination in three dimensions between the location of the different populations.
PLOS ONE, 2015
In recent years, there have been several attempts to improve the diagnosis of infection caused by... more In recent years, there have been several attempts to improve the diagnosis of infection caused by Helicobacter pylori. Fluorescence in situ hybridization (FISH) is a commonly used technique to detect H. pylori infection but it requires biopsies from the stomach. Thus, the development of an in vivo FISH-based method (FIVH) that directly detects and allows the visualization of the bacterium within the human body would significantly reduce the time of analysis, allowing the diagnosis to be performed during endoscopy. In a previous study we designed and synthesized a phosphorothioate locked nucleic acid (LNA)/ 2' O-methyl RNA (2'OMe) probe using standard phosphoramidite chemistry and FISH hybridization was then successfully performed both on adhered and suspended bacteria at 37°C. In this work we simplified, shortened and adapted FISH to work at gastric pH values, meaning that the hybridization step now takes only 30 minutes and, in addition to the buffer, uses only urea and probe at non-toxic concentrations. Importantly, the sensitivity and specificity of the FISH method was maintained in the range of conditions tested, even at low stringency conditions (e.g., low pH). In conclusion, this methodology is a promising approach that might be used in vivo in the future in combination with a confocal laser endomicroscope for H. pylori visualization.
Tumor Biology, 2015
Lung cancer is the leading cause of cancer-related death and accounts for approximately 30 % of a... more Lung cancer is the leading cause of cancer-related death and accounts for approximately 30 % of all cancer deaths. Despite the recent developments in personalized therapy, the prognosis in lung cancer is still very poor. Immunotherapy is now emerging as a new hope for patients with lung cancer. It is well known that standard chemotherapeutic regimens have devastating effects for the patient's immune system. Therefore, the aim of immunotherapy is to specifically enhance the immune response against the tumour. Recently, many trials addressed the role of such therapies for metastatic non-small cell lung cancer (NSCLC) treatment: ipilimumab, tremelimumab, nivolumab and pembrolizumab are immunotherapeutic agents of high relevance in this field. Anti-tumour vaccines, as well as dendritic cell-based therapies, have emerged as potent inducers of immune response against the tumour. Herein, we will review some of the most promising cancer immunotherapies, highlighting their advantages and try to understand, in an immunological perspective, the missteps associated with the current treatments for cancer.
Pharmacogenomics, 2015
HER2-targeted therapies have radically changed the prognosis of HER2-positive breast cancer over ... more HER2-targeted therapies have radically changed the prognosis of HER2-positive breast cancer over the last few years. However, resistance to these therapies has been a constant, leading to treatment-failure and new tumor progression. Recently, the kinase-impaired HER3 emerged as a pivotal player in oncogenic signaling, with an important role in both non-treated progression and treatment response. HER2/HER3 dimerization is required for full signaling potential and constitutes the key oncogenic unit. Also, when inhibiting PI3K/AKT pathway (as with anti-HER2 drugs) feedback mechanisms lead to a rebound in HER3 activity, which is one of the main roads to resistance. As current strategies to treat HER2-positive breast cancer are unable to inhibit this feedback response, two great promises emerged: the combination of targeted-therapies and drugs targeting HER3. In this article HER2 and HER3-targeted drugs and possible combinations between them, as well as the biomarkers to predict and monitor these drugs effect, are reviewed.
Applied Microbiology and Biotechnology, 2015
In the original publication, the names of the author's have been inverted. The correct presentati... more In the original publication, the names of the author's have been inverted. The correct presentation is given above.
Glyceraldehyde 3-phosphate dehydrogenases (GAPDH) are cytoplasmic glycolytic enzymes that, despit... more Glyceraldehyde 3-phosphate dehydrogenases (GAPDH) are cytoplasmic glycolytic enzymes that, despite lacking identifiable secretion signals, have been detected at the surface of several prokaryotic and eukaryotic organisms where they exhibit non-glycolytic functions including adhesion to host components. Group B Streptococcus (GBS) is a human commensal bacterium that has the capacity to cause life-threatening meningitis and septicemia in newborns. Electron microscopy and fluorescence-activated cell sorter (FACS) analysis demonstrated the surface localization of GAPDH in GBS. By addressing the question of GAPDH export to the cell surface of GBS strain NEM316 and isogenic mutant derivatives of our collection, we found that impaired GAPDH presence in the surface and supernatant of GBS was associated with a lower level of bacterial lysis. We also found that following GBS lysis, GAPDH can associate to the surface of many living bacteria. Finally, we provide
Carbohydrate Polymers, 2016
International Journal of Pharmaceutics, 2010
Journal of Bacteriology, 2003
The Journal of Immunology, 2007
Certain extracellular proteins produced by several pathogenic microorganisms interfere with the h... more Certain extracellular proteins produced by several pathogenic microorganisms interfere with the host immune system facilitating microbial colonization and were thus designated virulence-associated immunomodulatory proteins. In this study, a protein with B lymphocyte stimulatory activity was isolated from culture supernatants of Streptococcus agalactiae strain NEM316. This protein, with an apparent molecular mass of 45 kDa, was identified as GAPDH by N-terminal amino acid sequencing. The gapC gene was cloned and expressed in Escherichia coli for the production of a recombinant histidyl-tagged protein. The recombinant GAPDH (rGAPDH), purified in an enzymatically active form, induced in vitro an up-regulation of CD69 expression on B cells from normal and BCR transgenic mice. In addition, rGAPDH induced an increase in the numbers of total, but not of rGAPDH-specific, splenic Ig-secreting cells in C57BL/6 mice treated i.p. with this protein. These in vitro-and in vivo-elicited B cell responses suggest that the B cell stimulatory effect of rGAPDH is independent of BCR specificity. A S. agalactiae strain overexpressing GAPDH showed increased virulence as compared with the wild-type strain in C57BL/6 mice. This virulence was markedly reduced in IL-10deficient and anti-rGAPDH antiserum-treated mice. These results suggest that IL-10 production, which was detected at higher concentrations in the serum of rGAPDH-treated mice, is important in determining the successfulness of the host colonization by S. agalactiae and they highlight the direct role of GAPDH in this process. Taken together, our data demonstrate that S. agalactiae GAPDH is a virulence-associated immunomodulatory protein.
Oncotarget, Feb 20, 2018
Tyrosine kinase inhibition of the epidermal growth factor receptor (EGFR) is the standard in the ... more Tyrosine kinase inhibition of the epidermal growth factor receptor (EGFR) is the standard in the first line treatment of patients with advanced non-small-cell lung cancer (NSCLC) harbouring EGFR activating mutations. Here we aim to discern efficacy and toxicity measures through a meta-analysis of published studies that could aid treatment selection. We performed a meta-analysis of the main randomized clinical trials evaluating the currently approved EGFR-TKIs in first-line of treatment of EGFR-positive advanced NSCLC. Cochrane guidelines were used for statistical analysis. 3,179 patients were included. All EGFR TKIs showed improved outcomes with respect to ORR and PFS when compared to standard platinum-doublet chemotherapy. Comparative ORR for gefitinib, erlotinib and afatinib were 52.1%, 67.3% and 61.6% respectively. HRs for PFS were 0.62 (95% CI, 0.38-1.00) for gefitinib, 0.28 (95% CI, 0.17-0.45) for erlotinib and 0.40 (95% CI, 0.20-0.83) for afatinib. HRs for OS were not statisti...
Food Chemistry
An instant coffee fraction, rich in arabinogalactans, obtained by ultrafiltration, using 1 and 5k... more An instant coffee fraction, rich in arabinogalactans, obtained by ultrafiltration, using 1 and 5kDa membranes, has previously shown in vitro stimulatory activity on BALB/c mice spleen B lymphocytes. The compounds inducing lymphocytic activation were shown to elute, mainly near the void volume by size-exclusion chromatography, using Bio-Gel P6 (1-6kDa). Treatment of the compounds with chymotrypsin, a digestive protease, did not affect the induced B lymphocyte activation. On the contrary, hydrolysis with an α-l-arabinofuranosidase, removing single terminally-linked arabinose residues, decreased the extent of B cell activation. The immunostimulatory activity of these compounds was also observed by in vitro experiments, using bone marrow-derived macrophages and dendritic cells as responders. Altogether, these results show the relevance of single arabinose residues, present at the non-reducing end of polymeric compounds, to the coffee stimulatory activity in cells mediating innate and acquired immunity.
Biochemical Engineering Journal, 2016
Fluorescence in situ hybridization (FISH) is based on the use of fluorescent staining dyes, howev... more Fluorescence in situ hybridization (FISH) is based on the use of fluorescent staining dyes, however, the signal intensity of the images obtained by microscopy is seldom quantified with accuracy by the researcher. The development of innovative digital image processing programs and tools has been trying to overcome this problem, however, the determination of fluorescent intensity in microscopy images still has issues due to the lack of precision in the results and the complexity of existing software. This work presents FISHji, a set of new ImageJ methods for automated quantification of fluorescence in images obtained by epifluorescence microscopy. To validate the methods, results obtained by FISHji were compared with results obtained by flow cytometry. The mean correlation between FISHji and flow cytometry was high and significant, showing that the imaging methods are able to accurately assess the signal intensity of fluorescence images. FISHji are available for non-commercial use at http://paginas.fe. up.pt/∼nazevedo/.
Applied microbiology and biotechnology, Jan 12, 2016
Fluorescence in situ hybridization (FISH) employing nucleic acid mimics as probes is becoming an ... more Fluorescence in situ hybridization (FISH) employing nucleic acid mimics as probes is becoming an emerging molecular tool in the microbiology area for the detection and visualization of microorganisms. However, the impact that locked nucleic acid (LNA) and 2'-O-methyl (2'-OMe) RNA modifications have on the probe that is targeting microorganisms is unknown. In this study, the melting and hybridization efficiency properties of 18 different probes in regards to their use in FISH for the detection of the 16S rRNA of Helicobacter pylori were compared. For the same sequence and target, probe length and the type of nucleic acid mimics used as mixmers in LNA-based probes strongly influence the efficiency of detection. LNA probes with 10 to 15 mers showed the highest efficiency. Additionally, the combination of 2'-OMe RNA with LNA allowed an increase on the fluorescence intensities of the probes. Overall, these results have significant implications for the design and applications ...
Biomaterials, Jun 30, 2005
We have used self-assembled monolayers (SAMs) of alkanethiolates on gold to study the contributio... more We have used self-assembled monolayers (SAMs) of alkanethiolates on gold to study the contribution of methyl terminal functional groups in implant-triggered inflammation. The CH 3-coated biomaterials were inserted in an air-pouch cavity of the BALB/c mouse and the in situ inflammatory response was monitored 4, 24, 48 and 72 h later. Flow cytometry was applied to define surface expression of the adhesion receptor Mac-1 (CD11b/CD18), a marker of activated leukocytes, and also of CD3 and B220 antigens (T and B lymphocytes). The CH 3-coated surfaces caused a significant enhancement in the number of Mac-1 + cells in the implant. The only significant change in T and B lymphocytes was a transient increase in T cells detected 48 h after the implantation. Peak numbers of Mac-1 + phagocytes were observed 24 h after implantation. We conclude that if CH 3 is present at the surface of implants, this chemical group will trigger a significant enhancement of activated phagocytes involved in the inflammatory reaction, and this phenomenon may extend the local phlogistic event.
PloS one, 2015
Group B Streptococcus (GBS), a commensal organism, can turn into a life-threatening pathogen in n... more Group B Streptococcus (GBS), a commensal organism, can turn into a life-threatening pathogen in neonates and elderly, or in adults with severe underlying diseases such as diabetes. We developed a vaccine targeting the GBS glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme detected at the bacterial surface, which was proven to be effective in a neonatal mouse model of infection. Since this bacterium has emerged as an important pathogen in non-pregnant adults, here we investigated whether this vaccine also confers protection in an adult susceptible and in a diabetic mouse model of infection. For immunoprotection studies, sham or immunized adult mice were infected with GBS serotype Ia and V strains, the two most prevalent serotypes isolated in adults. Sham and vaccinated mice were also rendered diabetic and infected with a serotype V GBS strain. For toxicological (pre-clinical) studies, adult mice were vaccinated three times, with three concentrations of recombinant ...
Carbohydrate Polymers, 2015
Immunostimulatory polysaccharides are compounds capable of interacting with the immune system and... more Immunostimulatory polysaccharides are compounds capable of interacting with the immune system and enhance specific mechanisms of the host response. Glucans, mannans, pectic polysaccharides, arabinogalactans, fucoidans, galactans, hyaluronans, fructans, and xylans are polysaccharides with reported immunostimulatory activity. The structural features that have been related with such activity are the monosaccharide and glycosidic-linkage composition, conformation, molecular weight, functional groups, and branching characteristics. However, the establishment of structure-function relationships is possible only if purified and characterized polysaccharides are used and selective structural modifications performed. Aiming at contributing to the definition of the structure-function relationships necessary to design immunostimulatory polysaccharides with potential for preventive or therapeutical purposes or to be recognized as health-improving ingredients in functional foods, this review introduces basic immunological concepts required to understand the mechanisms that rule the potential claimed immunostimulatory activity of polysaccharides and critically presents a literature survey on the structural features of the polysaccharides and reported immunostimulatory activity.
Biochemical Engineering Journal, 2015
Multispecies biofilms are the dominant form of biofilms found in Nature. The application of fluor... more Multispecies biofilms are the dominant form of biofilms found in Nature. The application of fluorescence in situ hybridization (FISH)-based techniques to the discrimination of biofilm populations might contribute to the understanding of microorganism interactions in these structures, and might allow the development of efficient strategies to prevent or minimize biofilmassociated diseases. This work presents the first study that develops, optimizes and validates a multiplex FISH procedure using locked nucleic acid (LNA) and 2'-O-methyl RNA (2'OMe) oligonucleotides probes for the in vitro discrimination within mixed populations. As a case study, Escherichia coli, the major cause of urinary tract infections (UTIs), and three other atypical colonizers of urinary catheters (Delftia tsuruhatensis, Achromobacter xylosoxidans and Burkholderia fungorum) with unproven pathogenic potential, were selected. Specific probes for these species were designed and optimized for specific hybridization in multiplex experiments. Results showed that the LNA/2'OMe-FISH method performed well in multiplex experiments and presented a good correlation with total and cultivability counts, regardless of the cells physiological state. In fact, the method was also able to report variations of viable but noncultivable populations. Further analysis of mixed biofilm structures by confocal laser scanning microscopy provided a clear discrimination in three dimensions between the location of the different populations.
PLOS ONE, 2015
In recent years, there have been several attempts to improve the diagnosis of infection caused by... more In recent years, there have been several attempts to improve the diagnosis of infection caused by Helicobacter pylori. Fluorescence in situ hybridization (FISH) is a commonly used technique to detect H. pylori infection but it requires biopsies from the stomach. Thus, the development of an in vivo FISH-based method (FIVH) that directly detects and allows the visualization of the bacterium within the human body would significantly reduce the time of analysis, allowing the diagnosis to be performed during endoscopy. In a previous study we designed and synthesized a phosphorothioate locked nucleic acid (LNA)/ 2' O-methyl RNA (2'OMe) probe using standard phosphoramidite chemistry and FISH hybridization was then successfully performed both on adhered and suspended bacteria at 37°C. In this work we simplified, shortened and adapted FISH to work at gastric pH values, meaning that the hybridization step now takes only 30 minutes and, in addition to the buffer, uses only urea and probe at non-toxic concentrations. Importantly, the sensitivity and specificity of the FISH method was maintained in the range of conditions tested, even at low stringency conditions (e.g., low pH). In conclusion, this methodology is a promising approach that might be used in vivo in the future in combination with a confocal laser endomicroscope for H. pylori visualization.
Tumor Biology, 2015
Lung cancer is the leading cause of cancer-related death and accounts for approximately 30 % of a... more Lung cancer is the leading cause of cancer-related death and accounts for approximately 30 % of all cancer deaths. Despite the recent developments in personalized therapy, the prognosis in lung cancer is still very poor. Immunotherapy is now emerging as a new hope for patients with lung cancer. It is well known that standard chemotherapeutic regimens have devastating effects for the patient's immune system. Therefore, the aim of immunotherapy is to specifically enhance the immune response against the tumour. Recently, many trials addressed the role of such therapies for metastatic non-small cell lung cancer (NSCLC) treatment: ipilimumab, tremelimumab, nivolumab and pembrolizumab are immunotherapeutic agents of high relevance in this field. Anti-tumour vaccines, as well as dendritic cell-based therapies, have emerged as potent inducers of immune response against the tumour. Herein, we will review some of the most promising cancer immunotherapies, highlighting their advantages and try to understand, in an immunological perspective, the missteps associated with the current treatments for cancer.
Pharmacogenomics, 2015
HER2-targeted therapies have radically changed the prognosis of HER2-positive breast cancer over ... more HER2-targeted therapies have radically changed the prognosis of HER2-positive breast cancer over the last few years. However, resistance to these therapies has been a constant, leading to treatment-failure and new tumor progression. Recently, the kinase-impaired HER3 emerged as a pivotal player in oncogenic signaling, with an important role in both non-treated progression and treatment response. HER2/HER3 dimerization is required for full signaling potential and constitutes the key oncogenic unit. Also, when inhibiting PI3K/AKT pathway (as with anti-HER2 drugs) feedback mechanisms lead to a rebound in HER3 activity, which is one of the main roads to resistance. As current strategies to treat HER2-positive breast cancer are unable to inhibit this feedback response, two great promises emerged: the combination of targeted-therapies and drugs targeting HER3. In this article HER2 and HER3-targeted drugs and possible combinations between them, as well as the biomarkers to predict and monitor these drugs effect, are reviewed.
Applied Microbiology and Biotechnology, 2015
In the original publication, the names of the author's have been inverted. The correct presentati... more In the original publication, the names of the author's have been inverted. The correct presentation is given above.