Decoding human cardiac stem cells regenerative potential in acute myocardial infarction

Acute Myocardial Infarction (AMI) remains a leading cause of death worldwide. After AMI, clinical restauration of blood flow aggravates tissue damage (Ischemia/Reperfusion, I/R injury), critically decreasing the number of viable cardiomyocytes (CMs). Human myocardium harbors a population of endogenous cardiac stem/progenitor cells (CSCs) that is activated upon I/R injury, contributing to myocardial repair through the establishment of an auto/paracrine molecular crosstalk between CSCs and CMs in stress. Transplantation of CSCs is currently being tested in several clinical trials, and although some improvements have been reported regarding decrease of the infarcted area, it is still not enough to show benefit over pharmacological standard-of-care. Our work aims at combining the development of relevant I/R in vitro human cell models with implementation of advanced mass spectrometry (MS)-based proteomic tools to further characterize hCSC and unveil associated regenerative mechanisms upon AMI. hCSCs employed in the phase I/II clinical trial CARE-MI (NCT02439398) were used (allogeneic therapy). Different strategies were explored to recapitulate both phases of I/R injury in the human adult heart, including: the use of human adult/mature cells, 3D culture system and stirred-tank bioreactor technology. Firstly, we developed a transwell co-culture cell based I/R model, with human CSCs and human induced pluripotent stem cell derived CMs (hiPSC-CMs). Following this work, and aiming at further improving the relevance of the I/R injury in vitro setup, 3D hiPSC-CM aggregate cultures and bioreactors were combined, allowing the control/monitoring of environmental parameters such as pH and dissolved oxygen, critical in the context of I/R physiology. Important features of I/R injury were successfully captured in the two models, including hiPSC-CM death upon reperfusion, disruption of cell ultra-structure organization, as well as increased release of angiogenic and inflammatory cytokines, consistent with the described pathophysiology of AMI. hCSCs response to I/R was further probed using whole proteome analysis (including quantitative SWATH methodology), allowing us to propose new pathways in the hCSCs-mediated regenerative process along the different phases of I/R injury through the identification of more than 3800 proteins and quantification of 714 proteins. Our data shows that our AMI-setup up-regulates hCSC proteins associated with several pro-migratory, proliferation and stress response-related pathways. Moreover, our results reinforce the idea that paracrine-mediated mechanisms are a central response in hCSC activation, with the enrichment of several paracrine signaling and pro-angiogenic pathways. We also show for the first time increased CXCL6 secretion by hCSCs upon injury, suggesting a relevant role of this angiogenic cytokine in hCSC mediated myocardial regeneration. Overall, multiple strategies were used to develop novel and robust I/R injury in vitro models, recapitulating several features of the human adult myocardium. The systems established allowed to better characterize hCSC mechanisms of action in response to AMI contexts. The knowledge generated has the potential to be used in the development of novel strategies excelling endogenous and transplanted hCSCs regenerative potential

Influence of anaerobic conditions on vaginal microbiota recovery from bacterial vaginosis patients

Bacterial vaginosis (BV) is one of the most common infections in women of reproductive age. Clinical studies have shown an association among BV and abnormal pregnancy, pelvic inflammatory disease and increased risk of sexually transmitted infections, including HIV. This disorder was first described in 1914 by Curtis as a “white discharge” syndrome and despite the decades of research we have only limited, and clearly not conclusive, evidence of microbial cause of BV, mechanism of disease and effective treatment. The development of molecular techniques such as Denaturing Gradient Gel Electrophoresis (DGGE) and DNA sequencing produced a clearer picture of the complexities of the vaginal microbiota. It has also become more apparent that none of the microorganisms already isolated from the vagina are likely to be the sole pathogen responsible for BV. Thus, improved knowledge of the relationship between different species of bacteria and their host is critical to a better understanding of both women’s health and illness. In order to comprehend the dynamic interaction between bacteria–bacteria and bacteria–host it becomes necessary to isolate bacteria from vaginal samples and to use them in in vitro and in vivo assays. The human vagina has a very specific environment regarding pH, nutrient availability and oxygen tension, being populated by a large range of bacteria from facultative to obligated anaerobic species. To isolate bacteria from such a complex niche, rich media should be used in order to promote the growth of different species. By using Columbia Agar (CBA) with 5% horse blood and by incubating at 37°C in the presence of 5%, 10% CO or in anaerobic condition generated by AnaeroGenTM (Oxoid), we were able to recover different bacteria species, from each of the anaerobic conditions tested, from BV vaginal samples, including some species never reported, as far as we know, such as Brevibacterium ravenspurgense, Corynebacterium tuscaniense, Klebsiella variicola, Nosocomiicoccus ampullae, Staphylococcus warneri and Bacillus firmus. Despite the development of molecular methods and their unquestionable advantages in characterising the vaginal microbiota, classical bacteriology will nevertheless be required to elucidate the etiology of BV since the isolation of the pathogenic agent will be always required. Our findings revealed that slight differences in anaerobic conditions were crucial for the isolation of novel BV-associated organisms. Further in vitro studies of all the isolated bacteria will promote a better understanding of the dynamics of their interaction and will potentially reveal how BV develops and influences other sexual transmitted diseases.(undefined

Gardnerella vaginalis virulence potential outcompetes with 30 other microorganisms isolated from BV patients

Bacterial vaginosis (BV) is the most common vaginal disorder affecting millions of women every year, and is usually associated with several adverse health outcomes, including preterm birth and acquisition of sexually transmitted diseases. However, the etiology of BV is still under debate. Recently, new fastidious anaerobic bacteria have been associated with BV, but there are very few studies that comprehensively evaluate the virulence potential of these microorganisms, mainly due to difficult growth conditions. However, in order to find answers to some of the questions related to BV, isolation and characterization of such bacteria will be required. In this work, samples of vaginal exudate from BV women were collected and 31 different microorganisms were isolated, including 6 novel species never described in BV before. Each microorganism was characterized for their ability to adhere to HeLa epithelial cells and cause cytopathogenic changes; their intrinsic biofilm-forming capability; and finally their antimicrobial susceptibility tests for antibiotics commonly used in the treatment of BV. Despite the strong evidence that G. vaginalis outcompeted the other species in the defined virulence assays, our results also demonstrate that other bacteria showed significant biofilm-forming capability, but not initial adhesion, such as Actinomyces turicensis and Corynebacterium tuscaniense. This work supports the evidence that G. vaginalis is the main colonizer in multi-species related BV and further describes novel microorganisms with enhanced virulence potential. Finally, this is the first characterization of Portuguese microbiota associated with BV

Gardnerella vaginalis outcompetes 29 other bacterial species isolated from BV patients in an in vitro biofilm formation model

Despite the worldwide prevalence of bacterial vaginosis (BV), its etiology is still unknown. Although BV has been associated with the presence of biofilm, the ability of BV-associated bacteria to form biofilms is still largely unknown. Here, we isolated 30 BV-associated species and characterized their virulence, using an in vitro biofilm formation model. Our data suggests that Gardnerella vaginalis had the highest virulence potential, as defined by higher initial adhesion and cytotoxicity of epithelial cells, as well as the greater propensity to form a biofilm. Interestingly, we also demonstrated that most of the BV-associated bacteria had a tendency to grow as biofilms.This work was supported by the European Union (FEDER/COMPETE funds) and the Fundacao para a Ciencia e a Tecnologia (reference FCOMP-01-0124-FEDER-008991 [PTDC/BIA-MIC/098228/2008] and RECI/EBB-EBI/0179/2012 [FCOMP-01-0124-FEDER-027462])

Weight change in patients followed in a dyslipidemia

Objectivo: Avaliação da evolução do peso corporal dos doentes seguidos numa consulta de dislipidemias ao longo do tempo. Metodologia: Da população de doentes seguidos na nossa Consulta de Dislipidemias foi estudada uma amostra aleatória de 143 doentes, dos quais foram registados o peso inicial e a sua evolução aos 3 e 6 meses e 1,3 e 5 anos de seguimento; o subgrupo dos doentes a quem foi prescrita uma dieta de res- trição calórica quantifi cada foi analisado separadamente. Foram comparados os valores médios dos pesos nos vários períodos de avaliação para a amostra total e, posteriormente, realizadas comparações por quartis de índice de massa corporal (IMC), sendo ainda comparado o perfi l lipídico inicial e aos 3 meses. Resultados: Considerando a amostra total, não houve variação signifi cativa do peso para qualquer dos intervalos considerados. Na análise por quartis de IMC salienta-se, para o 4º quartil, uma redução estatisticamente signifi cativa dos 3 aos 6 meses (p=0,014) e, para o 3º quartil, uma redução dos 0 aos 6 meses, com signifi cado estatístico borderline (p=0,05). Nos primeiros 3 meses verifi cou-se uma redução estatisticamente signifi cativa do colesterol total e LDL e triglicerídeos. No grupo com restrição caló- rica individualmente adaptada (n=8) verifi cou-se, nos primeiros 3 meses, uma redução de peso de 2,18Kg (p=0,033), não havendo, no mesmo período, variação estatisticamente signifi cativa do perfi l lipídico, provavelmente pelo reduzido tamanho da amostra. Conclusão: A dieta de restrição calórica parece ser mais efi caz na redução de peso do que as medidas habituais de modifi cação do estilo de vida; estas têm, no entanto, efeito benéfi co no perfi l lipídico, pelo menos a curto prazo.Objective: To evaluate the evolution of body weight in patients followed in a dyslipidaemia outpatient clinic. Methods: We randomly selected a sample of 143 patients followed in our dyslipidaemia outpatient clinic and registered their initial weight, at 3 and 6 months and after 1,3 and 5 years of follow-up. A subgroup of patients was prescribed a quantifi ed caloric restriction diet and data from this group was analysed separately. We compared the mean weights of the total sample obtained at the different evaluation periods and also for each quartile of BMI; the lipid profi le was compared at times 0 and 3 months. Results: Considering the whole sample, there was no signifi cant weight variation at any interval considered. In the analysis by BMI quartiles, there was a statistically signifi cant reduction from 3 to 6 months (p=0.014) for the 4th quartile and a borderline signifi cant reduction from 0 to 6 months for the 3rd quartile (p=0.05). In the fi rst 3 months there was a statistically signifi cant reduc- tion of the total and LDL cholesterol and triglycerides. In the subgroup with individually adapted caloric restriction (n=8) there was, during the 1st 3 months, a weight reduction of 2.18 Kg (p=0.033); in the same period, there was no statistically signifi cant variation of the lipid profi le, probably due to the reduced sample size. Conclusion: The caloric restriction diet appears to be more effective in weight reduction than the usual lifestyle changes, which, in turn, have a favourable effect in the lipid profi le, at least in the short term

Isolation of Gardnerella vaginalis from BV patients and healthy women : analysis of virulence through adherence, biofilm formation and cytotoxicity assays

Bacterial vaginosis (BV) is one of the most common gynaecological disorder affecting women in the reproductive age. Microbiological analysis of BV has shown Gardnerella vaginalis to be the most frequent organism in BV. However, G. vaginalis colonization do not always lead to BV. This raised the question whether there are pathogenic and commensal lineages within this species. In an effort to understand the differences between G. vaginalis strains, we performed in vitro assays to compare virulence properties of recently isolated 14 G. vaginalis strains from Portuguese women with and without BV. G. vaginalis strains were characterised for their initial adhesion ability to a monolayer of HeLa cells by incubating the bacteria with this monolayer and quantifying the adhesion by staining with DAPI and fluorescence microscopy. These assays revealed that the BV isolates of G. vaginalis had a stronger initial adhesion capability than non-BV isolates. The biofilm-forming capacity was then assessed by allowing each of the strains to form biofilms under anaerobic conditions for 48 hours and using different growth media. It was possible to observe that BV isolates tend to growth preferentially as biofilms while non-BV isolates had a lower intrinsic tendency towards biofilm formation. In addition, BV isolates of G. vaginalis displayed robust cytotoxicity in the epithelial cells after 3 hours in the contact with a monolayer of HeLa cells. Thus, this study outlines two distinct variants of G. vaginalis, one apparently commensal and one pathogenic, and presents evidence for disparate virulence potentials

Can a specific sub-group of biofilm- forming Gardnerella vaginalis strains be the real causative agent of bacterial vaginosis?

In the past half century, bacterial vaginosis (BV) has been a controversial topic in medical microbiology, and despite the wealth of information on this topic, the etiological agent has not yet been definitively identified [1]. The first advances on BV pointed Gardnerella vaginalis as the infectious causative agent of BV [2] but soon after it was found that G. vaginalis was also present in healthy women [3]. Additionally, G. vaginalis was not able to cause BV consistently. Furthermore, other microorganisms started to be associated with BV, and this resulted in a shift in the paradigm to that of a multispecies infection. However, epidemiological data revealed inconsistencies with this latter theory [4]. A couple of years ago the first descriptions of multispecies biofilm communities were described in BV [5]. Interestingly, G. vaginalis was present in most cases and accounted for the majority of the biofilm biomass. Further studies demonstrated that biofilm-forming G. vaginalis presented higher tolerance to external stresses [6]. Taking these data into consideration, we hypothesized that strains of G. vaginalis that were able to form biofilms could be the causative agent of BV. To test our hypothesis, we isolated more than 30 bacterial species from BV patients and also several strains of G. vaginalis from healthy women, and tested biofilm forming ability, initial adhesion to human vaginal cells, cytotoxicity activity, antimicrobial resistance and gene expression of know virulent genes. Our results revealed that G. vaginalis outcompeted all the other bacterial species in the initial adhesion to the epithelial cells. Furthermore, when comparing BV-associated G. vaginalis strains to strains isolated from healthy women, we found that all 7strains from BV were more virulent than the 7 strains colonizing healthy women, as measured by the higher cytotoxicity and the higher initial adhesion to epithelial cells. No significant differences were found in antimicrobial resistance profiles. Interestingly, no significant differences in expression of known virulence genes were detected, suggesting that the higher virulence of the BV-associated G. vaginalis was due to a yet unknown virulence determinant. We then tested virulent G. vaginalis against other known BV-associated anaerobe pathogens, namely Mobiluncus mulieris, Atopobium vaginae, Prevotella bivia and Fusobacteria nucleatum in mixed biofilm formation quantification. Interestingly, while the other tested anaerobes did not reveal a higher initial adhesion, they did enhance biofilm formation by G. vaginalis. Overall, our data suggests that virulent variants of G. vaginalis have the potential to be the etiological agent of BV, while acknowledging that other anaerobes do enhance G. vaginalis virulence

New insights on mechanisms of sulfonamide bio-transformation by environmental bacteria

info:eu-repo/semantics/publishedVersio

Workshop Report

Climate is the planetary response of the atmospheric circulation to changes in its composition, the solar system configuration, Earth’s rotation, and the distribution of the oceans and continents. As a result, it is continuously evolving, expressed at a global scale by subsiding and uplifting convection cells. These changes have long been recognized and documented in geologic objects of all ages. There are climate signals in many rocks, different geologic features, fossil fragments and imprints, prehistoric remains, and historical reports that can be analyzed and interpreted in order to learn more about past climate changes. Lessons from the past support the view that change is the rule, not the exception, as evidenced by strongly contrasting and chaotic extremes, defined by the whole ensemble of extra-planetary, external, and internal geodynamic controls.Instituto Terra e Memória, Centro Geociências U.Coimbra_ UID_73info:eu-repo/semantics/publishedVersio

Deacetylation and Desuccinylation of the Fucose-Rich Polysaccharide Fucopol: Impact on Biopolymer Physical and Chemical Properties

FucoPol is an acylated polysaccharide with demonstrated valuable functional properties that include a shear thinning fluid behaviour, a film-forming capacity, and an emulsion forming and stabilizing capacity. In this study, the different conditions (concentration, temperature, and time) for alkaline treatment were investigated to deacylate FucoPol. Complete deacetylation and desuccinylation was achieved with 0.02 M NaOH, at 60 ºC for 15 min, with no significant impact on the biopolymer’s sugar composition, pyruvate content, and molecular mass distribution. FucoPol depyruvylation by acid hydrolysis was attempted, but it resulted in a very low polymer recovery. The effect of the ionic strength, pH, and temperature on the deacetylated/desuccinylated polysaccharide, d-FucoPol, was evaluated, as well as its emulsion and film-forming capacity. d-FucoPol aqueous solutions maintained the shear thinning behaviour characteristic of FucoPol, but the apparent viscosity decreased significantly. Moreover, contrary to FucoPol, whose solutions were not affected by the media’s ionic strength, the d-FucoPol solutions had a significantly higher apparent viscosity for a higher ionic strength. On the other hand, the d-FucoPol solutions were not affected by the pH in the range of 3.6–11.5, while FucoPol had a decreased viscosity for acidic pH values and for a pH above 10.5. Although d-FucoPol displayed an emulsification activity for olive oil similar to that of FucoPol (98 +- 0%) for an oil-to-water ratio of 2:3, the emulsions were less viscous. The d-FucoPol films were flexible, with a higher Young0s modulus (798 +- 152 MPa), a stress at the break (22.5 +- 2.5 MPa), and an elongation at the break (9.3 +- 0.7%) than FucoPol (458 +- 32 MPa, 15.5 +- 0.3 MPa and 8.1 +- 1.0%, respectively). Given these findings, d-FucoPol arises as a promising novel biopolymer, with distinctive properties that may render it useful for utilization as a suspending or emulsifier agent, and as a barrier in coatings and packaging filmsinfo:eu-repo/semantics/publishedVersio