Studies on the oxidative stress response of porphyromonas gingivalis

Porphyromonas gingivalis is a Gram-negative anaerobic cocco-bacillus strongly implicated in the aetiology of adult periodontitis. During the colonisation of the oral cavity it is likely that P. gingivalis encounters different sources of oxidative stress. Adaptation to this challenge is necessary for the microorganism to survive and establish in the periodontal environment. The aim of the present study was, therefore, to investigate the oxidative stress survival mechanisms of P. gingivalis. P. gingivalls was grown under different oxygenated environments in a continuous culture system under conditions of haemin-limitation and excess. Steady state was achieved under moderately oxygenated atmospheres, although a decrease in cell viability was observed as the oxygen concentration in the gas mixture increased. Haemin-excess conditions seemed to increase the ability of a culture to cope with a determined oxygen concentration. The main change in fermentation end-products characterising oxygen stressed cultures was an increase in the production of acetate. Scanning electron micrographs showed that oxygen triggers a change in the cell shape from a cocco-bacillary to a short rod. The effect of oxygen on the expression of cysteine proteinases, critical virulence factors of P. gingivalis, was assayed in supernatants and cell fractions and further analysed by 2-D gel electrophoresis. Both evaluations showed an increase in the cell-associated Arg-gingipain and a decrease in Lys-gingipain in oxygen stressed cells. Assays for NADH oxidase, NADH peroxidase and superoxide dismutase in cell extracts showed an increase in their activities as the environment became more oxygenated. The NADH oxidase activity was partially purified and characterised and, surprisingly, the isolated protein was identified as a 4-hydroxybutyryl-CoA dehydratase. This is the first report of NADH oxidase activity associated with this enzyme. The existence of other open reading frames in the P. gingivalls genome sequence that would encode for proteins with the potential for NADH oxidase/peroxidase activity was further investigated. The transcription product of the identified ORFs, encoding for a possible NADH oxidase (Nox) and an alkyl hydroperoxide reductase (AhpF-C), was analysed under anaerobic and oxygenated environments by northern blot hybridization. No mRNA for Nox was detected but AhpF-C was expressed constitutively in anaerobic cells and slightly increased under oxygenated conditions. Furthermore, the possibility of the existence of a common transcriptional switch for oxidative stress-related proteins was investigated. A homologue of OxyR, a redox-sensitive transcriptional activator, was identified in the P. gittgivalis genome sequence and an OxyR-disrupted mutant was constructed. Mutants exhibited decreased tolerance to air and hydrogen peroxide and were characterised by the absence of alkyl hydroperoxide reductase mRNA, suggesting a control of OxyR over its expression. The second part of this research project consisted of studies of P. gingivalis in co-culture with F. nucleatum, vnder oxygenated environments. These studies showed that not only does F. nucleatum have a much higher tolerance to oxygen than P. gingivalis but, in co-culture, it can protect the latter organism and increase its ability to survive under oxygenated conditions. Additionally, it was observed that F. nucleatum is able to generate a capnophilic environment essential for the growth of P. gingivalis. In conclusion, this study showed that P. gingivalis possesses basic mechanisms to cope with moderate or transient oxidative stress while it probably relies on the protection of other organisms, like F. nucleatum, to survive and replicate in highly oxygenated atmospheres.Thesis (Ph.D.) -- University of Adelaide, Dental School, 200

Vesicular-arbuscular mycorrhizal, dark septate endophytes and root anatomy in Fragaria ananassa var. Camino Real (Rosaceae) in the province of Tucumán, Argentina

En la provincia de Tucumán el cultivo de frutilla ocupa una superficie de 350 hectáreas. El mismo es invernal, con cosechas periódicas de fines de mayo a noviembre inclusive. Para evitar esta discontinuidad de producción, se incorporan al espectro de variedades precoces (Fortuna y Festival), variedades tardías, entre ellas Camino Real. Los objetivos de este trabajo fueron estudiar las micorrizas vesículo-arbusculares, los endófitos septados oscuros y caracterizar la anatomía radical en Fragaria ananassa var. Camino Real en cultivo comercial en la provincia de Tucumán. El muestreo fue realizado en el INTA-EEA Famaillá, Tucumán, Argentina. Se recolectaron los sistemas radicales correspondientes a un total de 20 individuos; los que fueron tratados con técnicas convencionales. Los sistemas radicales de la variedad Camino Real presentan las células del parénquima cortical colonizadas por micorrizas vesículo-arbusculares con dos tipos morfológicos simultáneos: Arum y Paris, siendo la morfología Arum la de mayor frecuencia. Además, el tejido cortical presenta endófitos septados oscuros. La anatomía radical de la estructura primaria presenta una histología típica con estelas de tipo diarca a tetrarca. Mientras que la estructura secundaria de la raíz muestra diferentes estadios de crecimiento, con restos de epidermis y parénquima cortical adheridos a la polidermis en formación. Se describen por primera vez, para Argentina, las micorrizas vesículoarbusculares, los endófitos septados oscuros y la anatomía radical en Fragaria ananassa var. Camino Real.In the province of Tucumán strawberry cultivation occupies an area of 350 hectares. It is a winter crop, with periodic harvests since late May to November inclusive. To avoid this discontinuity of production, a late variety (Camino Real) was added to the spectrum of early varieties (Fortuna and Festival). This paper aims to evaluate the vesicular-arbuscular mycorrhiza, dark septate endophytes and characterize the radical anatomy in Fragaria ananassa variety Camino Real in the province of Tucumán. The sampling was carried out at INTA-EEA Famaillá, Tucumán, Argentina. The radicals systems corresponding to 20 individuals were collected and treated with conventional techniques. The root systems of the variety Camino Real, show cortical parenchyma cells colonized by vesicular-arbuscular mycorrhizae which have two simultaneous morphological types: Arum and Paris. The Arum morphology is the most frequent. In addition, the cortical tissue has dark septate endophytes. The radical anatomy of the primary structure presents a typical histology with diarca to tetrarch stela, while the secondary root structure show different stages of growth with traces of epidermis and cortical parenchyma attached to the polidermis in development. The vesicular-arbuscular mycorrhizae, dark septate endophytes and radical anatomy of Fragaria ananassa var. ‘Camino Real’ were described for the first time for ArgentinaFil: Lizarraga, Sofía Valentina. Fundación Miguel Lillo; ArgentinaFil: Ruiz, A. I.. Fundación Miguel Lillo; ArgentinaFil: Salazar, Sergio Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Instituto Nacional de Tecnología Agropecuaria; ArgentinaFil: Diaz Ricci, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Albornoz, Patricia Liliana. Fundación Miguel Lillo; Argentin

Integrated Analysis of Clinical and Microbiome Risk Factors Associated with the Development of Oral Candidiasis during Cancer Chemotherapy.

Oral candidiasis is a common side effect of cancer chemotherapy. To better understand predisposing factors, we followed forty-five subjects who received 5-fluorouracil- or doxorubicin-based treatment, during one chemotherapy cycle. Subjects were evaluated at baseline, prior to the first infusion, and at three additional visits within a two-week window. We assessed the demographic, medical and oral health parameters, neutrophil surveillance, and characterized the salivary bacteriome and mycobiome communities through amplicon high throughput sequencing. Twenty percent of all subjects developed oral candidiasis. Using multivariate statistics, we identified smoking, amount of dental plaque, low bacteriome and mycobiome alpha-diversity, and the proportions of specific bacterial and fungal taxa as baseline predictors of oral candidiasis development during the treatment cycle. All subjects who developed oral candidiasis had baseline microbiome communities dominated by Candida and enriched in aciduric bacteria. Longitudinally, oral candidiasis was associated with a decrease in salivary flow prior to lesion development, and occurred simultaneously or before oral mucositis. Candidiasis was also longitudinally associated with a decrease in peripheral neutrophils but increased the neutrophil killing capacity of Candida albicans. Oral candidiasis was not found to be associated with mycobiome structure shifts during the cycle but was the result of an increase in Candida load, with C. albicans and Candida dubliniensis being the most abundant species comprising the salivary mycobiome of the affected subjects. In conclusion, we identified a set of clinical and microbiome baseline factors associated with susceptibility to oral candidiasis, which might be useful tools in identifying at risk individuals, prior to chemotherapy

Nintedanib Reduces Muscle Fibrosis and Improves Muscle Function of the Alpha-Sarcoglycan-Deficient Mice

Sarcoglycanopathies are a group of recessive limb-girdle muscular dystrophies, characterized by progressive muscle weakness. Sarcoglycan deficiency produces instability of the sarcolemma during muscle contraction, leading to continuous muscle fiber injury eventually producing fiber loss and replacement by fibro-adipose tissue. Therapeutic strategies aiming to reduce fibro-adipose expansion could be effective in muscular dystrophies. We report the positive effect of nintedanib in a murine model of alpha-sarcoglycanopathy. We treated 14 Sgca mice, six weeks old, with nintedanib 50 mg/kg every 12 h for 10 weeks and compared muscle function and histology with 14 Sgca mice treated with vehicle and six wild-type littermate mice. Muscle function was assessed using a treadmill and grip strength. A cardiac evaluation was performed by echocardiography and histological study. Structural analysis of the muscles, including a detailed study of the fibrotic and inflammatory processes, was performed using conventional staining and immunofluorescence. In addition, proteomics and transcriptomics studies were carried out. Nintedanib was well tolerated by the animals treated, although we observed weight loss. Sgca mice treated with nintedanib covered a longer distance on the treadmill, compared with non-treated Sgca mice, and showed higher strength in the grip test. Moreover, nintedanib improved the muscle architecture of treated mice, reducing the degenerative area and the fibrotic reaction that was associated with a reversion of the cytokine expression profile. Nintedanib improved muscle function and muscle architecture by reducing muscle fibrosis and degeneration and reverting the chronic inflammatory environment suggesting that it could be a useful therapy for patients with alpha-sarcoglycanopathy

Chemotherapy-induced oral mucositis is associated with detrimental bacterial dysbiosis.

BACKGROUND: Gastrointestinal mucosal injury (mucositis), commonly affecting the oral cavity, is a clinically significant yet incompletely understood complication of cancer chemotherapy. Although antineoplastic cytotoxicity constitutes the primary injury trigger, the interaction of oral microbial commensals with mucosal tissues could modify the response. It is not clear, however, whether chemotherapy and its associated treatments affect oral microbial communities disrupting the homeostatic balance between resident microorganisms and the adjacent mucosa and if such alterations are associated with mucositis. To gain knowledge on the pathophysiology of oral mucositis, 49 subjects receiving 5-fluorouracil (5-FU) or doxorubicin-based chemotherapy were evaluated longitudinally during one cycle, assessing clinical outcomes, bacterial and fungal oral microbiome changes, and epithelial transcriptome responses. As a control for microbiome stability, 30 non-cancer subjects were longitudinally assessed. Through complementary in vitro assays, we also evaluated the antibacterial potential of 5-FU on oral microorganisms and the interaction of commensals with oral epithelial tissues. RESULTS: Oral mucositis severity was associated with 5-FU, increased salivary flow, and higher oral granulocyte counts. The oral bacteriome was disrupted during chemotherapy and while antibiotic and acid inhibitor intake contributed to these changes, bacteriome disruptions were also correlated with antineoplastics and independently and strongly associated with oral mucositis severity. Mucositis-associated bacteriome shifts included depletion of common health-associated commensals from the genera Streptococcus, Actinomyces, Gemella, Granulicatella, and Veillonella and enrichment of Gram-negative bacteria such as Fusobacterium nucleatum and Prevotella oris. Shifts could not be explained by a direct antibacterial effect of 5-FU, but rather resembled the inflammation-associated dysbiotic shifts seen in other oral conditions. Epithelial transcriptional responses during chemotherapy included upregulation of genes involved in innate immunity and apoptosis. Using a multilayer epithelial construct, we show mucositis-associated dysbiotic shifts may contribute to aggravate mucosal damage since the mucositis-depleted Streptococcus salivarius was tolerated as a commensal, while the mucositis-enriched F. nucleatum displayed pro-inflammatory and pro-apoptotic capacity. CONCLUSIONS: Altogether, our work reveals that chemotherapy-induced oral mucositis is associated with bacterial dysbiosis and demonstrates the potential for dysbiotic shifts to aggravate antineoplastic-induced epithelial injury. These findings suggest that control of oral bacterial dysbiosis could represent a novel preventive approach to ameliorate oral mucositis

Prognostic models for mortality after cardiac surgery in patients with infective endocarditis: a systematic review and aggregation of prediction models.

Background There are several prognostic models to estimate the risk of mortality after surgery for active infective endocarditis (IE). However, these models incorporate different predictors and their performance is uncertain. Objective We systematically reviewed and critically appraised all available prediction models of postoperative mortality in patients undergoing surgery for IE, and aggregated them into a meta-model. Data sources We searched Medline and EMBASE databases from inception to June 2020. Study eligibility criteria We included studies that developed or updated a prognostic model of postoperative mortality in patient with IE. Methods We assessed the risk of bias of the models using PROBAST (Prediction model Risk Of Bias ASsessment Tool) and we aggregated them into an aggregate meta-model based on stacked regressions and optimized it for a nationwide registry of IE patients. The meta-model performance was assessed using bootstrap validation methods and adjusted for optimism. Results We identified 11 prognostic models for postoperative mortality. Eight models had a high risk of bias. The meta-model included weighted predictors from the remaining three models (EndoSCORE, specific ES-I and specific ES-II), which were not rated as high risk of bias and provided full model equations. Additionally, two variables (age and infectious agent) that had been modelled differently across studies, were estimated based on the nationwide registry. The performance of the meta-model was better than the original three models, with the corresponding performance measures: C-statistics 0.79 (95% CI 0.76–0.82), calibration slope 0.98 (95% CI 0.86–1.13) and calibration-in-the-large –0.05 (95% CI –0.20 to 0.11). Conclusions The meta-model outperformed published models and showed a robust predictive capacity for predicting the individualized risk of postoperative mortality in patients with IE. Protocol registration PROSPERO (registration number CRD42020192602).pre-print270 K

INRISCO: INcident monitoRing in Smart COmmunities

Major advances in information and communication technologies (ICTs) make citizens to be considered as sensors in motion. Carrying their mobile devices, moving in their connected vehicles or actively participating in social networks, citizens provide a wealth of information that, after properly processing, can support numerous applications for the benefit of the community. In the context of smart communities, the INRISCO [1] proposal intends for (i) the early detection of abnormal situations in cities (i.e., incidents), (ii) the analysis of whether, according to their impact, those incidents are really adverse for the community; and (iii) the automatic actuation by dissemination of appropriate information to citizens and authorities. Thus, INRISCO will identify and report on incidents in traffic (jam, accident) or public infrastructure (e.g., works, street cut), the occurrence of specific events that affect other citizens' life (e.g., demonstrations, concerts), or environmental problems (e.g., pollution, bad weather). It is of particular interest to this proposal the identification of incidents with a social and economic impact, which affects the quality of life of citizens.This work was supported in part by the Spanish Government through the projects INRISCO under Grant TEC2014-54335-C4-1-R, Grant TEC2014-54335-C4-2-R, Grant TEC2014-54335-C4-3-R, and Grant TEC2014-54335-C4-4-R, in part by the MAGOS under Grant TEC2017-84197-C4-1-R, Grant TEC2017-84197-C4-2-R, and Grant TEC2017-84197-C4-3-R, in part by the European Regional Development Fund (ERDF), and in part by the Galician Regional Government under agreement for funding the Atlantic Research Center for Information and Communication Technologies (AtlantTIC)

Stabilization of angiotensin-(1-7) by key substitution with a cyclic non-natural amino acid

Angiotensin-(1-7) [Ang-(1-7)], a heptapeptide hormone of the renin-angiotensin-aldosterone system (RAAS), is a promising candidate as a treatment for cancer that reflects its antiproliferative and anti-angiogenic properties. However, the peptide’s therapeutic potential is limited by the short half-life and low bioavailability resulting from rapid enzymatic metabolism by peptidases including angiotensin-converting enzyme (ACE) and dipeptidyl peptidase 3 (DPP 3). We report the facile assembly of three novel Ang-(1-7) analogues by solid-phase peptide synthesis which incorporates the cyclic non-natural δ-amino acid ACCA. The analogues containing the ACCA substitution at the site of ACE cleavage exhibit complete resistance to human ACE, while substitution at the DDP3 cleavage site provided stability against DPP 3 hydrolysis. Furthermore, the analogues retain the anti-proliferative properties of Ang-(1-7) against the 4T1 and HT-1080 cancer cell lines. These results suggest that ACCA-substituted Ang-(1-7) analogues which show resistance against proteolytic degradation by peptidases known to hydrolyze the native heptapeptide may be novel therapeutics in the treatment of cancer

Evolutionarily Conserved Substrate Substructures for Automated Annotation of Enzyme Superfamilies

The evolution of enzymes affects how well a species can adapt to new environmental conditions. During enzyme evolution, certain aspects of molecular function are conserved while other aspects can vary. Aspects of function that are more difficult to change or that need to be reused in multiple contexts are often conserved, while those that vary may indicate functions that are more easily changed or that are no longer required. In analogy to the study of conservation patterns in enzyme sequences and structures, we have examined the patterns of conservation and variation in enzyme function by analyzing graph isomorphisms among enzyme substrates of a large number of enzyme superfamilies. This systematic analysis of substrate substructures establishes the conservation patterns that typify individual superfamilies. Specifically, we determined the chemical substructures that are conserved among all known substrates of a superfamily and the substructures that are reacting in these substrates and then examined the relationship between the two. Across the 42 superfamilies that were analyzed, substantial variation was found in how much of the conserved substructure is reacting, suggesting that superfamilies may not be easily grouped into discrete and separable categories. Instead, our results suggest that many superfamilies may need to be treated individually for analyses of evolution, function prediction, and guiding enzyme engineering strategies. Annotating superfamilies with these conserved and reacting substructure patterns provides information that is orthogonal to information provided by studies of conservation in superfamily sequences and structures, thereby improving the precision with which we can predict the functions of enzymes of unknown function and direct studies in enzyme engineering. Because the method is automated, it is suitable for large-scale characterization and comparison of fundamental functional capabilities of both characterized and uncharacterized enzyme superfamilies