24 research outputs found
Stimuli-Induced Release of Compounds from Elastin Biomimetic Matrix
No full textStimuli-responsive hydrogel matrices have
attracted great attention in biomedical and biotechnological
fields for controlled delivery of bioactive compounds, as well as
a vehicle for therapeutic cell spreading. Elastin-derived
biomimetic polypeptides are recombinant macromolecules
suitable for the realization of smart biomaterials. In this study,
we explored the potential of an elastin biomimetic matrix to
realize proteolytic stimuli-responsive systems to control the
release of substances. Our approach showed that this matrix
was susceptible to elastolytic degradation, and it has been
successfully employed to obtain an efficient delivery of a model
protein. This setup will constitute a therapeutic agent delivery
platform to realize devices capable of responding and
interacting with biological systems at the molecular level
Evaluation of a biomimetic 3D substrate based on the Human Elastin-like Polypeptides (HELPs) model system for elastolytic activity detection
No full textElastin is a fibrous protein that confers elasticity to tissues such as skin, arteries and lung. It is extensively cross-linked, highly hydrophobic and insoluble. Nevertheless, elastin can be hydrolysed by bacterial proteases in infectious diseases, resulting in more or less severe tissue damage. Thus, development of substrates able to reliably and specifically detect pathogen-secreted elastolytic activity is needed to improve the in vitro evaluation of the injury that bacterial proteases may provoke. In this work, two human biomimetic elastin polypeptides, HELP and HELP1, as well as the matrices derived from HELP, have been probed as substrates for elastolytic activity detection. Thirty strains of Pseudomonas aeruginosa isolated from cystic fibrosis patients were analyzed in parallel with standard substrates, to detect proteolytic and elastolytic activity. Results point to the HELP-based 3D matrix as an interesting biomimetic model of elastin to assess bacterial elastolytic activity in vitro. Moreover, this model substrate enables to further elucidate the mechanism underlying elastin degradation at molecular level, as well as to develop biomimetic material-based devices responsive to external stimuli
HELP-based matrices for stimuli-responsive controlled release of bioactive compounds
No full textDirect delivery of bioactive
substances to the sites of injury represents a key
issue for therapies based on regenerative medicine
and tissue repair [1]. Protein derived hydrogels
represent an interesting system for this purpose
because they possess several features that make
them suitable to this purpose. A method for
preparation of hydrogel matrices based on Human
Elastin-like Polypeptide (HELP) has been set up
[2]. HELPs are a family of elastin-like
recombinant biopolymers modeled after the most
regularly repeated domain in human tropoelastin,
retaining peculiar properties as self-assembling
and thermoresponsive behavior [3]. In this study
we assayed two elastolytic activities from different
sources to test their potential to specifically
degrade the HELP matrix
Nephrogenic diabetes insipidus in a geriatric patient affected by SARS-CoV-2: complexity of a diagnosis, complexity of a virus
No full textBACKGROUND: Coronavirus disease 2019 (COVID-19) has an important impact on the kidney through direct and indirect damage mechanisms. Most previous studies have highlighted lesions caused by this virus in the early segments of the nephron. However, due to the antigenic characteristics of the virus, with almost ubiquitous receptors, and the molecular release it triggers, the distal segments of the nephron could also be affected.
METHODS: A 71 year-old-man with respiratory failure while suffering from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pneumonia presented with typical symptoms of diabetes insipidus after ~20 days of hospitalization. The water deprivation test led to the diagnosis of nephrogenic diabetes insipidus. The aetiological study was complex, in particular because of the patient’s previous lithium therapy.
RESULTS: The sequence of pathognomonic events typical of diabetes insipidus associated with anamnestic, clinical and laboratory evidence strongly supported the diagnosis of nephrogenic diabetes insipidus due to SARS-CoV-2 rather than other aetiologies.
CONCLUSIONS: The collecting duct could represent a target for SARS-CoV-2 infection, directly or indirectly, as a result of lesions of upstream portions of the nephron, which would cascade into the distal segment. Other molecules, besides angiotensin 2 converting enzyme, might be involved in facilitating the viral aggression. The complexity of the geriatric patient shows the importance of a comprehensive approach that integrates careful monitoring of clinical signs and symptoms and laboratory and instrumental tests. This is especially important in the context of SARS-CoV-2 infection and in the management of its unexpected complications
Fitness cost associated with the chromosomal integron In70.2 in Pseudomonas aeruginosa clinical isolates
No full textAn epidemiologic survey performed at the Trieste University Hospital (northeastern Italy) between 1999 and 2002 revealed a remarkable spread of an MDR Pseudomonas aeruginosa strain, named TS-832035, which carried the chromosomal integron In70.2 containing four gene cassettes (blaVIM-1, aacA4, aphA15 and aadA1) in its variable region and conferring resistance to ß-lactams, including carbapenems, and to several aminoglycosides. Moreover, some other P. aeruginosa isolates, strictly related to TS-832035 but lacking in the integron In70.2, were detected, but they remained a minor component within the cluster during the three years of surveillance.They showed an MDR phenotype like TS- 832035, differing only for the susceptibility level to carbapenems. The genomic relatedness between TS-832035 and TS-103 was investigated by random amplification of polymorphic DNA (RAPD) typing, pulsed-field gel electrophoresis (PFGE) analysis of SpeI-digested genomic DNA, and multilocus sequence typing (MLST). The cost of the integron In70.2 on the fitness of TS-832035 was determined by performing growth kinetics and direct competition assays against the clonal isolate TS-103 in three media differing for nutrient availability: a rich medium (Luria Bertani (LB) Broth) and a minimal medium (28 g/l K2HPO4, 12 g/l KH2PO4, 0.4 g/l MgSO4, 7H2O, 4 g/l (NH4)2SO4) added with a rich carbon source (0.4% w/v glucose) or with a poorer carbon source (0.4% w/v sodium acetate). Growth kinetic data were obtained by measuring optical density at 600 nm (OD600). For competition assays, the number of CFU/ml of each isolate was estimated by colony-hybridization. We proved the clonality of the two isolates by molecular investigations.The results of the growth kinetics showed the existence of a significant in vitro fitness cost associated with the integron In70.2, more evident in a poorer medium.The sensitivity of the two isolates to the antimicrobial agents tested was the same, except for the different levels of resistance to carbapenems (MIC 16 μg/ml versus 64-128 μg/ml). Although we can not exclude that other factors may have favoured the in vivo spread of TS-832035, our results suggest that the increased level of resistance to carbapenems has conferred on this isolate a selective advantage able to compensate metabolic cost associated to the integron
The biofilm produced by Burkholderia cepacia complex: molecular aspects and relationship with exopolysaccharides
No full textIntroduction. In cystic fibrosis patients, Burkholderia cepacia complex (Bcc) can cause serious pulmonary chronic infections thanks in part
to the ability to form biofilm, matrix rich in exopolysaccharides. In Bcc grown in the planktonic state, the main exopolysaccharide is cepacian
while in biofilm its presence is controversial.
Methods and Results. Two clinical isolates, named BTS7 and BTS2, were studied.
BTS7 produces abundant cepacian but not much biofilm (quantified by colorimetric method).At least two of the genes involved in cepacian
biosynthesis are not necessary for biofilm production as two BTS7 derivatives, bceB and bceQ knocked out by transposon mutagenesis,
produce biofilm levels comparable to the wild-type.
BTS2 sinthesyzes cepacian only if cultured on a specific medium. It has been colonizing a patient for almost ten years, showing a significant
reduction of biofilm production during this period.This reduction did not appear together with the lack of factors required for the initial
adhesion to the surface, or to differences in some of the Bcc genes involved in biofilm formation.
Moreover, sequencing of its bce locus revealed a bceX gene, absent in BTS7, coding for a trascriptional regulator. Its product may negatively
regulate the production of cepacian but not the one of other polysaccharides, promoting the formation of biofilm.
Conclusions. Cepacian seems to be marginal in the production of biofilm.The reduced ability to produce biofilm of BTS2 suggests possible
gene mutations occurred over time. Using custom arrays we will compare the gene expression of the BTS2 isolates, to identify the genes
responsible for the observed phenotypic changes
Study of class I integron in a Burkholderia cepacia complex strain isolated from blood colture
No full textThe Burkholderia cepacia complex (Bcc) consists of several species that cause lung infections in patients with cystic fibrosis but are also capable to colonize immunocompromised patients. Once established, the infection is usually difficult to eradicate, as Bcc is intrinsically resistant to many antibiotics. Besides, the acquisition of additional resistance determinants by horizontal gene transfer makes very difficult the therapeutic approach to these infections. Among horizontally acquired DNAs, integrons have been frequently reported in many Gramnegative bacteria that affect human health, but they have not been found frequently in Burkholderia isolates until now. In the present work we report on a Bcc isolate, recovered from the blood of an immunocompromised patient, that carries a 2.3 kb class I integron already described in a Salmonella enterica isolate eight years ago, coding for aacA4, aadA1 and catB2 in its cassette array
Characterization of integrons in Burkholderia cepacia clinical isolates
No full textBurkholderia cepacia is an opportunistic pathogen able to colonize the airways of Cystic Fibrosis (CF) patients, frequently developing chronic infections. In 20% of cases these infections cause severe and poorly controlled pathological situations because of the intrinsic antibiotic resistance expressed by the microorganism. CF patients are often subjected to antibiotic therapy: this facilitates the acquisition of antibiotic resistance determinants by the infecting bacteria. Integrons are mobile genetic elements that are widespread in bacterial populations and favor the acquisition of gene cassettes coding for these determinants.The presence of class 1 integrons was investigated by PCR with primers specific for the 5’ and 3’ ends in Burkholderia isolates recovered from patients in treatment at the CF center of Friuli Venezia Giulia. The same integron, carrying an uncommon allelic form (Ib) of the aacA4 gene in its cassette array and conferring resistance to some aminoglycosides, was found in two independent isolates (different RAPD profiles) infecting two different patients. In both isolates the integron was carried by plasmids and was still present 3 and 6 years later the first finding. Despite the exchange of integrons between bacterial pathogens is fully described, these items were not frequently found in Burkholderia isolates. Although the clinical relevance of the integron we identified is low (a single gene cassette encoding a widespread resistance),we feel concerned that these genetic elements begin to circulate in this bacterial species, as this could make more and more troublesome the treatment of infections notoriously difficult to eradicate
Genetic analysis of exopolysaccharide acetilation product by Burkholderia cepacia
No full textBacteria belonging to the Burkholderia cepacia complex are mainly isolated from the sputum of cystic fibrosis patients and frequently show a mucoid phenotype.Most of them produce an exopolysaccharide called cepacian, whose repeating unit consists of a branched heptasaccharide carrying from one to three acetyl esters. Two genetic loci, bceI and bceII, consisting of 11 and 9 genes respectively, are involved in cepacian biosynthesis.Three genes located in the bceII locus, named bceOSU, code for different acyltransferases. As the presence of acetyl groups influences the viscosity of cepacian, we compared three strains (two clinical isolates named BTS2 and BTS7, and the reference strain Burkholderia sp. 383) exhibiting differences both in the acetylation pattern and at the genomic level, for the presence of insertion sequences adjacent to bceU
Pseudomonas aeruginosa class I integron AAC (6')-Ib variant (aacA4) and CATB10-Ib variant (cat B10) genes, complete cds.
No full textNew chloramphenicol-acetyltransferases (CATB10) encoded by a class
1 integron from a multidrug resistant Pseudomonas aeruginosa
clinical isolat
