Stimuli-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.

A new, bifunctional recombinant protein was expressed as the fusion product of human elastin‐like polypeptide (HELP) and the bilirubin‐binding protein UnaG. The engineered product displays both the HELP‐specific property of forming a functional hydrogel matrix and the UnaG‐specific capacity of emitting green fluorescence upon ligand binding. The new fusion protein has been proven to be effective at detecting bilirubin in complex environments with high background noise. A cell culture model of the stress response, consisting of bilirubin released in the cell culture medium, was set up to assess the bilirubin‐sensing properties of the functional matrix obtained by cross‐linking the HELP moiety. Our engineered protein allowed us to monitor cell induction by the release of bilirubin in the culture medium on a nanomolar scale. This study shows that elastin‐like protein fusion represents a versatile platform for the development of novel and commercially viable analytical and biosensing devices.

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In vitro and in vivo studies have demonstrated that UCB (unconjugated bilirubin) is neurotoxic. Although previous studies suggested that both MRP1 (multidrug resistance-associated protein 1) and MDR1 (multidrug resistance protein 1) may protect cells against accumulation of UCB, direct comparison of their role in UCB transport was never performed. To this end, we used an inducible siRNA (small interfering RNA) expression system to silence the expression of MRP1 and MDR1 in human neuroblastoma SH-SY5Y cells. The effects of in vitro exposure to clinically-relevant levels of unbound UCB were compared between unsilenced (control) cells and cells with similar reductions in the expression of MRP1 or MDR1, documented by RT-PCR (reverse transcription-PCR) (mRNA), immunoblotting (protein), and for MDR1, the enhanced net uptake of a specific fluorescent substrate. Cytotoxicity was assessed by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] test. MRP1-deficient cells accumulated significantly more UCB and suffered greater cytotoxicity than controls. By contrast, MDR1-deficient cells exhibited UCB uptake and cytotoxicity comparable with controls. At intermediate levels of silencing, the increased susceptibility to UCB toxicity closely correlated with the decrease in the expression of MRP1, but not of MDR1. These data support the concept that limitation of cellular UCB accumulation, due to UCB export mediated by MRP1, but not MDR1, plays an important role in preventing bilirubin encephalopathy in the newborn.

The Pseudomonas aeruginosa isolate TS-832035 was responsible for an outbreak that occurred in an Italian hospital between 1999 and 2002. It exhibited a high-level resistance to carbapenems due to the contemporary presence of two independent mechanisms: the production of a carbapenemase, coded by a bla(VIM-1) determinant carried by the chromosomal class 1 integron In70.2 (containing also the aacA4, aphA15, and aadA1 genes in its cassette array), and the lack of the OprD porin. We compared TS-832035 with a strictly related isolate, TS-103, whose resistance to carbapenems was due to the lack of the OprD porin only, as it did not carry In70.2. We evaluated their growth kinetics, in both separate cultures and competition assays, under permissive conditions. These experiments highlighted a significant in vitro fitness cost associated with the integron. On the contrary, none of the resistance determinants other than the bla(VIM-1) seemed to confer a real selective advantage to its host. Comparison of these results with the in vivo behavior, showing that the In70.2-carrying isolates largely prevailed over the In70.2-lacking ones, besides the detection of similar integrons in other Italian clinical isolates, evidenced the need to investigate accurately the causes of their large distribution, as possible soft spots could exist in the ability of their hosts to adapt to the hospital settings.

Bacterial vaginosis involves the presence of a polymicrobial biofilm on the vaginal epithelium, guaranteeing immune escape and spread of antibiotic resistance. To spot known biofilm-forming bacteria, we profiled the vaginal microbiome of sixty-four symptomatic women suffering from a different grade of vaginal disorders and sixty asymptomatic healthy women. Specific microbial profiles distinguished symptomatic from asymptomatic women and characterized the grade of dysmicrobism within the symptomatic group. Lactobacillus crispatus and iners predominated on the healthy vaginal mucosa, while Lactobacillus gasseri predominated in the intermediate dysmicrobism. Furthermore, the intermediate grade of dysmicrobism was characterized by other lactic acid-producers species than Lactobacilli, able to rescue the microbial imbalance, and Ureaplasma parvum-serovar 3. The vaginosis group exhibited the overgrowth of Prevotella bivia, which is known to enhance the biofilm formation by Gardnerella vaginalis, and the presence of Streptococcus anginosus, which is emerging as a new cooperating player of the vaginal biofilm. Identifying specific microorganisms promoting or preventing the biofilm formation could increase the accuracy for a better definition of the vaginal dysmicrobism concept and therapeutic intervention.

Mycobacterium vaccae is a rapidly growing nonpathogenic species of the Mycobacteriaceae family of bacteria that can cause pulmonary and disseminated disease in particular in immunocompromised individuals. Here we describe a first case of matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass-spectrometry (MS) identification of this pathogen in a patient with non-Hodgkin’s lymphoma during chemoimmunotherapy salvage treatment, and its impact on clinical decision making.

Proprietà antimicrobiche di prodotti di origine vegetale per il trattamento di patologie infettive del basso tratto genitale femminile SUMMARYThe phlogistic-infectious diseases of the lower female genital tract are one of the most widespread obstetricgynecologic issues, due to treatment failures that cause frequent relapses and to the adverse effects of some commonly used drugs.The most common vaginal syndromes are due to uncontrolled growth of bacteria or fungi which replace the normal vaginal flora, causing phlogistic and infectious based diseases. These infections are treated with anti-inflammatory and antibiotic therapy; however, the emergence of resistant strains and the ability of many microorganisms to grow inside biofilms severely reduce the repertoire of useful agents.Thus, in the last years increasing interest has been focused toward compounds of plant origin with anti-microbial properties. In the present work, we studied the antimicrobial activity of fractions obtained from endemic plants of Sardinia towards microorganisms that frequently are involved in vaginal infectious diseases: Streptococcus agalactiae, Gardnerella vaginalis and Candida albicans.