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Although oocyte cryopreservation has great potentials in the field of reproductive technologies, it still is an open challenge in the majority of domestic animals and little is known on the biochemical transformation induced by this process in the different cellular compartments. Raman micro-spectroscopy allows the non-invasive evaluation of the molecular composition of cells, based on the inelastic scattering of laser photons by vibrating molecules. The aim of this work was to assess the biochemical modifications of both the zona pellucida and cytoplasm of vitrified/warmed in vitro matured bovine oocytes at different post-warming times. By taking advantage of Principal Component Analysis, we were able to shed light on the biochemical transformation induced by the cryogenic treatment, also pointing out the specific role of cryoprotective agents (CPs). Our results suggest that vitrification induces a transformation of the protein secondary structure from the α-helices to the β-sheet form, while lipids tend to assume a more packed configuration in the zona pellucida. Both modifications result in a mechanical hardening of this cellular compartment, which could account for the reduced fertility rates of vitrified oocytes. Furthermore, biochemical modifications were observed at the cytoplasmic level in the protein secondary structure, with α-helices loss, suggesting cold protein denaturation. In addition, a decrease of lipid unsaturation was found in vitrified oocytes, suggesting oxidative damages. Interestingly, most modifications were not observed in oocytes exposed to CPs, suggesting that they do not severely affect the biochemical architecture of the oocyte. Nevertheless, in oocytes exposed to CPs decreased developmental competence and increased reactive oxygen species production were observed compared to the control. A more severe reduction of cleavage and blastocyst rates after in vitro fertilization was obtained from vitrified oocytes. Our experimental outcomes also suggest a certain degree of reversibility of the induced transformations, which renders vitrified oocytes more similar to untreated cells after 2 h warming. [ABSTRACT FROM AUTHOR]

Bacterial spores are surrounded by the coat, a multilayered shell that contributes in protecting the genome during stress conditions. In Bacillus subtilis, the model organism for spore formers, the coat is composed by about seventy different proteins, organized into four layers by the action of several regulatory proteins. A major component of this regulatory network, CotE, is needed to assemble the outer coat and develop spores fully resistant to lysozyme and able to germinate efficiently. Another regulator, CotH, is controlled by CotE and is present in low amounts both during sporulation and in mature spores. In spite of this CotH controls the assembly of at least nine outer coat proteins and cooperates with CotE in producing fully resistant and efficiently germinating spores. In order to improve our understanding of CotH role in spore formation, we over-produced CotH by placing its coding region under the control of a promoter stronger than its own promoter but with a similar timing of activity during sporulation. Over-production of CotH in an otherwise wild type strain did not cause any major effect, whereas in a cotE null background a partial recovery of the phenotypes associated to the cotE null mutation was observed. Western blot, fluorescence microscopy and Surface-Enhanced Raman Scattering spectroscopy data indicate that, in the absence of CotE, over-production of CotH allowed the formation of spores overall resembling wild type spores and carrying in their coat some CotE−/CotH-dependant proteins. Our results suggest that the B. subtilis spore differentiation programme is flexible, and that an increase in the amount of a regulatory protein can replace a missing partner and partially substitute its function in the assembly of the spore coat. [ABSTRACT FROM AUTHOR]

Acanthamoeba keratitis is a rare but serious corneal disease, often observed in contact lens wearers. Clinical treatment of infected patients frequently involves the use of polyhexamethylene biguanide (PHMB), a polymer used as a disinfectant and antiseptic, which is toxic also for the epithelial cells of the cornea. Prompt and effective diagnostic tools are hence highly desiderable for both starting early therapy and timely suspension of the treatment. In this work we use Raman microspectroscopy to analyse in vitro a single Acanthamoeba cell in cystic phase. In particular, we investigate the effect of PHMB at the single-cell level, providing useful information on both the underlying biochemical mechanism and the time frame for Acanthamoeba eradication in ocular infections. Furthermore, we demonstrate that Raman spectroscopy, in conjunction with standard multivariate analysis methods, allows discriminating between live and dead Acanthamoebas, which is fundamental to optimizing patients’ treatment. [ABSTRACT FROM AUTHOR]

In cystic fibrosis (CF) patients airways mucus shows an increased viscoelasticity due to the concentration of high molecular weight components. Such mucus thickening eventually leads to bacterial overgrowth and prevents mucus clearance. The altered rheological behavior of mucus results in chronic lung infection and inflammation, which causes most of the cases of morbidity and mortality, although the cystic fibrosis complications affect other organs as well. Here, we present a quantitative study on the correlation between cystic fibrosis mucus viscoelasticity and patients clinical status. In particular, a new diagnostic parameter based on the correlation between CF sputum viscoelastic properties and the severity of the disease, expressed in terms of FEV1 and bacterial colonization, was developed. By using principal component analysis, we show that the types of colonization and FEV1 classes are significantly correlated to the elastic modulus, and that the latter can be used for CF severity classification with a high predictive efficiency (88%). The data presented here show that the elastic modulus of airways mucus, given the high predictive efficiency, could be used as a new clinical parameter in the prognostic evaluation of cystic fibrosis. [ABSTRACT FROM AUTHOR]