Thickness measurement of soft thin films on periodically patterned magnetic substrates by phase difference magnetic force microscopy.

The need for accurate measurement of the thickness of soft thin films is continuously encouraging the development of techniques suitable for this purpose. We propose a method through which the thickness of the film is deduced from the quantitative measurement of the contrast in the phase images of the sample surface acquired by magnetic force microscopy, provided that the film is deposited on a periodically patterned magnetic substrate. The technique is demonstrated by means of magnetic substrates obtained from standard floppy disks. Colonies of Staphylococcus aureus adherent to such substrates were used to obtain soft layers with limited lateral (a few microns) and vertical (hundreds of nanometers) size. The technique is described and its specific merits, limitations and potentialities in terms of accuracy and measurable thickness range are discussed. These parameters depend on the characteristics of the sensing tip/cantilever as well as of the substrates, the latter in terms of spatial period and homogeneity of the magnetic domains. In particular, with the substrates used in this work we evaluated an uncertainty of about 10%, a limit of detection of 50-100 nm and an upper detection limit (maximum measurable thickness) of 1 µm, all obtained with standard lift height values (50-100 nm). Nonetheless, these parameters can be easily optimized by selecting/realizing substrates with suitable spacing and homogeneity of the magnetic domains. For example, the upper detection limit can be increased up to 25-50 µm while the limit of detection can be reduced to a few tens of nanometers or a few nanometers.

Analytical techniques to study microbial biofilm on abiotic surfaces: pros and cons of the main techniques currently in use.

Biofilm is a bacterial lifestyle widespread in microbial world and represents a concern in health care. Despite the great life expectancy related to advanced health care, the increasing numbers of biofilm-mediated infections remain a significant public health challenge. Moreover, the problem of biofilm-mediated infections becomes much more severe when biofilm colonizes medical devices and biomaterials. The public health risk due to microbial biofilm-related infections is a concern that requires full attention. However, the complexity of biofilm makes difficult its exhaustive analysis. Although biofilm represents a major challenge in both microbiological and hygiene areas, at now methods aimed to analyse biofilm formation and development are not standardized yet. Different methods have been employed to qualitatively and quantitatively evaluate biofilm each of which is useful to estimate a peculiar aspect of biofilm lifestyle. In the present review, fifteen assays for the qualitative and quantitative evaluation of bacterial biofilm colonizing abiotic substrates, such as medical devices, prosthesis or surfaces for food production together with advantages and limitations of each method were described and compared. Some methods are suited to quantify biofilm matrix while others are capable to evaluate both living and dead cells or quantify exclusively viable cells in biofilm. In particular, colorimetric methods to evaluate biofilm matrix (crystal violet; 1,9-dimethyl methylen blue and fluorescein-di-acetate methods) or viable cells (LIVE/DEAD BacLight, BioTimer Assay, resazurin, tetrazolium hydroxide salt methods) and genetic methods to estimate the bacterial population (PCR and FISH) are reported. Moreover, a section is dedicated to examine the performances of advanced microscopic techniques employed to study microbial biofilms (mass spectrometry; confocal laser scanning microscopy; Raman spectroscopy and electron microscopy). Because of its complexity, an exhaustive study of biofilm requires a combination of different experimental approaches as biochemical, genetic or physical ones.

Body iron delocalization: the serious drawback in iron disorders in both developing and developed countries.

Over 2 billion people in both developing as well as developed countries - over 30% of the world's population - are anaemic. With the classical preconception that oral iron administration or the intake of foods rich in iron increase haemoglobin concentration and reduce the prevalence of anaemia, specific programs have been designed, but iron supplementations have been less effective than expected. Of note, this hazardous simplification on iron status neglects its distribution in the body. The correct balance of iron, defined iron homeostasis, involves a physiological ratio of iron between tissues/secretions and blood, thus avoiding its delocalization as iron accumulation in tissues/secretions and iron deficiency in blood. Changes in iron status can affect the inflammatory response in multiple ways, particularly in the context of infection, an idea that is worth remembering when considering the value of iron supplementation in areas of the world where infections are highly prevalent. The enhanced availability of free iron can increase susceptibility and severity of microbial and parasitic infections. The discovery of the hepcidin-ferroportin (Fpn) complex, which greatly clarified the enigmatic mechanism that supervises the iron homeostasis, should prompt to a critical review on iron supplementation, ineffective on the expression of the most important proteins of iron metabolism. Therefore, it is imperative to consider new safe and efficient therapeutic interventions to cure iron deficiency (ID) and ID anaemia (IDA) associated or not to the inflammation. In this respect, lactoferrin (Lf) is emerging as an important regulator of both iron and inflammatory homeostasis. Oral administration of Lf in subjects suffering of ID and IDA is safe and effective in significantly increasing haematological parameters and contemporary decreasing serum IL-6 levels, thus restoring iron localization through the direct or indirect modulation of hepcidin and ferroportin synthesis. Of note, the nuclear localization of Lf suggests that this molecule may be involved in the transcriptional regulation of some genes of host inflammatory response. We recently also reported that combined administration of oral and intravaginal Lf on ID and IDA pregnant women with preterm delivery threat, significantly increased haematological parameters, reduced IL-6 levels in both serum and cervicovaginal fluid, cervicovaginal prostaglandin PGF2α, and suppressed uterine contractility. Moreover, Lf combined administration blocked further the shortening of cervical length and the increase of foetal fibronectin, thus prolonging the length of pregnancy until the 37th-38th week of gestation. These new Lf functions effective in curing ID and IDA through the restoring of iron and inflammatory homeostasis and in preventing preterm delivery, could have a great relevance in developing countries, where ID and IDA and inflammation-associated anaemia represent the major risk factors of preterm delivery and maternal and neonatal death.

Lactoferrin decreases inflammatory response by cystic fibrosis bronchial cells invaded with Burkholderia cenocepacia iron-modulated biofilm.

In cystic fibrosis (CF) high iron concentration in airway secretion plays a pivotal role in bacterial multiplication and biofilm formation as well as in inflammatory response. Burkholderia cenocepacia, an opportunistic facultative pathogen responsible for chronic lung infections and cepacia syndrome, recurrently infects CF patients. Lactoferrin (Lf), an iron binding multifunctional glycoprotein synthesized by exocrine glands and neutrophils, has been found at higher concentration in the airway secretions of infected CF patients than in healthy subjects. Here the influence of milk derivative bovine lactoferrin (bLf), an emerging important regulator of iron and inflammatory homeostasis, on invasiveness of B. cenocepacia iron-modulated biofilm, as well as on inflammatory response by infected CF bronchial (IB3-1) cells, is reported. bLf did not significantly affect invasion efficacy by biofilmforming B. cenocepacia clinical strains. Conversely, the addition of bLf to cell monolayers during infection significantly decreased the pro-inflammatory Interleukin (IL)-1beta and increased the anti-inflammatory IL-11 expression compared to that observed in cells infected in the absence of bLf. The bLf ability to modulate genes expressed following B. cenocepacia infection seems related to its localization to the nucleus of infected IB3-1 cells. These results provide evidence for a role of bLf in the protection of infected CF cells from inflammation-related damage, thus extending the therapeutic potential of this multifunctional natural protein.

Streptococcus mutans and Streptococcus sobrinus are able to adhere and invade human gingival fibroblast cell line.

Streptococcus mutans and Streptococcus sobrinus, the principal etiologic agents of caries decay of teeth, are generally acquired in oral cavity at the moment of tooth eruption. However, as S. mutans has been detected in oral cavity of predentate children, the eruption of teeth seems not to be a necessary prerequisite, suggesting that this species may be not confined to dental plaque. Here, we evaluate the ability of S. mutans and S. sobrinus in planktonic and biofilm lifestyle to adhere, invade and survive within human gingival fibroblast (HGF-1) cells. Planktonic and biofilm streptococci adhered and invaded host cells to different extents, showing higher efficiencies of biofilm than planktonic counterparts. Moreover, planktonic and biofilm streptococci showed the same percentage of survival within host cells. Transmission electron and confocal microscopy observations confirmed intracellular localization of planktonic and biofilm bacteria. The adhesion, invasion and survival abilities within human oral cells may be considered S. mutans and S. sobrinus virulence mechanisms to colonize and persist in the oral cavity in the absence of tooth surface.