Valentina Palmieri

Biomimetic antimicrobial cloak by graphene-oxide agar hydrogel

Antibacterial surfaces have an enormous economic and social impact on the worldwide technological fight against diseases. However, bacteria develop resistance and coatings are often not uniform and not stable in time. The challenge is finding an antibacterial coating that is biocompatible, cost-effective, not toxic, and spreadable over large and irregular surfaces. Here we demonstrate an antibacterial cloak by laser printing of graphene oxide hydrogels mimicking the Cancer Pagurus carapace. We observe up to 90% reduction of bacteria cells. This cloak exploits natural surface patterns evolved to resist to microorganisms infection, and the antimicrobial efficacy of graphene oxide. Cell integrity analysis by scanning electron microscopy and nucleic acids release show bacteriostatic and bactericidal effect. Nucleic acids release demonstrates microorganism cutting, and microscopy reveals cells wrapped by the laser treated gel. A theoretical active matter model confirms our findings. The employment of biomimetic graphene oxide gels opens unique possibilities to decrease infections in biomedical applications and chirurgical equipment; our antibiotic-free approach, based on the geometric reduction of microbial adhesion and the mechanical action of Graphene Oxide sheets, is potentially not affected by bacterial resistance.

The graphene oxide contradictory effects against human pathogens

Standing out as the new wonder bidimensional material, graphene oxide (GO) has aroused an exceptional interest in biomedical research by holding promise for being the antibacterial of future. First, GO possesses a specific interaction with microorganisms combined with a mild toxicity for human cells. Additionally, its antibacterial action seems to be directed to multiple targets in pathogens, causing both membranes mechanical injury and oxidative stress. Lastly, compared to other carbon materials, GO has easy and low-cost processing and is environment-friendly. This remarkable specificity and multi-targeting antibacterial activity come at a time when antibiotic resistance represents the major health challenge. Unfortunately, a comprehensive framework to understand how to effectively utilize this material against microorganisms is still lacking. In the last decade, several groups tried to define the mechanisms of interaction between GO flakes and pathogens but conflicting results have been reported. This review is focused on all the contradictions of GO antimicrobial properties in solution. Flake size, incubation protocol, time of exposure and species considered are examples of factors influencing results. These parameters will be summarized and analyzed with the aim of defining the causes of contradictions, to allow fast GO clinical application.

Time evolution of noise induced oxidation in outer hair cells: Role of NAD(P)H and plasma membrane fluidity

BACKGROUND Noise exposure impairs outer hair cells (OHCs). The common basis for OHC dysfunction and loss by acoustic over-stimulation is represented by reactive oxygen species (ROS) overload that may affect the membrane structural organization through generation of lipid peroxidation. METHODS Here we investigated in OHC different functional zones the mechanisms linking metabolic functional state (NAD(P)H intracellular distribution) to the generation of lipid peroxides and to the physical state of membranes by two photon fluorescence microscopy. RESULTS In OHCs of control animals, a more oxidized NAD(P)H redox state is associated to a less fluid plasma membrane structure. Acoustic trauma induces a topologically differentiated NAD(P)H oxidation in OHC rows, which is damped between 1 and 6h. Peroxidation occurs after ~4h from noise insult, while ROS are produced in the first 0.2h and damage cells for a period of time after noise exposure has ended (~7.5h) when a decrease of fluidity of OHC plasma membrane occurs. OHCs belonging to inner rows, characterized by a lower metabolic activity with respect to other rows, show less severe metabolic impairment. CONCLUSIONS Our data indicate that plasma membrane fluidity is related to NAD(P)H redox state and lipid peroxidation in hair cells. GENERAL SIGNIFICANCE Our results could pave the way for therapeutic intervention targeting the onset of redox umbalance.

Biomechanical investigation of colorectal cancer cells

The nanomechanical properties of SW480 colon cancer cells were investigated using Atomic Force Microscopy. SW480 cells are composed of two sub-populations with different shape and invasiveness. These two cells populations showed similar adhesion properties while appeared significantly different in term of cells stiffness. Since cell stiffness is related to invasiveness and growth, we suggest elasticity as a useful parameter to distinguish invasive cells inside the colorectal tumor bulk and the high-resolution mechanical mapping as a promising diagnostic tool for the identification of malignant cells.

Recent advances in superhydrophobic surfaces and their relevance to biology and medicine.

By mimicking naturally occurring superhydrophobic surfaces, scientists can now realize artificial surfaces on which droplets of a few microliters of water are forced to assume an almost spherical shape and an extremely high contact angle. In recent decades, these surfaces have attracted much attention due to their technological applications for anti-wetting and self-cleaning materials. Very recently, researchers have shifted their interest to investigate whether superhydrophobic surfaces can be exploited to study biological systems. This research effort has stimulated the design and realization of new devices that allow us to actively organize, visualize and manipulate matter at both the microscale and nanoscale levels. Such precise control opens up wide applications in biomedicine, as it allows us to directly manipulate objects at the typical length scale of cells and macromolecules. This progress report focuses on recent biological and medical applications of superhydrophobicity. Particular regard is paid to those applications that involve the detection, manipulation and study of extremely small quantities of molecules, and to those that allow high throughput cell and biomaterial screening.

Self-assembling of large ordered DNA arrays using superhydrophobic patterned surfaces

In this paper we present a simple and robust method to realize highly ordered arrays of stretched and suspended DNA molecules over the millimeter length scale. To this end we used an ad hoc designed superhydrophobic surface made of high aspect-ratio silicon pillars, where we deposited a droplet containing genomic DNA. A precise positioning of DNA strands was achieved by shaping the silicon pillars so that sharpened features resembling tips were included. Such features allowed us to accurately control the droplet de-wetting dynamics, pinning DNA strands in a well-defined position above pillars. The proposed technique has the potential to positively impact on the development of novel DNA chips for genetic analysis.

The future development of bacteria fighting medical devices: the role of graphene oxide.

ABSTRACT Introduction: The clinical challenge that research on antibacterial coatings faces nowadays is the need of reduction of resistant bacterial infections, major source of implant rejection and repeated surgery. In order to avoid microorganisms attachment and biofilm formation, coating materials on medical devices have been developed with shortcomings represented by short-term durability and induction of new mechanisms of bacterial resistance. Graphene-based films and hydrogel could represent the next generation protective coatings due to their excellent mechanical, chemical and thermal properties, high nanoparticle adsorption and antibacterial action. Areas covered: In this short commentary, we will report the recent developments of graphene oxide based coatings. Graphene oxide is a water-soluble derivative of graphene that allows high drug loading and miscibility with polymers, making it mouldable in any desired shape. Recent applications in wound healing and tissue engineering will be discussed as well as critical issues prior to clinical use of graphene oxide coatings. Expert commentary: The current evidence is insufficient to establish the efficacy of Graphene Oxide against bacteria and the durability of coatings. Further studies should clarify how to control Graphene Oxide antibacterial mechanism.

Synthesis and characterization of different immunogenic viral nanoconstructs from rotavirus VP6 inner capsid protein

In order to deliver low-cost viral capsomeres from a large amount of soluble viral VP6 protein from human rotavirus, we developed and optimized a biotechnological platform in Escherichia coli. Specifically, three different expression protocols were compared, differing in their genetic constructs, ie, a simple native histidine-tagged VP6 sequence, VP6 fused to thioredoxin, and VP6 obtained with the newly described small ubiquitin-like modifier (SUMO) fusion system. Our results demonstrate that the histidine-tagged protein does not escape the accumulation in the inclusion bodies, and that SUMO is largely superior to the thioredoxin-fusion tag in enhancing the expression and solubility of VP6 protein. Moreover, the VP6 protein produced according to the SUMO fusion tag displays well-known assembly properties, as observed in both transmission electron microscopy and atomic force microscopy images, giving rise to either VP6 trimers, 60 nm spherical virus-like particles, or nanotubes a few microns long. This different quaternary organization of VP6 shows a higher level of immunogenicity for the elongated structures with respect to the spheres or the protein trimers. Therefore, the expression and purification strategy presented here – providing a large amount of the viral capsid protein in the native form with relatively simple, rapid, and economical procedures – opens a new route toward large-scale production of a more efficient antigenic compound to be used as a vaccination tool or as an adjuvant, and also represents a top-quality biomaterial to be further modified for biotechnological purposes.

Stearoyl-CoA desaturase 1 and paracrine diffusible signals have a major role in the promotion of breast cancer cell migration induced by cancer-associated fibroblasts

Background:Despite the recognised contribution of the stroma to breast cancer development and progression, the effective targeting of the tumor microenvironment remains a challenge to be addressed. We previously reported that normal fibroblasts (NFs) and, notably, breast cancer-associated fibroblasts (CAFs) induced epithelial-to-mesenchymal transition and increases in cell membrane fluidity and migration in well- (MCF-7) and poorly-differentiated (MDA-MB-231) breast cancer cells. This study was designed to better define the role played, especially by CAFs, in promoting breast tumor cell migration.Methods:Fibroblast/breast cancer cell co-cultures were set up to investigate the influence of NFs and CAFs on gene and protein expression of Stearoyl-CoA desaturase 1 (SCD1), the main enzyme regulating membrane fluidity, as well as on the protein level and activity of its transcription factor, the sterol regulatory element-binding protein 1 (SREBP1), in MCF-7 and MDA-MB-231 cells. To assess the role of SREBP1 in the regulation of SCD1 expression, the desaturase levels were also determined in tumor cells treated with an SREBP1 inhibitor. Migration was evaluated by wound-healing assay in SCD1-inhibited (by small-interfering RNA (siRNA) or pharmacologically) cancer cells and the effect of CAF-conditioned medium was also assessed. To define the role of stroma-derived signals in cancer cell migration speed, cell-tracking analysis was performed in the presence of neutralising antibodies to hepatocyte growth factor, transforming growth factor-β or basic fibroblast growth factor.Results:A two to three fold increase in SCD1 mRNA and protein expression has been induced, particularly by CAFs, in the two cancer cell lines that appear to be dependent on SREBP1 activity in MCF-7 but not in MDA-MB-231 cells. Both siRNA-mediated and pharmacological inhibition of SCD1 impaired tumor cells migration, also when promoted by CAF-released soluble factors. Fibroblast-triggered increase in cancer cell migration speed was markedly reduced or abolished by neutralising the above growth factors.Conclusion:These results provide further insights in understanding the role of CAFs in promoting tumor cell migration, which may help to design new stroma-based therapeutic strategies.

Effect of Alginate Lyase on Biofilm-Grown Helicobacter pylori Probed by Atomic Force Microscopy

Helicobacter pylori (H. pylori) is a microorganism with a pronounced capability of adaptation under environmental stress solicitations. Its persistence and antimicrobial resistance to the drugs commonly used in the anti-H. pylori therapy are associated with the development of a biofilm mainly composed of DNA, proteins, and polysaccharides. A fundamental step to increase the success of clinical treatments is the development of new strategies and molecules able to interfere with the biofilm architecture and thus able to enhance the effects of antibiotics. By using Atomic Force Microscopy and Scanning Electron Microscopy we analyzed the effects of the alginate lyase (AlgL), an enzyme able to degrade a wide class of polysaccharides, on the H. pylori shape, surface morphology, and biofilm adhesion properties. We demonstrated that AlgL generates a noticeable loss of H. pylori coccoid form in favor of the bacillary form and reduces the H. pylori extracellular polymeric substances (EPS).