A general approach to the encapsulation of glycoenzymes chains inside calcium alginate gel beads

In this work an enzyme encapsulation general approach, based on the use of calcium alginate hydrogels, is reported. Alginate gels are biodegradable and low cost and have been found to provide a good matrix for the entrapment of sensitive biomolecules. Alginate is an anionic polymer whose gelation occurs by an exchange of sodium ions from the polymer chains with multivalent cations, resulting in the formation of a three dimensional gel network. For gelation alginate is dripped into a calcium chloride solution. The cations diffuse from the continuous phase to the interior of the alginate droplets and form a gelled matrix. By means of this “external gelation method” beads with a diameter of few millimeters can be obtained (see figure 1). The entrapment of enzymes in alginate beads suffers some disadvantages, like as low enzyme loading efficiency with reduction of the immobilization yields and reusability, related to the enzyme leakage from the large beads pores (cut off of about 100 kDa). Please click Additional Files below to see the full abstract

Study of radiation damage and substrate resistivity effects from beam test of silicon microstrip detectors using LHC readout electronics

We present the beam test results of single-sided silicon microstrip detectors, with different substrate resistivities. The effects of radiation damage are studied for a detector irradiated to a fluence of 2.4 multiplied by 10**1**4 n/cm**2. The detectors are read out with the APV6 chip, which is compatible with the 40 MHz LHC clock. The performance of different detectors and readout modes are studied in terms of signal-to-noise ratio and efficiency

Clinical Features, Cardiovascular Risk Profile, and Therapeutic Trajectories of Patients with Type 2 Diabetes Candidate for Oral Semaglutide Therapy in the Italian Specialist Care

Introduction: This study aimed to address therapeutic inertia in the management of type 2 diabetes (T2D) by investigating the potential of early treatment with oral semaglutide. Methods: A cross-sectional survey was conducted between October 2021 and April 2022 among specialists treating individuals with T2D. A scientific committee designed a data collection form covering demographics, cardiovascular risk, glucose control metrics, ongoing therapies, and physician judgments on treatment appropriateness. Participants completed anonymous patient questionnaires reflecting routine clinical encounters. The preferred therapeutic regimen for each patient was also identified. Results: The analysis was conducted on 4449 patients initiating oral semaglutide. The population had a relatively short disease duration (42%  60% of patients, and more often than sitagliptin or empagliflozin. Conclusion: The study supports the potential of early implementation of oral semaglutide as a strategy to overcome therapeutic inertia and enhance T2D management

Photodynamic Inactivation of Bacterial Contaminations: Application to Biofilms

In 1999, it was discovered that bacteria prefer to live within complex communities that are attached to interfaces, called biofilms. Since this discovery, the interest of the scientific community to explore this new bacterial lifestyle aroused. Among the different types of contaminations, bacterial biofilms are considered to be the most difficult to eradicate since the extracellular polymeric substances, self-produced by the bacterial cells, act as a barrier to external agents. Due to the emerging problems related to the growing ability of microorganisms to tolerate common antimicrobial strategies (e.g., chemicals, antibiotics), innovative and unconventional technologies are required today for the fight against microbial contaminations. There is also a growing desire of the industry and consumers towards methods that are fast, easy to apply, characterized by low costs for both maintenance and purchase, safe to use and environmentally friendly. In this context that provides challenges in dealing with bacterial contaminations, an emerging technology that has shown positive outcomes is photodynamic inactivation (PDI). The technology makes use of light within the visible spectrum in the presence of oxygen-containing species to tackle microorganisms. Two different methods of applying PDI are possible: (i) one exploits the photosensitizers (PS) produced by bacteria that are intrinsically present within their cells, i.e., endogenous PSs, (ii) the second requires the external addition of exogenous PSs. Both methods have demonstrated promising results and effectiveness for the inactivation of bacterial contaminations within different sectors such as the food industry and clinical settings. This PhD research aims to determine the impact of the PDI as a decontamination technology for bacteria with special attention towards biofilms. In order to assess this overall goal, three main objectives are achieved. To achieve the first objective, a protocol for obtaining a reproducible biofilm on a flat, wide polystyrene surface was developed for Pseudomonas fluorescens, a Gram-negative bacterium. This process gave insight into the evolution of the biofilms in terms of viable cells and biomass over 24 hours. The protocol was also successfully validated for a Gram-positive bacterium, Staphylococcus epidermidis. To meet the second objective, the impact of the PDI with endogenous PSs was studied on the two bacteria P. fluorescens and S. epidermidis. The aims of this second part are twofold: first, to provide an understanding of PDI applied to different types of bacterial contaminations and, second, to examine the effect of PDI when biofilms are considered. Biofilms can be found both hydrated and not hydrated, the way they are affected by PDI can be different. For this reason both of them were considered for the research in Chapter 5 and 6, respectively, since both can be found on equipment, tools and in industrial water tanks. First, the effect of violet, blue, green and red light-emitting diode irradiation was investigated on three different types of contaminations of P. fluorescens. A short treatment of 30 minutes was tested on the planktonic form, single cells inoculated on a surface, and hydrated biofilms. Among the different colours, violet and blue light were found to inactivate some types of contaminations at different extents. (i) For planktonic cells, violet light slightly inactivated the population. (ii) For individual cells on a surface, violet light caused a consistent reduction below the detection limit, while blue light was effective when salt stress was added to the system. (iii) Hydrated biofilms were not affected by any of the light sources. Second, the effect of five different colours, i.e., violet, blue, green, yellow and red, and three or four different irradiance conditions (2.5, only for violet light, 25.0, 75.0 and 100.0%) were tested on biofilms of P. fluorescens and S. epidermidis. (i) Violet light caused a high reduction of both species, with 6.80 and 3.69 log reductions, respectively, (ii) blue light was only effective against P. fluorescens using harsher conditions (100% of maximum irradiance) and (iii) green, yellow and red irradiation did not have any effect. Finally, to fulfil the third objective, the PDI was investigated using exogenous PSs and adjuvants excited by the means of white light both on P. fluorescens and S. epidermidis. Three surfactants (non-ionic, anionic and cationic) are used as adjuvants for PDI on the Gram-negative P. fluorescens and the Gram-positive S. epidermidis, while chelating agents are used only against P. fluorescens. White light in combination with exogenous PSs promotes a bactericidal effect within 30 minutes of irradiation on S. epidermidis, but not on P. fluorescens. Moreover, among all the adjuvants, some of them enhance the inactivation of S. epidermidis. But none of them enhances the effect on P. fluorescens. In conclusion, this PhD thesis demonstrates that PDI both with endogenous PSs and exogenous PSs is a promising technology that can be used for different types of bacterial contaminations, even against biofilms, which are the most recalcitrant to several kinds of remedies. The findings of this dissertation are encouraging and will serve as a base for future research in the field of PDI.status: publishe

General Approach to the Immobilization of Glycoenzyme Chains Inside Calcium Alginate Beads for Bioassay

A general method to obtain the efficient entrapment of mixtures of glyco-enzymes in calcium alginate hydrogel is proposed in this paper. As a proof of principle, three glyco-enzymes acting in series (trehalase, glucose oxidase and horseradish peroxidase) have been co-immobilized in calcium alginate beads. The release of the enzymes from the hydrogel mesh (leakage) is avoided by exploiting the enzymes aggregation induced by the concanavalin A. The aggregation process has been monitored by dynamic light scattering technique, while both enzyme encapsulation efficiency and leakage have spectrophotometrically been quantified. Obtained data show an encapsulation efficiency above 95% and a negligible leakage from the beads when enzyme aggregates are larger than 300 nm. Operational stability of “as prepared” beads has been largely improved by a coating of alternated shells of polycation poly(diallyldimethylammonium chloride) and of alginate. As a test for the effectiveness of the overall procedure, analytical bio-assays exploiting the enzyme containing beads have been developed for the optical determination of glucose and trehalose and Limit of Detection values of 0.2 uM and of 40 uM respectively have been obtained

Atmospheric Pressure Cold Plasma: A Friendly Environment for Dry Enzymes

A comprehensive study of the interaction of cold atmospheric pressure plasmas (CAPs) with dry enzymes is conducted to identify the experimental conditions that allow preserving enzyme functionality. Glucose oxidase (GOx) dry deposits are exposed to dielectric barrier discharges fed with pure helium, helium–oxygen, and helium–ethylene mixtures. The GOx loses functionality upon exposure to He/O2 CAP due to dry etching. X-ray photoelectron spectroscopy reveals that, in parallel with the ablation, there are modifications of the chemical structure of the surface of the enzyme deposit that decrease the etching rate and eventually lead to a “crust” with a strong resistance against the ablation, responsible of process termination. Interestingly, the customary conditions used for plasma-enhanced chemical vapor deposition using helium–ethylene mixtures (applied voltage <1.1 kVrms and exposure time ≤10 min) are friendly for GOx. It is therefore possible to immobilize enzymes by overcoating with a plasma-deposited thin film, fully retaining enzyme functionality

Visible Light as an Antimicrobial Strategy for Inactivation of Pseudomonas fluorescens and Staphylococcus epidermidis Biofilms

The increase of antimicrobial resistance is challenging the scientific community to find solutions to eradicate bacteria, specifically biofilms. Light-Emitting Diodes (LED) represent an alternative way to tackle this problem in the presence of endogenous or exogenous photosensitizers. This work adds to a growing body of research on photodynamic inactivation using visible light against biofilms. Violet (400 nm), blue (420 nm), green (570 nm), yellow (584 nm) and red (698 nm) LEDs were used against Pseudomonas fluorescens and Staphylococcus epidermidis. Biofilms, grown on a polystyrene surface, were irradiated for 4 h. Different irradiance levels were investigated (2.5%, 25%, 50% and 100% of the maximum irradiance). Surviving cells were quantified and the inactivation kinetic parameters were estimated. Violet light could successfully inactivate P. fluorescens and S. epidermidis (up to 6.80 and 3.69 log10 reduction, respectively), while blue light was effective only against P. fluorescens (100% of maximum irradiance). Green, yellow and red irradiation neither increased nor reduced the biofilm cell density. This is the first research to test five different wavelengths (each with three intensities) in the visible spectrum against Gram-positive and Gram-negative biofilms. It provides a detailed study of the potential of visible light against biofilms of a different Gram-nature.status: publishe

A Reproducible Method for Growing Biofilms on Polystyrene Surfaces: Biomass and Bacterial Viability Evolution ofPseudomonas fluorescensandStaphylococcus epidermidis

status: publishe

Beam test results on n+ on n type silicon microstrip detectors before and after neutron irradiation

We present beam test results on AC-coupled, single-sided, n+ on n type silicon microstrip detectors. We have tested the detectors before and after irradiation at a fluence of 8.3E13 n/cm2, at different temperatures and bias voltages. The detectors signal-to-noise ratio, spatial resolution, charge collection and overall efficiency have been measured