Ultrasound delivery of Surface Enhanced InfraRed Absorption active gold-nanoprobes into fibroblast cells: a biological study via Synchrotron-based InfraRed microanalysis at single cell level.

Ultrasound (US) induced transient membrane permeabilisation has emerged as a hugely promising tool for the delivery of exogenous vectors through the cytoplasmic membrane, paving the way to the design of novel anticancer strategies by targeting functional nanomaterials to specific biological sites. An essential step towards this end is the detailed recognition of suitably marked nanoparticles in sonoporated cells and the investigation of the potential related biological effects. By taking advantage of Synchrotron Radiation Fourier Transform Infrared micro-spectroscopy (SR-microFTIR) in providing highly sensitive analysis at the single cell level, we studied the internalisation of a nanoprobe within fibroblasts (NIH-3T3) promoted by low-intensity US. To this aim we employed 20 nm gold nanoparticles conjugated with the IR marker 4-aminothiophenol. The significant Surface Enhanced Infrared Absorption provided by the nanoprobes, with an absorbance increase up to two orders of magnitude, allowed us to efficiently recognise their inclusion within cells. Notably, the selective and stable SR-microFTIR detection from single cells that have internalised the nanoprobe exhibited clear changes in both shape and intensity of the spectral profile, highlighting the occurrence of biological effects. Flow cytometry, immunofluorescence and murine cytokinesis-block micronucleus assays confirmed the presence of slight but significant cytotoxic and genotoxic events associated with the US-nanoprobe combined treatments. Our results can provide novel hints towards US and nanomedicine combined strategies for cell spectral imaging as well as drug delivery-based therapies.

Long-term physical evolution of an elastomeric ultrasound contrast microbubble.

HYPOTHESIS: One of the main assets of crosslinked polymer-shelled microbubbles (MBs) as ultrasound-active theranostic agents is the robustness of the shells, combined with the chemical versatility in modifying the surface with ligands and/or drugs. Despite the long shelf-life, subtle modifications occur in the MB shells involving shifts in acoustic, mechanical and structural properties. EXPERIMENTS: We carried out a long-term morphological and acoustic evolution analysis on elastomeric polyvinyl-alcohol (PVA)-shelled MBs, a novel platform accomplishing good acoustic and surface performances in one agent. Confocal laser scanning microscopy, acoustic spectroscopy and AFM nanomechanics were integrated to understand the mechanism of PVA MBs ageing. The changes in the MB acoustic properties were framed in terms of shell thickness and viscoelasticity using a linearised oscillation theory, and compared to MB morphology and to nanomechanical analysis. FINDINGS: We enlightened a novel, intriguing ageing time evolution of the PVA MBs with double behaviour with respect to a crossover time of ∼50 days. Before, significant changes occur in MB stiffness and shell thickness, mainly due to a massive release of entangled PVA chains. Then, the MB resonance frequency increases together with shell thickening and softening. Our benchmark study is of general interest for emerging viscoelastomeric bubbles towards personalised medicine.

Differential effects on membrane permeability and viability of human keratinocyte cells undergoing very low intensity megasonic fields.

Among different therapeutic applications of Ultrasound (US), transient membrane sonoporation (SP) - a temporary, non-lethal porosity, mechanically induced in cell membranes through US exposure - represents a compelling opportunity towards an efficient and safe drug delivery. Nevertheless, progresses in this field have been limited by an insufficient understanding of the potential cytotoxic effects of US related to the failure of the cellular repair and to the possible activation of inflammatory pathway. In this framework we studied the in vitro effects of very low-intensity US on a human keratinocyte cell line, which represents an ideal model system of skin protective barrier cells which are the first to be involved during medical US treatments. Bioeffects linked to US application at 1 MHz varying the exposure parameters were investigated by fluorescence microscopy and fluorescence activated cell sorting. Our results indicate that keratinocytes undergoing low US doses can uptake drug model molecules with size and efficiency which depend on exposure parameters. According to sub-cavitation SP models, we have identified the range of doses triggering transient membrane SP, actually with negligible biological damage. By increasing US doses we observed a reduced cells viability and an inflammatory gene overexpression enlightening novel healthy relevant strategies.

Liquid hydridosilane precursor prepared from cyclopentasilane via sonication at low temperatures without the action of light.

We report on a liquid hydridosilane precursor ink prepared via the ultrasonically induced ring-opening polymerisation of cyclopentasilane (Si5H10) without irradiation by ultraviolet light. The sonication is carried out in N2 atmosphere at temperatures between 20 and 75°C. We use size exclusion chromatography (SEC) to show polymer growth and estimate molecular mass with increasing sonication time. In combination with UV-vis transmission measurements, further SEC analysis is used to compare solutions subjected to either purely thermal or ultrasonic treatment at the same process temperature and for the same duration. Our findings provide strong evidence showing that the initiation of the polymerisation is sonocatalytic in nature and not thermic due to the macroscopic temperature of the solution. The liquid precursor is used to produce homogeneous hydrogenated amorphous silicon (a-Si:H) thin films via spin coating and pyrolytic conversion. The optoelectronic properties of the films are subsequently improved by hydrogen radical treatment. Fourier transform infrared spectroscopy (FTIR) is used to determine a compact film morphology and electrical conductivity measurements show that the layers attain a light-to-dark photosensitivity ratio of 2×103 making them suitable for application in optoelectronic devices.

Clinical and Microbiological Characteristics of Visceral Leishmaniasis Outbreak in a Northern Italian Nonendemic Area: A Retrospective Observational Study.

Background. Visceral leishmaniasis (VL) caused by Leishmania infantum is endemic in the Mediterranean area. In the last decades a northward spread of the parasite has been observed in Italy. This paper describes a VL outbreak in Modena province (Emilia-Romagna, Northern Italy) between 2012 and 2015. Methods. Retrospective, observational study to evaluate epidemiological, microbiological characteristics, and clinical management of VL in patients referring to Policlinico Modena Hospital. Results. Sixteen cases of VL occurred in the study period. An immunosuppressive condition was present in 81.3%. Clinical presentation included anemia, fever, leukopenia, thrombocytopenia, and hepatosplenomegaly. Serology was positive in 73.3% of cases, peripheral blood PCR in 92.3%, and bone marrow blood PCR in 100%. Culture was positive in 3/6 cases (50%) and all the isolates were identified as L. infantum by ITS1/ITS2 sequencing. The median time between symptom onset and diagnosis was 22 days (range 6-131 days). All patients were treated with liposomal amphotericin b. 18.8% had a VL recurrence and were treated with miltefosine. Attributable mortality was 6.3%. Conclusions. VL due to L. infantum could determine periodical outbreaks, as the one described; thus it is important to include VL in the differential diagnosis of fever of unknown origin, even in low-endemic areas.

Multidrug-resistant tuberculosis outbreak in an Italian prison: tolerance of pyrazinamide plus levofloxacin prophylaxis and serial interferon gamma release assays.

The optimal treatment for latent tuberculosis infection (LTBI) in subjects exposed to multidrug-resistant (MDR) tuberculosis (TB) remains unclear, and the change in response of the QuantiFERON-TB Gold In-Tube (QTB-IT) test during and after treatment is unknown. Between May 2010 and August 2010, 39 prisoners at the 'Casa Circondariale' of Modena, Italy, were exposed to a patient with active pulmonary MDR TB. All contacts were tested with the tuberculin skin test and QTB-IT. Upon exclusion of active TB, subjects positive to both tests were offered 6 months' treatment with pyrazinamide (PZA) and levofloxacin (LVX). QTB-IT testing was repeated at 3 and 6 months after initial testing in all subjects who were offered LTBI treatment. Seventeen (43.5%) of 39 subjects tested positive to both tuberculin skin test and QTB-IT test, and 12 (70.5%) agreed to receive therapy with PZA and LVX at standard doses. Only five (41.6%) of 12 subjects completed 6 months' treatment. Reasons for discontinuation were asymptomatic hepatitis, gastritis and diarrhoea. The QTB-IT values decreased in all subjects who completed the treatment, in two (33%) of six of those who received treatment for less than 3 months and in one (50%) of two patients who discontinued therapy after 3 months. The QTB-IT test results never turned negative. Despite the small number of subjects, the study confirmed that PZA plus LVX is a poorly tolerated option for MDR LTBI treatment. We observed a large degree of variation in the results of the QTB-IT test results among participants. The study confirmed that the interferon gamma release assay is not a reliable tool for monitoring the treatment of MDR LTBI in clinical practice.

Use of EPR and FTIR to detect biological effects of ultrasound and microbubbles on a fibroblast cell line.

Structural and functional effects of exposing murine fibroblasts (NIH 3T3) to therapeutic ultrasound at 1 MHz frequency are described. These bioeffects can be attributed to the formation of free radical species by sonolysis of water. When cavitation occurs, dissociation of water vapor into H atoms and OH radicals is observed; these H atoms and OH radicals combine to form H(2), H(2)O(2), and HO(2). The radicals can chemically modify biomolecules, for example enzymes, DNA, and lipids. Generation of free radicals during exposure to ultrasound with or without encapsulated microbubbles (contrast agents) was studied by use of electron paramagnetic resonance with DMPO spin trapping. Recently the potential for possible use of these microbubbles in gene therapy has been investigated, because of the ability of the stabilized microbubbles to release their content when exposed to ultrasound. Structural changes were studied by Fourier-transform infrared spectroscopy, and induction of possible genotoxic damage by exposure of the cells to therapeutic ultrasound at 1 MHz frequency with our experimental device was verified by use of the cytokinesis-block micronucleus assay.

Structural changes induced in proteins by therapeutic ultrasounds.

The structural effect induced by therapeutic ultrasound on proteins in aqueous solution has been investigated with FTIR spectroscopy, UV-VIS spectroscopy, circular dichroism and light scattering. Six proteins (cytochrome, lysozyme, myoglobin, bovine serum albumin, trypsinogen, and alpha-chymotrypsinogen A) with different molecular weight and secondary structure have been studied. The experiment has been performed using an ultrasound source at resonant frequency of 1 MHz and sonication times of 10, 20, 30, 40, 50, and 60 min. A different behaviour of proteins under sonication depends on the dominant secondary structure type (alpha-helix or beta-sheets) and on the grade of the ordered structure. The results suggest that the free radicals, produced by water sonolysis, have an important role in the changes of structural order.

[Information and communication on the electromagnetic fields: analysis of the Italian Internet sites]. / Informazione e comunicazione sui campi elettromagnetici: analisi dei siti internet italiani.

The aim of this study is to evaluate the presence of contents related to communication and information on the exposure to the electromagnetic fields (emf) in the first 100 Italian Internet sites, carried out using the search engine Google with the key words "emf" and "emf and health". Each Internet site has been evaluated using 10 selected indicators: (1) Definition of electric, magnetic and electromagnetic fields; (2) Description of the physical effects of the emf; (3) Description of biological and health effects of the emf; (4) Description of the environmental sources; (5) Description of the environmental levels produced by the different sources; (6) Main legislation; (7) Risk perception; (8) Frequently asked questions (FAQ); (9) Links; (10) Forum for discussion. The sites, obtained for each search, have been classified into 6 main categories: (1) Public Research Institutes; (2) Health and Environmental Authorities; (3) Local Authorities; (4) Associations; (5) Commercial sites; (6) Other. The results show lack of information and communication on the emf in the analysed Italian Internet sites. A need for a design of any scientific Internet information and communication on this topic is shown.

Gram-positive bloodstream infections in liver transplant recipients: incidence, risk factors, and impact on survival.

The objective of the study was to assess the incidence, risk factors, and survival of gram-positive bloodstream infections (GP-BSI(s)) among liver transplant recipients during the first year after transplantation. Between October 2000 and September 2006, 42 episodes of GP-BSI(s) occurred in 205 patients with an overall incidence of 0.20 episodes/patient. Coagulase-negative staphylococci were detected in 45.2% of cases, Enterococcus species in 42.9% (E faecalis, eight; E faecium, seven; E avium, two; E gallinarum, one) and Staphylococcus aureus in 11.9%. Retransplantation was the only independent risk factor for GP-BSI (odds ratio [OR], 0.253; 95% confidence interval (CI), 0.089 to 0.715; P = .009). Thirty-day mortality rate was 28.5% and S aureus infections were related to a poorer outcome. It is noteworthy that all the isolates of S aureus were methicillin-resistant. Ampicillin was inactive against all the strains of E faecium and 50% of E avium isolates, but active against all E faecalis and E gallinarum strains. All the isolates were glycopeptide-susceptible. No significant differences in mortality rate were observed in relation to sex, etiologies of end-stage liver disease, cytomegalovirus infection/reinfection, type of donor, rejection, or retransplantation. GP-BSI, the only independent risk factor for death (OR, 0.262; 95% CI, 0.106 to 0.643; P = .003), reduced the survival rate by 26% in the first year posttransplant. In conclusion, GP-BSI(s) impact significantly on morbidity and mortality posttransplant, particularly among retransplantations. Control measures are required to reduce the incidence of GP-BSI(s) in liver transplant recipients. These findings must be considered when empirical antimicrobial therapy is indicated while awaiting blood-culture results.

Clastogenicity and aneuploidy in newborn and adult mice exposed to 50 Hz magnetic fields.

PURPOSE: To detect possible clastogenic and aneugenic properties of a 50 Hz, 650 muT magnetic field. MATERIALS AND METHODS: The micronucleus test with CREST (Calcinosis, Raynaud's phenomenon, Esophageal dismotility, Sclerodactility, Telangectasia) antibody staining was performed on liver and peripheral blood sampled from newborn mice exposed to an ELF (Extremely Low Frequency) magnetic field during the whole intra-uterine life (21 days), and on bone marrow and peripheral blood sampled from adult mice exposed to the same magnetic field for the same period. RESULTS: Data obtained in newborn mice show a significant increase in micronuclei frequencies. In absolute terms, most of the induced micronuclei were CREST-negative (i.e., formed by a chromosome fragment). However, in relative terms, ELF exposure caused a two-fold increase in CREST-negative micronuclei and a four-fold increase in CREST-positive micronuclei (i.e., formed by a whole chromosome). No significant effect was recorded on exposed adults. CONCLUSIONS: These findings suggest the need for investigation of aneugenic properties of ELF magnetic fields in order to establish a possible relationship to carcinogenesis.

Epidemiology of candidaemia and antifungal susceptibility patterns in an Italian tertiary-care hospital.

The epidemiological and antifungal susceptibility data for 94 episodes of candidaemia in an Italian tertiary-care hospital between January 2000 and August 2003 were evaluated by prospective laboratory-based surveillance. The incidence of fungaemia was 0.90 episodes/10 000 patient-days, and the most common species isolated were Candida albicans (40.4%), Candida parapsilosis (22.3%), Candida tropicalis (16.0%) and Candida glabrata (12.8%). Among 24 patients who received antifungal prophylaxis, non-albicans Candida spp. were more prevalent than C. albicans (p 0.012). The 30-day mortality rate was high (38.2%), particularly for haematological (71.4%) and solid-organ transplant patients (50.0%), and in individuals with C. tropicalis and C. glabrata bloodstream infections (60.0% and 50.0%, respectively). In-vitro susceptibility tests demonstrated that 95% of the isolates were susceptible to amphotericin B (MIC < 2 mg/L), 98.1% to posaconazole (MIC < 1 mg/L), 95.8% to flucytosine (MIC < 32 mg/L) and fluconazole (MIC < 64 mg/L), and 94.7% to itraconazole (MIC < 1 mg/L). Posaconazole was active (MIC 0.5 mg/L) against all three isolates of Candida krusei, which had reduced susceptibility to both fluconazole and itraconazole. Overall, non-albicans Candida spp. accounted for 60% of the episodes of candidaemia, which could be related to the use of antifungal prophylaxis. Resistance is still uncommon in Candida spp. recovered from blood cultures. The in-vitro activity of posaconazole is encouraging, and this agent could play an important role in the management of invasive candidiasis, including episodes caused by inherently less susceptible species such as C. krusei.

Synthesis and biological evaluation of 6-bromo-6-substituted penicillanic acid derivatives as beta-lactamase inhibitors.

The synthesis of a selected set of 6-bromopenicillanic acid derivatives with an additional C6 substituent is reported. All these substances were tested as inhibitors of class A and C beta-lactamase enzymes derived from Escherichia coli (TEM-1) and E. cloacae (P99). As 6-(1-hydroxyethyl) derivatives 4c and 6c were found to be weak beta-lactamase inhibitors, they were further investigated in combination with amoxicillin against a series of beta-lactamase-producing bacterial strains. Some structure-activity relationships are discussed.

Synthesis of new C-6 alkyliden penicillin derivatives as beta-lactamase inhibitors.

New penicillin, penicillin sulfone and sulfoxide derivatives bearing a C-6-alkyliden substituent were prepared. Their chemical synthesis, in vitro antibacterial activity and inhibition properties against two selected enzymes representing Class A and C beta-lactamases are reported. Compounds 3a-c, 7a-c were able to inhibit either TEM-1 (a Class A enzyme, from Escherichia coli) or P-99 (a Class C enzyme, from E. cloacae), or both enzymes, when tested in competition experiments using nitrocefin as the reporter substrate. However, when tested in combination with amoxicillin, the same compounds did not show synergistic effects against E. coli and E. cloacae strains producing TEM-1 and P99 enzymes, respectively. This finding is most likely related to poor penetration through the bacterial cell wall, as shown by using a more permeable isogenic E. coli strain. Interestingly, a synergistic effect against a strain of S. aureus which produces PC1-enzyme (a Class A beta-lactamase) was observed for compound 3a when used in combination with amoxicillin.

An observational study on patients treated or not for acute HIV infection.

BACKGROUND: A vast majority of HIV-infected subjects who experience HIV acute seroconversion actually receive treatment. Open questions are how can we identify patients who will be slow progressors or long-term non progressors, and, as a consequence, do not require treatment. METHODS: An observational retrospective study on patients who experienced acute HIV seroconversion from August 1995 to June 2001, who are still alive and followed as outpatients at the Clinic of Infectious Diseases of Modena, Italy. RESULTS: Twelve patients were studied. Five patients (45.4%) were treated during acute seroconversion, while 7 were not treated. Two of these seven subjects received antiretroviral treatment 12 and 26 weeks after acute seroconversion. All the untreated patients were in good viro-immunological condition 6 months after seroconversion, and 2 of them also after 3 and 7 years. Patients who were treated showed a significant daily increase in CD4/CD8 T cell ratio with longer time spent on therapy (0.04% increase per day longer on antiretroviral therapy, p=0.02). CONCLUSIONS: This study suggests that treatment during primary HIV infection should not be considered in all patients. Randomized clinical trials enrolling patients with an asymptomatic primary HIV infection, with a high CD4 count and low HIV plasma viremia are needed to evaluate the indications for treatment in this subgroup of patients. On the other hand, this study confirms the good viro-immunological response obtained after treating patients during primary HIV infection.

Synthesis, pharmacological characterization and QSAR studies on 2-substituted indole melatonin receptor ligands.

A number of 6-methoxy-1-(2-propionylaminoethyl)indoles, carrying properly selected substituents at the C-2 indole position, were prepared and tested as melatonin receptor ligands. Affinities and intrinsic activities for the human cloned mt1 and MT2 receptors were examined and compared with those of some 2-substituted melatonin derivatives recently described by us. A quantitative structure activity relationship (QSAR) study of the sixteen 2-substituted indole compounds, 5a-k, 1, 8-11, using partial least squares (PLS) and multiple regression analysis (MRA) revealed the existence of an optimal range of lipophilicity for the C2 indole substituent. There are also indications that planar, electron-withdrawing substituents contribute to the affinity by establishing additional interactions with the binding pocket. No mt1/MT2 subtype selectivity was observed, with the relevant exception of the 2-phenethyl derivative 5e, which exhibited the highest selectivity for the h-MT2 receptor among all the compounds tested (MT2/mt1 ratio of ca. 50). Conformational analysis and superposition of 5e to other reported selective MT2 ligands revealed structural and conformational similarities that might account for the MT2/mt1 selectivity of 5e.

Extrapleural pneumonectomy for sarcomas report of two cases.

AIMS AND BACKGROUND: Extrapleural pneumonectomy (EPP), which is a very uncommon surgical procedure, is electively indicated only in patients with early stages of malignant pleural mesothelioma, a rare condition. Two adults suffering from sarcomas and treated with EPP are described here. METHODS: A 29-year-old male with four left-sided lung metastases and ipsilateral pleural effusion from a chondrosarcoma of the mandibula and a 64-year old woman with a megamass in the left chest due to a local recurrence of a hemangiopericytoma underwent EPP. RESULTS: Extra-EPP-field multiorgan progression was diagnosed 14 months following surgery in the first patient who died at the 24th postoperative month but remained free of disease at the site of surgery. The second patient had a chest wall relapse at the forty-third month following EPP, which was treated by partial resection of the second and third ribs. She is alive and disease-free at the twelfth postoperative month. DISCUSSION: EPP may be considered for salvage treatment in selected patients with intrathoracic sarcomas not amenable to other effective therapies to achieve mid- to long-term disease control, even in the case of advanced spread.