Survey of laboratory-acquired infections around the world in biosafety level 3 and 4 laboratories.

Laboratory-acquired infections due to a variety of bacteria, viruses, parasites, and fungi have been described over the last century, and laboratory workers are at risk of exposure to these infectious agents. However, reporting laboratory-associated infections has been largely voluntary, and there is no way to determine the real number of people involved or to know the precise risks for workers. In this study, an international survey based on volunteering was conducted in biosafety level 3 and 4 laboratories to determine the number of laboratory-acquired infections and the possible underlying causes of these contaminations. The analysis of the survey reveals that laboratory-acquired infections have been infrequent and even rare in recent years, and human errors represent a very high percentage of the cases. Today, most risks from biological hazards can be reduced through the use of appropriate procedures and techniques, containment devices and facilities, and the training of personnel.

Functional Interaction Between the ESCRT-I Component TSG101 and the HSV-1 Tegument Ubiquitin Specific Protease.

Similar to phosphorylation, transient conjugation of ubiquitin to target proteins (ubiquitination) mediated by the concerted action of ubiquitin ligases and de-ubiquitinating enzymes (DUBs) can affect substrate function. As obligate intracellular parasites, viruses rely on different cellular pathways for their own replication and the well conserved ubiquitin conjugating/de-conjugating system is not an exception. Viruses not only usurp the host proteins involved in the ubiquitination/de-ubiquitination process, but they also encode their own ubiquitin ligases and DUBs. Here we report that an N-terminal variant of the herpes simplex virus (HSV) type-1 large tegument protein VP1/2 (VP1/2(1-767)), encompassing an active DUB domain (herpesvirus tegument ubiquitin specific protease, htUSP), and TSG101, a component of the endosomal sorting complex required for transport (ESCRT)-I, functionally interact. In particular, VP1/2(1-767) modulates TSG101 ubiquitination and influences its intracellular distribution. Given the role played by the ESCRT machinery in crucial steps of both cellular pathways and viral life cycle, the identification of TSG101 as a cellular target for the HSV-1 specific de-ubiquitinating enzyme contributes to the clarification of the still under debate function of viral encoded DUBs highly conserved throughout the Herpesviridae family.

Vaginal microbiome and metabolome highlight specific signatures of bacterial vaginosis.

In this study, we sought to find novel bacterial and metabolic hallmarks for bacterial vaginosis (BV). We studied the vaginal microbiome and metabolome of vaginal fluids from BV-affected patients (n = 43) and healthy controls (n = 37) by means of an integrated approach based on quantitative polymerase chain reaction (qPCR) and proton nuclear magnetic resonance ((1)H-NMR). The correlations between the clinical condition and vaginal bacterial communities were investigated by principal component analysis (PCA). To define the metabolomics signatures of BV, 100 discriminant analysis by projection on latent structure (PLS-DA) models were calculated. Bacterial signatures distinguishing the health condition and BV were identified by qPCR. Lactobacillus crispatus strongly featured the healthy vagina, while increased concentrations of Prevotella, Atopobium and Mycoplasma hominis specifically marked the infection. (1)H-NMR analysis has led to the identification and quantification of 17 previously unreported molecules. BV was associated with changes in the concentration of metabolites belonging to the families of amines, organic acids, short chain fatty acids, amino acids, nitrogenous bases and monosaccharides. In particular, maltose, kynurenine and NAD(+) primarily characterised the healthy status, while nicotinate, malonate and acetate were the best metabolic hallmarks of BV. This study helps to better understand the role of the vaginal microbiota and metabolome in the development of BV infection. We propose a molecular approach for the diagnosis of BV based on quantitative detection in the vaginal fluids of Atopobium, Prevotella and M. hominis, and nicotinate, malonate and acetate by combining qPCR and (1)H-NMR.

Gene transfer of integration defective anti-HSV-1 meganuclease to human corneas ex vivo.

Corneal graft rejection is a major problem in chronic herpetic keratitis (HK) patients with latent infection. A new class of antiviral agents targeting latent and active forms of herpes simplex virus type 1 (HSV-1) is importantly required. Meganucleases are sequence-specific homing endonucleases capable of inducing DNA double-strand breaks. A proof-of-concept experiment has shown that tailor-made meganucleases are efficient against HSV-1 in vitro. To take this work a step forward, we hypothesized that the pre-treatment of human corneas in eye banks using meganuclease-encoding vectors will allow HK patients to receive a medicated cornea to resist the recurrence of the infection and the common graft rejection problem. However, this strategy requires efficient gene delivery to human corneal endothelium. Using recombinant adeno-associated virus, serotype 2/1 (rAAV2/1), efficient gene delivery of a reporter gene was demonstrated in human corneas ex vivo. The optimum viral dose was 3.7 × 10(11) VG with an exposure time of 1 day, followed by 6 days incubation in de-swelling medium. In addition, 12 days incubation can result in transgene expression in excess of 70%. Using similar transduction conditions, meganuclease transgene expression was detected in 39.4% of the endothelial cells after 2 weeks in culture. Reduction of the total viral load in the media and the endothelial cells of corneas infected with HSV-1 was shown. Collectively, this work provides information about the optimum conditions to deliver genetic material to the cornea, and demonstrates for the first time the expression of meganuclease in human corneas ex vivo and its antiviral activity. In conclusion, we demonstrate that the treatment of human corneas in eye banks before transplantation is a new approach to address the unmet clinical needs in corneal diseases.

Azithromycin-loaded liposomes and niosomes for the treatment of skin infections: Influence of excipients and preparative methods on the functional properties.

The aim of this study was to develop azithromycin (AZT)-loaded liposomes (LP) and niosomes (NS) useful for the treatment of bacterial skin infections and acne. LP based on phosphatidylcholine from egg yolk (EPC) or from soybean lecithin (SPC), and NS composed of sorbitan monopalmitate (Span 40) or sorbitan monostearate (Span 60) were prepared through the thin film hydration (TFH) and the ethanol injection (EI) methods. The formulations were subsequently characterized for their physico-chemical and functional properties. Vesicles prepared through TFH showed higher average sizes than the corresponding formulations obtained by EI. All the vesicles presented adequate encapsulation efficiency and a negative ζ potential, which assured good stability during the storage period (except for LP-SPC). Formulations prepared with TFH showed a more prolonged AZT release than those prepared through EI, due to their lower surface area and multilamellar structure, as confirmed by atomic force microscopy nanomechanical characterization. Finally, among all the formulations, NS-Span 40-TFH and LP-EPC-TFH allowed the highest drug accumulation in the skin, retained the antimicrobial activity and did not alter fibroblast metabolism and viability. Overall, they could ensure to minimize the dosing and the administration frequency, thus representing promising candidates for the treatment of bacterial skin infections and acne.

Analysis of human immunodeficiency virus type 1 vector cis- and trans-acting elements production by means of Semliki Forest virus.

Recombinant Semliki Forest virus (SFV) is an attractive viral vector system owing to its ability to allow high efficiency of viral protein expression. To produce recombinant pseudotyped human immunodeficiency virus type 1 (HIV-1) virions, we designed a chimeric SFV/HIV vector system that contains both the HIV-1 cis- and trans-acting elements under the transcriptional control of the SFV replicase and investigated the ability of the hybrid SFV/HIV system to produce lentiviral particles capable of transducing target cells. Co-transfection of target cells with the two helper SFV packaging system RNAs along with each SFV/Gag-Pol, SFV/VSV(G) as well as SFV/HIV-1 vector unit replicon led to the generation of efficient transducing competent recombinant SFV/HIV particles. In contrast, co-transduction of target cells with the SFV/HIV chimeric virions produced recombinant particles with low transducing ability. Our data suggest that both the genomic and the subgenomic RNAs containing the HIV-1 vector unit were negatively selected for incorporation into recombinant particles, despite the fact that the SFV-driven HIV-1 vector replicon was the only one containing a lentiviral packaging sequence. The results of this study provide insights relevant to the design of chimeric lentiviral vectors.

Modulation of apoptotic signalling by 9-hydroxystearic acid in osteosarcoma cells.

9-hydroxystearic acid (9-HSA) belongs to the class of endogenous lipid peroxidation by-products that greatly diminish in tumors, causing as a consequence the loss of one of the control mechanisms on cell division. We have previously shown that 9-HSA controls cell growth and differentiation by inhibiting histone deacetylase 1 (HDAC1) activity. In this paper our attention has not only been focused on HDAC1 inhibition but also on the hyperacetylation of other substrates such as p53, that is involved in inducing cell cycle arrest and/or apoptosis, and whose activity and stability are known to be regulated by posttranslational modifications, particularly by acetylation at the C-terminus region. 9-HSA administration to U2OS, an osteosarcoma cell line p53 wt, induces a growth arrest of the cells in G2/M and apoptosis via a mitochondrial pathway. In particular hyperacetylation of p53 induced by the HDAC1 inhibitory activity of 9-HSA has been demonstrated to increase Bax synthesis both at the transcriptional and the translational level. The subsequent translocation of Bax to the mitochondria is associated to a significant increase in caspase 9 activity. Our data demonstrate that the effects of 9-HSA on U2OS correlate with posttranslational modifications of p53.

Interaction of vaginal Lactobacillus strains with HeLa cells plasma membrane.

Vaginal lactobacilli offer protection against recurrent urinary and vaginal infections. The precise mechanisms underlying the interaction between lactobacilli and the host epithelium remain poorly understood at the molecular level. Deciphering such events can provide valuable information on the mode of action of commensal and probiotic bacteria in the vaginal environment. We investigated the effects exerted by five Lactobacillus strains of vaginal origin (Lactobacillus crispatus BC1 and BC2, Lactobacillus gasseri BC9 and BC11 and Lactobacillus vaginalis BC15) on the physical properties of the plasma membrane in a cervical cell line (HeLa). The interaction of the vaginal lactobacilli with the cervical cells determined two kinds of effects on plasma membrane: (1) modification of the membrane polar lipid organisation and the physical properties (L. crispatus BC1 and L. gasseri BC9); (2) modification of α5ß1 integrin organisation (L. crispatus BC2, L. gasseri BC11 and L. vaginalis BC15). These two mechanisms can be at the basis of the protective role of lactobacilli against Candida albicans adhesion. Upon stimulation with all Lactobacillus strains, we observed a reduction of the basal oxidative stress in HeLa cells that could be related to modifications in physical properties and organisation of the plasma membrane. These results confirm the strictly strain-specific peculiarities of Lactobacillus and deepen the understanding of the mechanisms underlying the health-promoting role of this genus within the vaginal ecosystem.

Histone proteins determined in a human colon cancer by high-performance liquid chromatography and mass spectrometry.

The application of reversed-phase high-pressure liquid chromatography under gradient conditions and electrospray ion trap mass spectrometry (LC-ESI-MS) to the analysis of global modification levels of core histones is described. The optimised LC-ESI-MS method was applied for the first time to the characterisation of histones extracted from HT29, a human colon cancer cell line. Eight histones (H1-1, H1-2, H2A-1, H2A-2, H2B, H3-1, H3-2, H4) were separated on a C4 stationary phase with complete resolution, never reached in previous HPLC-MS methods, by using a gradient elution with the combined presence of heptafluorobutyric acid and formic acid as acidic modifiers in the mobile phase. Heptafluorobutyric acid was found to improve selectivity, whereas the presence of formic acid decreased ion suppression. Histones eluted from the column were detected with an ion trap mass spectrometer with an electrospray source. The peak averaged mass spectra were reconstructed by Mag Tran 1.0 software and the mass of the various isoforms of histones were derived. Method validation was conducted by performing the same sample analysis by coupling LC-ESI to a quadrupole-time-of-flight mass spectrometer (Q-TOF). The number of histone forms and their mass were found to differ not significantly from those obtained by ion trap mass spectrometer. Also the relative modifications abundance within the same histone type was found following the same trend as the two mass analysers. This method was then applied to the characterisation of changes in histone modification in HT29, never analysed by LC-MS before, treated with histone deacetylase inhibitors such as valproate and sodium butyrate, also used in preclinical trials as anticancer drugs. In particular, both the inhibitors produced a significant increase in H4 histone acetylated forms: 89% increase of the diacetyl dimethyl H4 form was observed with 1mM valproate supplementation, whereas 5 mM butyrate led to a 68% increase of the same form. Triacetyl monomethyl H4 (11,377 Da) and triacetyl dimethyl H4 (11,390 Da) were found only in cells treated with butyrate. Selective changes of H3 histone were detected with butyrate, in agreement with recently reported western blotting studies. Modifications in the H2A histone degree of acetylation were revealed by treatment of the cells with butyrate (H2A-1, H2A-2) and valproate (H2A-2). The results of the proposed methodology confirmed that inhibition of histone deacetylases caused histone hyperacetylation, responsible for decondensation and reorganization of interphase dynamic chromatin. This method resulted in selective and sensitive method to monitor variations in the acetylation and methylation state of histones after treatment of HT29 with inhibitors, and is therefore suitable for further application in new drug discovery for tumour therapy.

Aloe-emodin is a new type of anticancer agent with selective activity against neuroectodermal tumors.

Here we report that aloe-emodin (AE), a hydroxyanthraquinone present in Aloe vera leaves, has a specific in vitro and in vivo antineuroectodermal tumor activity. The growth of human neuroectodermal tumors is inhibited in mice with severe combined immunodeficiency without any appreciable toxic effects on the animals. The compound does not inhibit the proliferation of normal fibroblasts nor that of hemopoietic progenitor cells. The cytotoxicity mechanism consists of the induction of apoptosis, whereas the selectivity against neuroectodermal tumor cells is founded on a specific energy-dependent pathway of drug incorporation. Taking into account its unique cytotoxicity profile and mode of action, AE might represent a conceptually new lead antitumor drug.

Microparticles based on chitosan/carboxymethylcellulose polyelectrolyte complexes for colon delivery of vancomycin.

The aim of this work was to prepare polyelectrolyte complexes based on chitosan (CH) and carboxymethylcellulose (CMC) for colon delivery of vancomycin (VM). Various batches of polyelectrolyte complexes, using three different CH/CMC weight ratios (3:1, 1:1 and 1:3), were prepared and collected as microparticles by spray-drying process. Microparticles were characterized in terms of yield, encapsulation efficiency, drug loading, morphology and mucoadhesion properties. Microparticles water-uptake and VM release as well as its protection against gastric pepsin degradation were also investigated. Finally, the antibacterial activity against Staphylococcus aureus, a Gram-positive model strain, was evaluated. The best formulation CH/CMC 1:3 was selected based on the encapsulation efficiency, water-uptake and drug release rate. Moreover, microparticles were able to prevent VM degradation and showed a good antibacterial activity against S. aureus. Finally, to improve the release of VM in the colon the selected formulation was coated with lauric acid.

Autochthonous Hookworm-Related Cutaneous Larva Migrans Disease in Northeastern Italy: A Case Report.

Here we report the case of a 42-yr-old patient who presented himself to us with a serpiginous erythematous lesion from the wrist of the right forearm up the arm to the right shoulder A similar lesion of a smaller size was also present in the left forearm. On the basis of clinical manifestations and progression of the lesion, combined with previous treatments and different diagnostic investigations, hookworm-related cutaneous larva migrans (HrCLM) disease was hypothesized. Albendazole was employed as treatment and the resolution of the symptoms confirmed the diagnosis. The relevance of the reported case relies on 3 main aspects: the acquisition of the disease in Italy, the initial treatment with topical corticosteroids that sped up the progression of the cutaneous trail, and the uncommon location of the lesions. Furthermore, the anamnestic data and the laboratory/clinical investigations strongly suggested an occupational exposure to the etiological agent. As illustrated here, HrCLM might represent a challenge for Western physicians in terms of diagnosis, treatment, and ways of acquisition. Describing the clinical presentation and the treatment of cases of cutaneous larva migrans might contribute to early and correct diagnosis, to an increase of our knowledge on this disease, and to an update on its epidemiology.

Chitosan based micro- and nanoparticles for colon-targeted delivery of vancomycin prepared by alternative processing methods.

The aim of this work was to prepare chitosan (CH) based particulate formulations for colon delivery of vancomycin (VM). Chitosan microparticles (MPs) and nanoparticles (NPs) loaded with VM were prepared using different CH/tripolyphosphate (TPP) molar ratios and different technological processes. In particular, nanoparticles were prepared by ionic gelation and freeze-drying to recover these particles, or, alternatively, by spray-drying method. Microparticles were prepared using a different spray-dryer. Micro- and nanoparticles were characterized in terms of size distributions by photon correlation spectroscopy (PCS), while encapsulation and drug loading efficiencies were studied using a dialysis method. Fourier Transform Infrared Spectroscopy (FT-IR) was employed to determine the surface composition of the micro- and nanoparticles respectively, and the morphologies of the developed systems were studied by scanning electron microscopy (SEM). Water uptake as well as drug release profiles were also measured. Antibacterial activity against Staphylococcus aureus, a Gram-positive model strain, was evaluated. FT-IR results suggested an electrostatic interaction between VM and CH/TPP particles. Moreover, the particles were found to hold a positive zeta-potential, indicating the presence of CH on the particle surfaces. Particle size and encapsulation efficiency were mainly influenced by the different manufacturing processes employed. Nanoparticles obtained by spray-drying showed the best results in terms of water uptake and drug release rate. Moreover, they showed a good bactericidal activity against S. aureus.

6-Aminoquinolones as new potential anti-HIV agents.

A series of 6-aminoquinolone compounds were evaluated for their in vitro activity against human immunodeficiency virus type 1 (HIV-1). Compound 12a, bearing a methyl substituent at the N-1 position and a 4-(2-pyridyl)-1-piperazine moiety at the C-7 position, was the most active in inhibiting HIV-1 replication on de novo infected C8166 human lymphoblastoid cell lines. The 12a EC(50) value was 0.1 microM, a 7-20-fold lower concentration relative to that for compounds 8a and 7a containing a cyclopropyl and tert-butyl substituent at the N-1 position, respectively. When the C-6 amino group was replaced with a fluorine atom, a decreased antiviral effect was observed. The observed effects are selective, since potency is substantially reduced when testing the compounds against the herpes simplex virus type 1 (HSV-1). Active quinolone derivatives very efficiently interact with TAR RNA, which suggests a nucleic acid-targeted mechanism of action.

Acyclovir resistance in herpes simplex virus type 1: biochemical and functional studies on the thymidine kinase of the highly resistant R100 strain.

The biochemical and functional properties of the thymidine kinase (TK) of the herpes simplex virus type 1 mutant R100, that is highly resistant to 9-(2-hydroxyethoxymethyl)guanine (acyclovir), are reported in comparison with the properties of its parental strain, wt. The mutant induced the production of a TK activity that accounted for only 10% of the wt one. This feature was not apparently related to a defective expression of the TK gene but it was rather connected to some functional characteristics of R100 enzyme. Although affinities of this enzyme for ATP and thymidine were unchanged, apparent Vmax values for thymidine were much reduced. In addition, affinities for antiviral analogues acyclovir, 9-(1,3-dihydroxymethyl)guanine (DHPG), 5-(2-bromovinyl)2'-deoxyuridine (BVdU), and 5-iodo-2'deoxycytidine (IdCyd) were drastically diminished (between 50-fold and more than 100-fold). This mutation therefore seems to affect the active site of the enzyme which is involved in the catalytic conversion of thymidine and in the binding of the analogues. The above features of HSV-1 R100 seem quite distinct from those of previously described HSV-1 resistant mutants.

Relevance of ionic effects on norfloxacin uptake by Escherichia coli.

The uptake of the quinolone drug norfloxacin by Escherichia coli was investigated at initial rate kinetics at different pH and monovalent/divalent metal ion concentration. The results support a simple diffusion mechanism for quinolone incorporation into cells. The uptake process decreases under acidic conditions. The presence of Na+ or K+ ions does not affect the results to an appreciable extent, whereas divalent ions cause a dramatic decrease in drug incorporation. The antibacterial activity, evaluated under identical experimental conditions, shows a direct relationship with the uptake data. As a general explanation for the above results it is suggested that the ability of the drug to penetrate into cells is a function of its net charge. The molecule in the zwitterionic form exhibits maximum permeation properties, whereas the uptake is remarkably reduced when the drug bears a net charge as a result of ionization or complex formation with bivalent ions. These results allow further insight into the mechanism of quinolone access to the intracellular compartment.

Cellular uptake of phosphonylmethoxyalkylpurine derivatives.

The cellular uptake of the phosphonylmethoxyalkylpurine derivatives HPMPA and PMEA has been studied in H9 cells. The two compounds exhibited an identical pattern of permeation in this cell line. Uptake did not occur via the nucleoside transport system, but through a different mechanism which, for its slow kinetics and temperature-dependence, is compatible with an endocytosis-like process. The amount of cell-associated drug increased up to one hour post-incubation.

The effect of the minor groove binding agent DAPI (4,6-diamidino-2-phenyl-indole) on DNA-directed enzymes: an attempt to explain inhibition of plasmid expression in Escherichia coli [corrected].

The activity of DAPI, on a number of DNA-directed enzymes involved in DNA topology, transcription, replication and repair, is reported in this paper. DAPI was always more inhibitory than ethidium bromide, in particular against RNA polymerase and DNA ligase, which seemed to be specifically affected. While the effect on RNA polymerase is likely due to a preferential occupancy of the promoter region, that on DNA ligase could rely upon a mechanism of steric hindrance in the minor groove. These phenomena, independently from an alteration of the tertiary structure of DNA by the ligand, can account for the previously reported inhibition of plasmid expression in Escherichia coli.