Identification of influenza PA-Nter endonuclease inhibitors using pharmacophore- and docking-based virtual screening

Searching for new antiviral agents, we focused our interest on the influenza PA-Nter endonuclease. Therefore, we developed a three-dimensional pharmacophore model which contains the binding features addressed to the metal-chelating active site. The obtained hypothesis has been fruitfully employed to select three “hit compounds” through an in silico screening campaign on our in-house database of small molecules. We studied the binding poses of these hit compounds using molecular docking, and subjected them to an enzymatic assay with recombinant PA-Nter endonuclease. Compound 20 proved the most active inhibitor of the endonucleolytic cleavage reaction, with an IC₅₀ value of 12?μM.     

Segmented Bayesian calibration approach for estimating age in forensic science

Forensic age estimation is receiving growing attention from researchers in the last few years. Accurate estimates of age are needed both for identifying real age in individuals without any identity document and assessing it for human remains. The methods applied in such context are mostly based on radiological analysis of some anatomical districts and entail the use of a regression model. However, estimating chronological age by regression models leads to overestimated ages in younger subjects and underestimated ages in older ones. We introduced a full Bayesian calibration method combined with a segmented function for age estimation that relied on a Normal distribution as a density model to mitigate this bias. In this way, we were also able to model the decreasing growth rate in juveniles. We compared our new Bayesian‐segmented model with other existing approaches. The proposed method helped producing more robust and precise forecasts of age than compared models while exhibited comparable accuracy in terms of forecasting measures. Our method seemed to overcome the estimation bias also when applied to a real data set of South‐African juvenile subjects.     

The significance of conversion of skin reactivity to efficacy of bacillus Calmette-Guérin (BCG) vaccinations given immediately after radical surgery in stage II melanoma patients

Summary A group of 668 stage II melanoma patients was entered into a randomized prospective study aimed at evaluating the efficacy of adjuvant BCG, 5-(dimethyltriazeno)imidazole-4-carboxamide (DTIC), or a combination of the two, given immediately after radical lymph node dissection. Of these, 176 patients received BCG and 164 BCG plus DTIC. These 340 patients had histologically proven metastatic nodes and 156 had a negative skin reactivity to BCG at the beginning of treatment. The distribution of known prognostic factors (sex, age, number of positive nodes, extracapsular invasion) was balanced in the groups of patients either with initially negative or with positive skin reactivity. All patients who were initially non-reactive to BCG developed skin reactivity after 6.7±9 BCG vaccinations. Disease-free and overall survival of patients receiving BCG or BCG + DTIC with an initially negative skin reactivity to BCG was significantly (P=7×10^–3) better than that observed in patients with an initial positive skin reactivity. This finding was still evident after adjustment for other known prognostic criteria (P=0.02). It seems likely that the initial BCG skin reactivity as such marks the prognosis; however, some therapeutic effect of BCG treatment in patients having initially no skin reactivity to BCG, can not be ruled out.This study has been partially funded by the Associazione Italiana per la Ricerca sul Cancro, and by a grant (NO1-CM-43726) from the National Cancer Institute of Bethesda, USAWriting committee of the W. H. O. Melanoma Programme (Chairman: U. Veronesi). The following researchers also took part in the study and are co-authors of this paper: J. Adamus, Oncological Institute, Gliwice (Poland); C. Aubert, Institut J. Paoli Calmettes, Marseille (France); E. Bajetta, G. Beretta, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan (Italy); G. Cocconi, Centro Medico Oncologico, Ospedali Riuniti, Parma (Italy); J. Durand, Fondation Curie, Paris (France); J. De Marsillac, Instituto Nacional de Cancer, Rio de Janeiro (Brasil); R. L. Ikonopisov, Dr. T. Tsanov, Oncological Research Institute, Sofia (Bulgaria); B. Kiss, State Institute of Oncology, Budapest (Hungary); F. Lejeune, Institut Jules Bordet, Brussels (Belgium); G. Madej, Oncological Institute, Warsaw (Poland); H. Mulder, Rotterdamsch Radiotherapeutisch Instituut, Rotterdam (The Netherlands); Z. Mechl, Oncological Institute, Brno (Czechoslovakia); G. W. Milton, Sydney Hospital, Sydney (Australia); H. Peter, P. von Wussow, Abteilung für Klinische Immunologie, Medizinische Hochschule, Hannover (FRG); J. Priario, Hospital de Clinicas M. Quintela, Montevideo (Uruguay); E. Paul, Abteilung für Klinische und Experimentelle Dermatologie, Giessen (FRG); R. Sertoli, Istituto di Oncologia, Genoa (Italy); R. Tomin, Institute of Oncology, Belgrade (Yugoslavia)     

5-Arylidene-2-imino-4-thiazolidinones: design and synthesis of novel anti-inflammatory agents

The synthesis and pharmacological activity of 5-arylidene-2-imino-4-thiazolidinones (3a-8a) are described. All derivatives exhibited significant activity levels in models of acute inflammation such as carrageenan-induced paw and pleurisy edema in rats. In particular, 5-(3-methoxyphenylidene)-2-phenylimino-3-propyl-4-thiazolidinone (3a) displayed high levels of carrageenan-induced paw edema inhibition comparable to those of indomethacin. In addition the ability of such a new class of anti-inflammatory agents to inhibit COX isoforms was assessed in murine monocyte/macrophage J774 cell line assay. 5-(4-Methoxyphenylidene)-2-phenylimino-3-propyl-4-thiazolidinone (6a), the most interesting compound in such an experiment, was docked in the known active site of COX-2 protein and showed that its 4-methoxyarylidene moiety can easily occupy the COX-2 secondary pocket considered as the critical interaction for COX-2 selectivity.     

Cadmium induces an apoptotic response in sea urchin embryos

Cadmium is a heavy metal toxic for living organisms even at low concentrations. It does not have any biological role, and since it is a permanent metal ion, it is accumulated by many organisms. In the present paper we have studied the apoptotic effects of continuous exposure to subacute/sublethal cadmium concentrations on a model system: Paracentrotus lividus embryos. We demonstrated, by atomic absorption spectrometry, that the intracellular amount of metal increased during exposure time. We found, using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, that long treatments with cadmium triggered a severe DNA fragmentation. We demonstrated, by immunocytochemistry on whole-mount embryos, that treatment with cadmium causes activation of caspase-3 and cleavage of death substrates alpha-fodrin and lamin A. Incubating the embryos since fertilization with Z-DEVD FMK, a caspase-3 inhibitor, we found, by immunocytochemistry, that cleavage by caspase-3 and cleavage of death substrates were inactivated.     

The aberrant expression in epithelial cells of the mesenchymal isoform of FGFR2 controls the negative crosstalk between EMT and autophagy

Signalling of the epithelial splicing variant of fibroblast growth factor receptor 2 (FGFR2b) triggers both differentiation and autophagy, while the aberrant expression of the mesenchymal FGFR2c isoform in epithelial cells induces impaired differentiation, inhibition of autophagy as well as the induction of the epithelial-mesenchymal transition (EMT). In light of the widely proposed negative loop linking autophagy and EMT in the early steps of carcinogenesis, here we investigated the possible involvement of FGFR2c aberrant expression and signalling in orchestrating this crosstalk in human keratinocytes. Biochemical, molecular, quantitative immunofluorescence analysis and in vitro invasion assays, coupled to the use of specific substrate inhibitors and transient or stable silencing approaches, showed that AKT/MTOR and PKCε are the two hub signalling pathways, downstream FGFR2c, intersecting with each other in the control of both the inhibition of autophagy and the induction of EMT and invasive behaviour. These results indicate that the expression of FGFR2c, possibly resulting from FGFR2 isoform switch, could represent a key upstream event responsible for the establishment of a negative interplay between autophagy and EMT, which contributes to the assessment of a pathological oncogenic profile in epithelial cells.     

Molecular Cloning and Functional Characterization of Components of the Capsule Biosynthesis Complex of Neisseria meningitidis Serogroup A: TOWARD IN VITRO VACCINE PRODUCTION*

The human pathogen Neisseria meningitidis (Nm) is a leading cause of bacterial meningitis and sepsis globally. A major virulence factor of Nm is the capsular polysaccharide (CPS), which in Nm serogroup A consists of N-acetyl-mannosamine-1-phosphate units linked together by phosphodiester linkages [→6)-α-d-ManNAc-(1→OPO₃⁻→]_n. Acetylation in O-3 (to a minor extent in O-4) position results in immunologically active polymer. In the capsule gene cluster (cps) of Nm, region A contains the genetic information for CPSA biosynthesis. Thereby the open reading frames csaA, -B, and -C are thought to encode the UDP-N-acetyl-d-glucosamine-2-epimerase, poly-ManNAc-1-phosphate-transferase, and O-acetyltransferase, respectively. With the aim to use a minimal number of recombinant enzymes to produce immunologically active CPSA, we cloned the genes csaA, csaB, and csaC and functionally characterized the purified recombinant proteins. If recombinant CsaA and CsaB were combined in one reaction tube, priming CPSA-oligosaccharides were efficiently elongated with UDP-GlcNAc as the donor substrate, confirming that CsaA is the functional UDP-N-acetyl-d-glucosamine-2-epimerase and CsaB the functional poly-ManNAc-1-phosphate-transferase. Subsequently, CsaB was shown to transfer ManNAc-1P onto O-6 of the non-reducing end sugar of priming oligosaccharides, to prefer non-O-acetylated over O-acetylated primers, and to efficiently elongate the dimer of ManNAc-1-phosphate. The in vitro synthesized CPSA was purified, O-acetylated with recombinant CsaC, and proven to be identical to the natural CPSA by ¹H NMR, ³¹P NMR, and immunoblotting. If all three enzymes and their substrates were combined in a one-pot reaction, nature identical CPSA was obtained. These data provide the basis for the development of novel vaccine production protocols.     

A new topology of the human Y chromosome haplogroup E1b1 (E-P2) revealed through the use of newly characterized binary polymorphisms

Haplogroup E1b1, defined by the marker P2, is the most represented human Y chromosome haplogroup in Africa. A phylogenetic tree showing the internal structure of this haplogroup was published in 2008. A high degree of internal diversity characterizes this haplogroup, as well as the presence of a set of chromosomes undefined on the basis of a derived character. Here we make an effort to update the phylogeny of this highly diverse haplogroup by including seven mutations which have been newly discovered by direct resequencing. We also try to incorporate five previously-described markers which were not, however, reported in the 2008 tree. Additionally, during the process of mapping, we found that two previously reported SNPs required a new position on the tree. There are three key changes compared to the 2008 phylogeny. Firstly, haplogroup E-M2 (former E1b1a) and haplogroup E-M329 (former E1b1c) are now united by the mutations V38 and V100, reducing the number of E1b1 basal branches to two. The new topology of the tree has important implications concerning the origin of haplogroup E1b1. Secondly, within E1b1b1 (E-M35), two haplogroups (E-V68 and E-V257) show similar phylogenetic and geographic structure, pointing to a genetic bridge between southern European and northern African Y chromosomes. Thirdly, most of the E1b1b1* (E-M35*) paragroup chromosomes are now marked by defining mutations, thus increasing the discriminative power of the haplogroup for use in human evolution and forensics.     

Role of the blood service in cellular therapy

Cellular therapy is a novel form of medical or surgical treatment using cells in place of or in addition to traditional chemical drugs. The preparation of cellular products - called advanced therapy medicinal products - ATMP in Europe, requires compliance with good manufacturing practices (GMP). Based on long-term experience in blood component manufacturing, product traceability and hemovigilance, selected blood services may represent ideal settings for the development and experimental use of ATMP. International harmonization of the protocols and procedures for the preparation of ATMP is of paramount importance to facilitate the development of multicenter clinical trials with adequate sample size, which are urgently needed to determine the clinical efficacy of ATMP. This article describes European regulations on cellular therapy and summarizes the activities of the 'Franco Calori' Cell Factory, a GMP unit belonging to the department of regenerative medicine of a large public university hospital, which acquired a certification for the GMP production of ATMP in 2007 and developed nine experimental clinical protocols during 2003-2011.