Molecular epidemiology of ESbetaL producing P. mirabilis strains from a long-term care and rehabilitation facility in Italy

We report the detection of multidrug resistant ESbetaL producing Proteus mirabilis isolates from a long-term care and rehabilitation facility (LTCRF) in Northern Italy. 53% of the collected P. mirabilis strains were ESbetaL producers. PCR and sequencing techniques confirmed the presence of the bla(TEM-92) and bla(CMY-16) resistance genes in 23/26 (88.5%) and 3/26 (11.5%) of the ESbetaL producers respectively. PFGE showed that the TEM-92 beta-lactamase producing isolates were not clonally related, indicating the presence of at least four different clonal lineages (A, B, C, D), whereas all the CMY-16 enzyme producers belonged in the same lineage. The bla(TEM-92) and bla(CYY-16) determinants were distributed in seven different wards, but in three of them they coexisted. Our results show that the most patients are co-colonized by ESbetaLs producing P. mirabilis strains at the time of admission to an LTCRF. An effective strategy to curtail the spread of ESbetaLs mediated resistance in LTCRFs could be to activate sourveillance programs to monitor routinely the entry of resistant bacteria.     

5'-Modified G-quadruplex forming oligonucleotides endowed with anti-HIV activity: synthesis and biophysical properties

Oligodeoxyribonucleotides of sequence d(5'TGGGAG3') carrying bulky aromatic groups at the 5' end were found to exhibit potent anti-HIV activity [Hotoda, H., et al. (1998) J. Med. Chem. 41, 3655-3663 and references therein]. Structure-activity relationship investigations indicated that G-quadruplex formation, as well as the presence of large aromatic substituents at the 5'-end, were both essential for their antiviral activity. In this work, we synthesized some representative examples of the anti-HIV active Hotoda's 6-mers and analyzed the resulting G-quadruplexes by CD, DSC, and molecular modeling studies, in comparison with the unmodified oligonucleotide. In the case of the sequence carrying the 3,4-dibenzyloxybenzyl (DBB) group, identified as the best candidate for further drug optimization, we developed an alternative protocol to synthesize the 5'-DBB-thymidine phosphoramidite building block in higher yields. The thermodynamic and kinetic parameters for the association/dissociation processes of the 5'-conjugated quadruplexes, determined with respect to the unmodified one, were discussed in light of the molecular modeling studies. The aromatic groups at the 5' position of d(5'TGGGAG3') dramatically enhance both the equilibrium and the rate of formation of the quadruplex complexes. The overall stability of the investigated quadruplexes was found to correlate with the reported IC50 values, thus furnishing quantitative evidence for the hypothesis that the G-quadruplex structures are the ultimate active species, effectively responsible for the biological activity.     

Transplantation of Foetal Neural Stem Cells into the Rat Hippocampus During Trimethyltin-Induced Neurodegeneration

The present study investigates the survival and fate of neural stem cells/progenitor cells (NSC/NPCs) homografted into the hippocampus of rats treated with trimethyltin (TMT), a potent neurotoxicant considered a useful tool to obtain a well characterized model of neurodegeneration, to evaluate their possible role in the reparative mechanisms that accompany neurodegenerative events. NSC/NPCs expressing eGFP by lentivirus-mediated infection were stereotaxically grafted into the hippocampus of TMT-treated animals and controls. Two weeks after transplantation surviving NSC/NPCs were detectable in 60% of TMT-treated animals and 30% of controls, while 30 days after transplantation only 40% of TMT-treated animals showed surviving grafted cells, which were undetectable in controls. At both times investigated, while grafted NSC/NPCs differentiated into neurons or astrocytes could be observed in addition to undifferentiated NSC/NPCs, we did not find evidence of structural integration of grafted cells into the main site of hippocampal lesion leading to appreciable repair. Maria Concetta Geloso and Stefano Giannetti contributed equally to this work.     

Heterozygous Reelin Mutations Cause Autosomal-Dominant Lateral Temporal Epilepsy

Autosomal-dominant lateral temporal epilepsy (ADLTE) is a genetic epilepsy syndrome clinically characterized by focal seizures with prominent auditory symptoms. ADLTE is genetically heterogeneous, and mutations in LGI1 account for fewer than 50% of affected families. Here, we report the identification of causal mutations in reelin (RELN) in seven ADLTE-affected families without LGI1 mutations. We initially investigated 13 ADLTE-affected families by performing SNP-array linkage analysis and whole-exome sequencing and identified three heterozygous missense mutations co-segregating with the syndrome. Subsequent analysis of 15 small ADLTE-affected families revealed four additional missense mutations. 3D modeling predicted that all mutations have structural effects on protein-domain folding. Overall, RELN mutations occurred in 7/40 (17.5%) ADLTE-affected families. RELN encodes a secreted protein, Reelin, which has important functions in both the developing and adult brain and is also found in the blood serum. We show that ADLTE-related mutations significantly decrease serum levels of Reelin, suggesting an inhibitory effect of mutations on protein secretion. We also show that Reelin and LGI1 co-localize in a subset of rat brain neurons, supporting an involvement of both proteins in a common molecular pathway underlying ADLTE. Homozygous RELN mutations are known to cause lissencephaly with cerebellar hypoplasia. Our findings extend the spectrum of neurological disorders associated with RELN mutations and establish a link between RELN and LGI1, which play key regulatory roles in both the developing and adult brain.     

Spatiotopic coding and remapping in humans

How our perceptual experience of the world remains stable and continuous in the face of continuous rapid eye movements still remains a mystery. This review discusses some recent progress towards understanding the neural and psychophysical processes that accompany these eye movements. We firstly report recent evidence from imaging studies in humans showing that many brain regions are tuned in spatiotopic coordinates, but only for items that are actively attended. We then describe a series of experiments measuring the spatial and temporal phenomena that occur around the time of saccades, and discuss how these could be related to visual stability. Finally, we introduce the concept of the spatio-temporal receptive field to describe the local spatiotopicity exhibited by many neurons when the eyes move.     

Design, synthesis, biophysical and biological studies of trisubstituted naphthalimides as G-quadruplex ligands

A series of trisubstituted naphthalimides have been synthesized and evaluated as telomeric G-quadruplex ligands by biophysical methods. Affinity for telomeric G-quadruplex AGGG(TTAGGG)(3) binding was first screened by fluorescence titrations. Subsequently, the interaction of the telomeric G-quadruplex with compounds showing the best affinity has been studied by isothermal titration calorimetry and UV-melting experiments. The two best compounds of the series tightly bind the telomeric quadruplex with a 2:1 drug/DNA stoichiometry. These derivatives have been further evaluated for their ability to inhibit telomerase by a TRAP assay and their pharmacological properties by treating melanoma (M14) and human lung cancer (A549) cell lines with increasing drug concentrations. A dose-dependent inhibition of cell proliferation was observed for all cellular lines during short-term treatment.     

A large set of estrogen receptor β-interacting proteins identified by tandem affinity purification in hormone-responsive human breast cancer cell nuclei

Estrogen receptors α (ER-α) and β (ER-β) play distinct biological roles in onset and progression of hormone-responsive breast cancer, with ER-β exerting a modulatory activity on ER-α-mediated estrogen signaling and stimulation of cell proliferation by mechanisms still not fully understood. We stably expressed human ER-β fused to a tandem affinity purification-tag in estrogen-responsive MCF-7 cells and applied tandem affinity purification and nanoLC-MS/MS to identify the ER-β interactome of this cell type. Functional annotation by bioinformatics analyses of the 303 proteins that co-purify with ER-β from nuclear extracts identify several new molecular partners of this receptor subtype that represents nodal points of a large protein network controlling multiple processes and functions in breast cancer cells.     

Retrospective investigation of an influenza A/H1N1pdm outbreak in an Italian military ship cruising in the Mediterranean Sea, May-September 2009

Background

Clinical surveillance may have underestimated the real extent of the spread of the new strain of influenza A/H1N1, which surfaced in April 2009 originating the first influenza pandemic of the 21^st century. Here we report a serological investigation on an influenza A/H1N1pdm outbreak in an Italian military ship while cruising in the Mediterranean Sea (May 24-September 6, 2009).

Methods

The contemporary presence of HAI and CF antibodies was used to retrospectively estimate the extent of influenza A/H1N1pdm spread across the crew members (median age: 29 years).

Findings

During the cruise, 2 crew members fulfilled the surveillance case definition for influenza, but only one was laboratory confirmed by influenza A/H1N1pdm-specific RT-PCR; 52 reported acute respiratory illness (ARI) episodes, and 183 reported no ARI episodes. Overall, among the 211 crew member for whom a valid serological result was available, 39.3% tested seropositive for influenza A/H1N1pdm. The proportion of seropositives was significantly associated with more crowded living quarters and tended to be higher in those aged <40 and in those reporting ARI or suspected/confirmed influenza A/H1N1pdm compared to the asymptomatic individuals. No association was found with previous seasonal influenza vaccination.

Conclusions

These findings underline the risk for rapid spread of novel strains of influenza A in confined environment, such as military ships, where crowding, rigorous working environment, physiologic stress occur. The high proportion of asymptomatic infections in this ship-borne outbreak supports the concept that serological surveillance in such semi-closed communities is essential to appreciate the real extent of influenza A/H1N1pdm spread and can constitute, since the early stage of a pandemic, an useful model to predict the public health impact of pandemic influenza and to establish proportionate and effective countermeasures.