The cell wall binding domain of Listeria bacteriophage endolysin PlyP35 recognizes terminal GlcNAc residues in cell wall teichoic acid

The cell wall binding domains (CBD) of bacteriophage endolysins target the enzymes to their substrate in the bacterial peptidoglycan with extraordinary specificity. Despite strong interest in these enzymes as novel antimicrobials, little is known regarding their interaction with the bacterial wall and their binding ligands. We investigated the interaction of Listeria phage endolysin PlyP35 with carbohydrate residues present in the teichoic acid polymers on the peptidoglycan. Biochemical and genetic analyses revealed that CBD of PlyP35 specifically recognizes the N-acetylglucosamine (GlcNAc) residue at position C4 of the polyribitol-phosphate subunits. Binding of CBDP35 could be prevented by removal of wall teichoic acid (WTA) polymers from cell walls, and inhibited by addition of purified WTAs or acetylated saccharides. We show that Listeria monocytogenes genes lmo2549 and lmo2550 are required for decoration of WTAs with GlcNAc. Inactivation of either gene resulted in a lack of GlcNAc glycosylation, and the mutants failed to bind CBDP35. We also report that the GlcNAc-deficient phenotype of L. monocytogenes strain WSLC 1442 is due to a small deletion in lmo2550, resulting in synthesis of a truncated gene product responsible for the glycosylation defect. Complementation with lmo2550 completely restored display of characteristic serovar 1/2 specific WTA and the wild-type phenotype.     

Regeneration of sensory axons within the injured spinal cord induced by intraganglionic cAMP elevation

The peripheral branch of primary sensory neurons regenerates after injury, but there is no regeneration when their central branch is severed by spinal cord injury. Here we show that microinjection of a membrane-permeable analog of cAMP in lumbar dorsal root ganglia markedly increases the regeneration of injured central sensory branches. The injured axons regrow into the spinal cord lesion, often traversing the injury site. This result mimics the effect of a conditioning peripheral nerve lesion. We also demonstrate that sensory neurons exposed to cAMP in vivo, when subsequently cultured in vitro, show enhanced growth of neurites and an ability to overcome inhibition by CNS myelin. Thus, stimulating cAMP signaling increases the intrinsic growth capacity of injured sensory axons. This approach may be useful in promoting regeneration after spinal cord injury.     

Synthesis, characterization, crystal structures and in vitro antistaphylococcal activity of organotin(IV) derivatives with 5,7-disubstituted-1,2,4-triazolo[1,5-a]pyrimidine

New organotin(IV) complexes of 5,7-ditertbutyl-1,2,4-triazolo[1,5-a]pyrimidine (dbtp) and 5,7-diphenyl-1,2,4-triazolo[1,5-a]pyrimidine (dptp) with 1:1 and/or 1:2 stoichiometry were synthesized and investigated by X-ray diffraction, FT-IR and ¹¹⁹Sn Mössbauer in the solid state and by ¹H and ¹³C NMR spectroscopy, in solution. Moreover, the crystal and molecular structures of Et₂SnCl₂(dbtp)₂ and Ph₂SnCl₂(EtOH)₂(dptp)₂ are reported. The complexes contain hexacoordinated tin atoms: in Et₂SnCl₂(dbtp)₂ two 5,7-ditertbutyl-1,2,4-triazolo[1,5-a]pyrimidine molecules coordinate classically the tin atom through N(3) atom and the coordination around the tin atom shows a skew trapezoidal structure with axial ethyl groups. In Ph₂SnCl₂(EtOH)₂(dptp)₂ two ethanol molecules coordinate tin through the oxygen atom and the 5,7-diphenyl-1,2,4-triazolo[1,5-a]pyrimidine molecules are not directly bound to the metal center but strictly H-bonded, through N(3), to the OH group of the ethanol moieties; Ph₂SnCl₂(EtOH)₂(dptp)₂ has an all-trans structure and the C-Sn-C fragment is linear. On the basis of Mössbauer data, the 1:2 diorganotin(IV) complexes are advanced to have the same structure of Et₂SnCl₂(dbtp)₂, while Me₂SnCl₂(dptp)₂ to have a regular all-trans octahedral structure. A distorted cis-R₂ trigonal bipyramidal structure is assigned to 1:1 diorganotin(IV) complexes. The in vitro antibacterial activities of the synthesized complexes have been tested against a group of reference pathogen micro-organisms and some of them resulted active with MIC values of 5 μg/mL, most of all against staphylococcal strains, which shows their inhibitory effect.     

Molecular conformation changes along the malignancy revealed by optical nanosensors

An interdisciplinary approach employing functionalized nanoparticles and ultrasensitive spectroscopic techniques is reported here to track the molecular changes in early stage of malignancy. Melanoma tissue tracking at molecular level using both labelled and unlabelled silver and gold nanoparticles has been achieved using surface enhanced Raman scattering (SERS) technique. We used skin tissue from ex vivo mice with induced melanoma. Raman and SERS molecular characterization of melanoma tissue is proposed here for the first time. Optical nanosensors based on Ag and Au nanoparticles with chemisorbed cresyl violet molecular species as labels revealed sensitive capability to tissues tagging and local molecular characterization. Sensitive information originating from surrounding native biological molecules is provided by the tissue SERS spectra obtained either with visible or NIR laser line. Labelled nanoparticles introduced systematic differences in tissue response compared with unlabelled ones, suggesting that the label functional groups tag specific tissue components revealed by proteins or nucleic acids bands. Vibrational data collected from tissue are presented in conjunction with the immunohistochemical analysis. The results obtained here open perspectives in applied plasmonic nanoparticles and SERS for the early cancer diagnostic based on the appropriate spectral databank.     

Multiple Delivery of siRNA against Endoglin into Murine Mammary Adenocarcinoma Prevents Angiogenesis and Delays Tumor Growth

Endoglin is a transforming growth factor-β (TGF- β) co-receptor that participates in the activation of a signaling pathway that mediates endothelial cell proliferation and migration in angiogenic tumor vasculature. Therefore, silencing of endoglin expression is an attractive approach for antiangiogenic therapy of tumors. The aim of our study was to evaluate the therapeutic potential of small interfering RNA (siRNA) molecules against endoglin in vitro and in vivo. Therapeutic potential in vitro was assessed in human and murine endothelial cells (HMEC-1, 2H11) by determining endoglin expression level, cell proliferation and tube formation. In vivo, the therapeutic potential of siRNA molecules was evaluated in TS/A mammary adenocarcinoma growing in BALB/c mice. Results of our study showed that siRNA molecules against endoglin have a good antiangiogenic therapeutic potential in vitro, as expression of endoglin mRNA and protein levels in mouse and human microvascular endothelial cells after lipofection were efficiently reduced, which resulted in the inhibition of endothelial cell proliferation and tube formation. In vivo, silencing of endoglin with triple electrotransfer of siRNA molecules into TS/A mammary adenocarcinoma also significantly reduced the mRNA levels, number of tumor blood vessels and the growth of tumors. The obtained results demonstrate that silencing of endoglin is a promising antiangiogenic therapy of tumors that could not be used as single treatment, but as an adjunct to the established cytotoxic treatment approaches.     

Bayesian Modeling of the Yeast SH3 Domain Interactome Predicts Spatiotemporal Dynamics of Endocytosis Proteins

SH3 domains are peptide recognition modules that mediate the assembly of diverse biological complexes. We scanned billions of phage-displayed peptides to map the binding specificities of the SH3 domain family in the budding yeast, Saccharomyces cerevisiae. Although most of the SH3 domains fall into the canonical classes I and II, each domain utilizes distinct features of its cognate ligands to achieve binding selectivity. Furthermore, we uncovered several SH3 domains with specificity profiles that clearly deviate from the two canonical classes. In conjunction with phage display, we used yeast two-hybrid and peptide array screening to independently identify SH3 domain binding partners. The results from the three complementary techniques were integrated using a Bayesian algorithm to generate a high-confidence yeast SH3 domain interaction map. The interaction map was enriched for proteins involved in endocytosis, revealing a set of SH3-mediated interactions that underlie formation of protein complexes essential to this biological pathway. We used the SH3 domain interaction network to predict the dynamic localization of several previously uncharacterized endocytic proteins, and our analysis suggests a novel role for the SH3 domains of Lsb3p and Lsb4p as hubs that recruit and assemble several endocytic complexes.     

Nitrous oxide and methane fluxes from soils of the Orinoco savanna under different land uses

The study investigates the effect of land‐use change on nitrous oxide (N₂O) and methane (CH₄) fluxes from soil, in savanna ecosystems of the Orinoco region (Venezuela). Gas fluxes were measured by closed static chambers, in the wet and dry season, in representative systems of land management of the region: a cultivated pasture, an herbaceous savanna, a tree savanna and a woodland (control site). Higher N₂O emissions were observed in the cultivated pasture and in the herbaceous savanna compared with the tree savanna and the woodland, and differences were mainly related to fine soil particle content and soil volumetric water content measured in the studied sites. Overall N₂O emissions were quite low in all sites (0–1.58 mg N₂O‐N m^?2 day^?1). The cultivated pasture and the woodland savanna were on average weak CH₄ sinks (?0.05±0.07 and ?0.08±0.05 mg CH₄ m^?2 day^?1, respectively), whereas the herbaceous savanna and the tree savanna showed net CH₄ production (0.23±0.05 and 0.19±0.05 mg CH₄ m^?2 day^?1, respectively). Variations of CH₄ fluxes were mainly driven by variation of soil water‐filled pore space (WFPS), and a shift from net CH₄ consumption to net CH₄ production was observed at around 30% WFPS. Overall, the data suggest that conversion of woodland savanna to managed landscape could alter both CH₄ and N₂O fluxes; however, the magnitude of such variation depends on the soil characteristics and on the type of land management before conversion.     

Speciation of Al in human serum by convective-interaction media fast-monolithic chromatography with inductively coupled plasma mass spectrometric detection

A new analytical procedure using anion-exchange separation support based on convective-interaction media (CIM) was developed for the speciation of Al in human serum. The separation of proteins was performed on a weak anion-exchange CIM diethylamine (DEAE) fast-monolithic disk. To prevent co-elution of low molecular mass (LMM) Al species with high molecular mass (HMM) Al compounds on CIM disk serum proteins were first separated from LMM-Al species by the use of size exclusion chromatography (SEC). For this purpose 1 mL of serum was injected onto SEC (Superdex 75 HR 10/30) column. Isocratic elution using 0.05 M TRIS-HCl+0.03 M NaHCO(3) was applied and separation of proteins was followed by UV detection at 278 nm. It was experimentally proven that proteins were eluted in 5.5 mL peak that was collected into a polyethylene cup. A 0.1 mL of the sample aliquot was then injected onto the CIM DEAE disk. The separation of serum proteins was obtained in 10 min by applying linear gradient elution from 100% buffer A (0.05 M TRIS-HCl+0.03 M NaHCO(3)) to 100% buffer B (A+1M NH(4)Cl) and followed by UV detection at 278 nm. Separated Al species were detected on-line by inductively coupled plasma mass spectrometry (ICP-MS). Well-resolved protein peaks were obtained. It was experimentally proven that 90+/-3% of Al in spiked serum of renal patient was eluted under the transferrin peak. The proposed speciation procedure removes LMM-Al species and enables reliable determination of the concentration and composition of Al bound to proteins by CIM DEAE-ICP-MS when the concentration of Al in serum is higher than 5 ng mL(-1). In comparison to chromatographic columns CIM disks enable faster separation and simpler manipulation during cleaning procedure and coupling to ICP-MS.     

Human blood-brain barrier disruption by retroviral-infected lymphocytes: role of myosin light chain kinase in endothelial tight-junction disorganization

The blood-brain barrier (BBB), which constitutes the interface between blood and cerebral parenchyma, has been shown to be disrupted during retroviral associated neuromyelopathies. Human T cell leukemia virus (HTLV-1)-associated myelopathy/tropical spastic paraparesis is a slowly progressive neurodegenerative disease, in which evidence of BBB breakdown has been demonstrated by the presence of lymphocytic infiltrates in the CNS and plasma protein leakage through cerebral endothelium. Using an in vitro human BBB model, we investigated the cellular and molecular mechanisms involved in endothelial changes induced by HTLV-1-infected lymphocytes. We demonstrate that coculture with infected lymphocytes induces an increase in paracellular endothelial permeability and transcellular migration, via IL-1alpha and TNF-alpha secretion. This disruption is associated with tight junction disorganization between endothelial cells, and alterations in the expression pattern of tight junction proteins such as zonula occludens 1. These changes could be prevented by inhibition of the NF-kappaB pathway or of myosin light chain kinase activity. Such disorganization was confirmed in histological sections of spinal cord from an HTLV-1-associated myelopathy/tropical spastic paraparesis patient. Based on this BBB model, the present data indicate that HTLV-1-infected lymphocytes can induce BBB breakdown and may be responsible for the CNS infiltration that occurs in the early steps of retroviral-associated neuromyelopathies.