A preparation of 3alpha-methyl-5alpha-cholestane-2beta,3beta-diol.

A simple preparation of 3α-methyl-5α-cholestane-2β, 3β-diol (1a) by a four-step synthesis from 2α, 3α-epoxy-5α-cholestane is described.     

High yield purification and first structural characterization of the full‐length bacterial toxin CNF1

The Cytotoxic Necrotizing Factor 1 (CNF1) is a bacterial toxin secreted by certain Escherichia coli strains causing severe pathologies, making it a protein of pivotal interest in toxicology. In parallel, the CNF1 capability to influence important neuronal processes, like neuronal arborization, astrocytic support, and efficient ATP production, has been efficiently used in the treatment of neurological diseases, making it a promising candidate for therapy. Nonetheless, there are still some unsolved issues about the CNF1 mechanism of action and structuration probably caused by the difficulty to achieve sufficient amounts of the full‐length protein for further studies. Here, we propose an efficient strategy for the production and purification of this toxin as a his‐tagged recombinant protein from E. coli extracts (CNF1‐H8). CNF1‐H8 was expressed at the low temperature of 15°C to diminish its characteristic degradation. Then, its purification was achieved using an immobilized metal affinity chromatography (IMAC) and a size exclusion chromatography so as to collect up to 8 mg of protein per liter of culture in a highly pure form. Routine dynamic light scattering (DLS) experiments showed that the recombinant protein preparations were homogeneous and preserved this state for a long time. Furthermore, CNF1‐H8 functionality was confirmed by testing its activity on purified RhoA and on HEp‐2 cultured cells. Finally, a first structural characterization of the full‐length toxin in terms of secondary structure and thermal stability was performed by circular dichroism (CD). These studies demonstrate that our system can be used to produce high quantities of pure recombinant protein for a detailed structural analysis. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:150–159, 2018     

Activation of Human Toll-like Receptor 4 (TLR4)·Myeloid Differentiation Factor 2 (MD-2) by Hypoacylated Lipopolysaccharide from a Clinical Isolate of Burkholderia cenocepacia

Lung infection by Burkholderia species, in particular Burkholderia cenocepacia, accelerates tissue damage and increases post-lung transplant mortality in cystic fibrosis patients. Host-microbe interplay largely depends on interactions between pathogen-specific molecules and innate immune receptors such as Toll-like receptor 4 (TLR4), which recognizes the lipid A moiety of the bacterial lipopolysaccharide (LPS). The human TLR4·myeloid differentiation factor 2 (MD-2) LPS receptor complex is strongly activated by hexa-acylated lipid A and poorly activated by underacylated lipid A. Here, we report that B. cenocepacia LPS strongly activates human TLR4·MD-2 despite its lipid A having only five acyl chains. Furthermore, we show that aminoarabinose residues in lipid A contribute to TLR4-lipid A interactions, and experiments in a mouse model of LPS-induced endotoxic shock confirmed the proinflammatory potential of B. cenocepacia penta-acylated lipid A. Molecular modeling combined with mutagenesis of TLR4-MD-2 interactive surfaces suggests that longer acyl chains and the aminoarabinose residues in the B. cenocepacia lipid A allow exposure of the fifth acyl chain on the surface of MD-2 enabling interactions with TLR4 and its dimerization. Our results provide a molecular model for activation of the human TLR4·MD-2 complex by penta-acylated lipid A explaining the ability of hypoacylated B. cenocepacia LPS to promote proinflammatory responses associated with the severe pathogenicity of this opportunistic bacterium.     

Subchondral bone response to injected adipose-derived stromal cells for treating osteoarthritis using an experimental rabbit model

Although articular cartilage is the target of osteoarthritis (OA), its deterioration is not always clearly associated with patient symptoms. Because a functional interaction between cartilage and bone is crucial, the pathophysiology of OA and its treatment strategy must focus also on subchondral bone. We investigated whether adipose-derived stromal cells (ASCs) injected into a joint at two different concentrations could prevent subchondral bone damage after the onset of mild OA in a rabbit model. We measured both volumetric and densitometric aspects of bone remodeling. Although OA can stimulate bone remodeling either catabolically or anabolically over time, the accelerated turnover does not allow complete mineralization of new bone and therefore gradually reduces its density. We measured changes in morphometric and densitometric bone parameters using micro-CT analysis and correlated them with the corresponding parameters in cartilage and meniscus. We found that ASCs promoted cartilage repair and helped counteract the accelerated bone turnover that occurs with OA.     

Structural analysis of a novel putative capsular polysaccharide from Pseudomonas (Burkholderia) caryophylli strain 2151.

A novel putative capsular polysaccharide consisting of D-Glcp and D-Fruf in the molar ratio of 1:1 was isolated as minor constituent from the lipopolysaccharide (LPS) fraction of Pseudomonas (Burkholderia) caryophylli. Its structure was determined, using mainly one- and two-dimensional NMR spectroscopy, as: -->6)-alpha-D-Glcp-(1-->1)-beta-D-Fruf-(2-->.     

Investigating the Role of the Host Multidrug Resistance Associated Protein Transporter Family in Burkholderia cepacia Complex Pathogenicity Using a Caenorhabditis elegans Infection Model

This study investigated the relationship between host efflux system of the non-vertebrate nematode Caenorhabditis elegans and Burkholderia cepacia complex (Bcc) strain virulence. This is the first comprehensive effort to profile host-transporters within the context of Bcc infection. With this aim, two different toxicity tests were performed: a slow killing assay that monitors mortality of the host by intestinal colonization and a fast killing assay that assesses production of toxins. A Virulence Ranking scheme was defined, that expressed the toxicity of the Bcc panel members, based on the percentage of surviving worms. According to this ranking the 18 Bcc strains were divided in 4 distinct groups. Only the Cystic Fibrosis isolated strains possessed profound nematode killing ability to accumulate in worms’ intestines. For the transporter analysis a complete set of isogenic nematode single Multidrug Resistance associated Protein (MRP) efflux mutants and a number of efflux inhibitors were interrogated in the host toxicity assays. The Bcc pathogenicity profile of the 7 isogenic C. elegans MRP knock-out strains functionality was classified in two distinct groups. Disabling host transporters enhanced nematode mortality more than 50% in 5 out of 7 mutants when compared to wild type. In particular mrp-2 was the most susceptible phenotype with increased mortality for 13 out 18 Bcc strains, whereas mrp-3 and mrp-4 knock-outs had lower mortality rates, suggesting a different role in toxin-substrate recognition. The use of MRP efflux inhibitors in the assays resulted in substantially increased (>40% on average) mortality of wild-type worms.     

Identification and structural determination of the capsular polysaccharides from two Acinetobacter baumannii clinical isolates, MG1 and SMAL

The structures of the capsular polysaccharides (CPSs) of the two clinical isolates Acinetobacter baumannii SMAL and MG1 were elucidated. Hot phenol/water extractions of the dry biomasses, followed by enzymatic digestions and repeated ultracentrifugations led to the isolation of polysaccharides that were negative in Western blot analysis utilizing an anti-lipid A antibody, thus proving that they were not the LPS O-antigens but CPSs. Their structures were established on the basis of NMR spectroscopy and GC-MS analyses. The A. baumannii MG1 CPS consisted of a linear aminopolysaccharide with acyl substitution heterogeneity at the N-4 amino group of QuipN4N:4)-α-d-GlcpNAc-(1→4)-α-l-GalpNAcA-(1→3)-β-d-QuipNAc4NR-(1→R = 3-hydroxybutyrryl or acetyl.The repeating unit of the CPS produced by strain SMAL is a pentasaccharide, already reported for the O-antigen moiety from A. baumannii strain ATCC 17961:     

Structural investigation on the lipooligosaccharide fraction of psychrophilic Pseudoalteromonas haloplanktis TAC 125 bacterium.

The core structure of the cell-wall lipooligosaccharide (LOS) fraction of an Antarctic Gram-negative bacterium, Pseudoalteromonas haloplanktis TAC 125 strain, was determined to be deacetylated alditols. These were obtained from native LOS fraction by O-deacylation, dephosphorylation, reduction and finally N-deacylation. Two novel structures were detected, the more highly represented molecule consisting of the following hexasaccharide chain: alpha-D-ManpNH(2)-(1-->3)-beta-D-Galp-(1-->4)-alpha-L-glycero-D-manno-Hepp-(1-->5)-alpha-D-Kdo-(2-->6)-beta-D-GlcpNH(2)-(1-->6)-D-GlcNH(2)(ol) while the corresponding pentasaccharide, lacking the ManpNH(2) residue, was less abundant. To the best of our knowledge, the structural investigation presented here, mainly performed by NMR and MS methods, is the first report of the lipopolysaccharide fraction of a psychrophilic bacterium.