A combined fermentative-chemical approach for the scalable production of pure E. coli monophosphoryl lipid A

Lipid A is the lipophilic region of lipopolysaccharides and lipooligosaccharides, the major components of the outer leaflet of most part of Gram-negative bacteria. Some lipid As are very promising immunoadjuvants. They are obtained by extraction from bacterial cells or through total chemical synthesis. A novel, semisynthetic approach to lipid As is ongoing in our laboratories, relying upon the chemical modification of a natural lipid A scaffold for the fast obtainment of several other lipid As and derivatives thereof. The first requisite for this strategy is to have this scaffold available in large quantities through a scalable process. Here, we present an optimized fed-batch fermentation procedure for the gram-scale production of lipid A from Escherichia coli K4 and a suitable phenol-free protocol for its purification. A study for regioselective de-O-phosphorylation reaction was then performed to afford pure monophosphoryl lipid A with an attenuated endotoxic activity, as evaluated by cytokine production in human monocytic cell line THP-1 in vitro. The reported method for the large-scale obtainment of monophoshoryl lipid A from the fed-batch fermentation broth of a recombinant strain of E. coli may permit the access to novel semisynthetic lipid A immunoadjuvant candidates.     

A Multi-Level Geographical Study of Italian Political Elections from Twitter Data

In this paper we present an analysis of the behavior of Italian Twitter users during national political elections. We monitor the volumes of the tweets related to the leaders of the various political parties and we compare them to the elections results. Furthermore, we study the topics that are associated with the co-occurrence of two politicians in the same tweet. We cannot conclude, from a simple statistical analysis of tweet volume and their time evolution, that it is possible to precisely predict the election outcome (or at least not in our case of study that was characterized by a “too-close-to-call” scenario). On the other hand, we found that the volume of tweets and their change in time provide a very good proxy of the final results. We present this analysis both at a national level and at smaller levels, ranging from the regions composing the country to macro-areas (North, Center, South).     

NMR and MS evidences for a random assembled O-specific chain structure in the LPS of the bacterium Xanthomonas campestris pv. Vitians. A case of unsystematic biosynthetic polymerization.

Xanthomonas campestris pv. vitians is a Gram-negative plant-associated bacterium that acts as causative agent of bacterial leaf spot and headrot in lettuce. The lipopolysaccharide of this bacterium is suspected to be an important molecule for adhesion to and infection of the plants. The lipopolysaccharide has been isolated from the phenol phase and the O-specific chain characterized by compositional analysis, high field NMR and MALDI-TOF MS. It consists of a nonrepetitive branched polysaccharide with a rhamnan backbone to which Fuc3NAc is linked. The NMR and MS approach led to the characterization of the fine structure of the polymer, which is randomly assembled. The rhamnan backbone is built up of beta-Rhap and alpha-Rhap, this last is present in one, two or three adjacent units and branched by an alpha-Fucp3NAc unit. This is a real case of a random constituted O-specific chain, therefore biosynthetic studies towards the comprehension of this irregular biosynthesis are needed.     

Recruitment of phospholipase Cγ1 to the non-structural membrane protein pK15 of Kaposi Sarcoma-associated herpesvirus promotes its Src-dependent phosphorylation

Kaposi Sarcoma-associated herpesvirus (KSHV) causes three human malignancies, Kaposi Sarcoma (KS), Primary Effusion Lymphoma (PEL) and the plasma cell variant of multicentric Castleman’s Disease (MCD), as well as an inflammatory cytokine syndrome (KICS). Its non-structural membrane protein, pK15, is among a limited set of viral proteins expressed in KSHV-infected KS tumor cells. Following its phosphorylation by Src family tyrosine kinases, pK15 recruits phospholipase C gamma 1 (PLCγ1) to activate downstream signaling cascades such as the MEK/ERK, NFkB and PI3K pathway, and thereby contributes to the increased proliferation and migration as well as the spindle cell morphology of KSHV-infected endothelial cells. Here, we show that a phosphorylated Y⁴⁸¹EEVL motif in pK15 preferentially binds into the PLCγ1 C-terminal SH2 domain (cSH2), which is involved in conformational changes occurring during the activation of PLCγ1 by receptor tyrosine kinases. We determined the crystal structure of a pK15 12mer peptide containing the phosphorylated pK15 Y⁴⁸¹EEVL motif in complex with a shortened PLCγ1 tandem SH2 (tSH2) domain. This structure demonstrates that the pK15 peptide binds to the PLCγ1 cSH2 domain in a position that is normally occupied by the linker region connecting the PLCγ1 cSH2 and SH3 domains. We also show that longer pK15 peptides containing the phosphorylated pK15 Y⁴⁸¹EEVL motif can increase the Src-mediated phosphorylation of the PLCγ1 tSH2 region in vitro. This pK15-induced increase in Src-mediated phosphorylation of PLCγ1 can be inhibited with the small pK15-derived peptide which occupies the PLCγ1 cSH2 domain. Our findings thus suggest that pK15 may act as a scaffold protein to promote PLCγ1 activation in a manner similar to the cellular scaffold protein SLP-76, which has been shown to promote PLCγ1 activation in the context of T-cell receptor signaling. Reminiscent of its positional homologue in Epstein-Barr Virus, LMP2A, pK15 may therefore mimic aspects of antigen-receptor signaling. Our findings also suggest that it may be possible to inhibit the recruitment and activation of PLCγ1 pharmacologically.     

The structures of the cell wall teichoic acids from the thermophilic microorganism Geobacillus thermoleovorans strain Fango

The structures of two teichoic acid fractions (TA1 and TA2) isolated from the thermophilic gram-positive bacterium Geobacillus thermoleovorans strain Fango were investigated by means of chemical and NMR spectroscopic methods. The most abundant species (TA1) exhibited a rather regular structure comprising two different repeating units of 1,3-glycerol phosphate nonstoichiometrically substituted by terminal-alpha-D-Gal p (t-alpha-D-Gal p). The second molecular species (TA2) presented a higher structural variability and t-alpha-D-Glc p and the disaccharides t-alpha-D-Glc pNAc-(1-->2)-alpha-D-Glc p and t-alpha-D-Glc pNAc-(1-->3)-alpha-D-Glc p were also present as minor substituents at O-2 of the glycerol phosphate residues. Minor substitution by alanine could also be detected.     

Core oligosaccharide structure from the highly phytopathogenic Agrobacterium tumefaciens TT111 and conformational analysis of the putative rhamnan epitope

The structure of the complex mixture of the core oligosaccharide components of the lipooligosaccharide (LOS) fraction of Agrobacterium tumefaciens strain TT111 was determined directly on the deacetylated products by means of spectroscopical methods. The rhamnan oligosaccharide elongating the inner Kdo residue shares structural features with other polysaccharides from well-known plant pathogenic bacteria. Its conformation was determined through extensive molecular dynamic (MD) analysis and presents an epitope similar to that recognized from the plant defense system.     

The structures of glycolipids isolated from the highly thermophilic bacterium Thermus thermophilus Samu-SA1

Thermophiles constitute a class of microorganisms able to grow at extremely elevated temperatures. Some of these species are classified as Gram-negative bacteria, because of the presence of an outer membrane in the cell envelope, which is located on the top of a thick murein layer. Unlike typical Gram-negative bacteria, the outer membranes of Thermus species are not composed of lipopolysaccharides but of peculiar glycolipids (GL), whose structures seem to be strictly involved in the adaptation to high temperatures. In this work, the complete structures of the major GL components from the cell envelope of the thermophilic bacterium Thermus thermophilus Samu-SA1 are presented. Protocols conventionally adopted for Gram-negative bacteria were used, and, for the first time, GL from Thermus were analyzed in their native form. Two GL and one phosphoglycolipid (PGL) were detected and characterized. The two GL, analyzed by nuclear magnetic resonance (NMR) spectroscopy and electrospray ionization Fourier transform ion cyclotron resonance (ESI FT-ICR) mass spectrometry, possessed the same tetrasaccharide structure linked to a glycerol unit or, alternatively, to a long-chain diol. Moreover, a PGL from Thermus was characterized for the first time, in which N-glyceroyl-heptadecaneamine was present. These molecules are chemically related to other GL from thermophile bacteria, in which they play a crucial role in the adaptation of cell membranes to heat.     

Large-scale population analysis challenges the current criteria for the molecular diagnosis of fascioscapulohumeral muscular dystrophy

Facioscapulohumeral muscular dystrophy (FSHD) is a common hereditary myopathy causally linked to reduced numbers (≤8) of 3.3 kilobase D4Z4 tandem repeats at 4q35. However, because individuals carrying D4Z4-reduced alleles and no FSHD and patients with FSHD and no short allele have been observed, additional markers have been proposed to support an FSHD molecular diagnosis. In particular a reduction in the number of D4Z4 elements combined with the 4A(159/161/168)PAS haplotype (which provides the possibility of expressing DUX4) is currently used as the genetic signature uniquely associated with FSHD. Here, we analyzed these DNA elements in more than 800 Italian and Brazilian samples of normal individuals unrelated to any FSHD patients. We find that 3% of healthy subjects carry alleles with a reduced number (4–8) of D4Z4 repeats on chromosome 4q and that one-third of these alleles, 1.3%, occur in combination with the 4A161PAS haplotype. We also systematically characterized the 4q35 haplotype in 253 unrelated FSHD patients. We find that only 127 of them (50.1%) carry alleles with 1–8 D4Z4 repeats associated with 4A161PAS, whereas the remaining FSHD probands carry different haplotypes or alleles with a greater number of D4Z4 repeats. The present study shows that the current genetic signature of FSHD is a common polymorphism and that only half of FSHD probands carry this molecular signature. Our results suggest that the genetic basis of FSHD, which is remarkably heterogeneous, should be revisited, because this has important implications for genetic counseling and prenatal diagnosis of at-risk families.     

Synthesis and characterization of new polyamino-cyclodextrin materials

With the aim of the synthesis of chemically modified cyclodextrins bearing polyamine pendant groups, potentially useful as capping agents for the preparation of nanosized metal systems or as auxiliaries for gene transfection, the reaction between the heptakis-(6-iodo)-(6-deoxy)-β-cyclodextrin and various polyamines has been explored. This synthetic approach allows obtaining materials constituted by mixtures of cyclodextrins, having different degrees of substitution, which were satisfactorily characterized by means of various complementary techniques (ESI-MS, NMR, potentiometric titration). The products obtained were successfully subjected to preliminary tests for their binding abilities towards suitable organic guests and as capping agents for the preparation of stable silver nanoparticles.     

Preparation and NMR characterization of glucosamine oligomers bearing an azide function using chitosan

In this study, a procedure to produce glucosamine oligomers with the amino functions transformed into azido groups was optimized, and HPLC purification afforded to the isolation of nine different oligosaccharides derivatives, with the reducing end transformed in alditol. These oligomers differed for the degree of polymerization and for the type of alditol at the reducing end. The first group comprehended species from di- to hexasaccharide, with all the amino functions converted into an azido group. The second and the third groups were isolated in minor yields, and were both constituted from tri- and tetrasaccharides; the difference between the two groups regarded exclusively the type of alditol found at the reducing end, which was a glucosaminitol in the first case, or a N-acetylglucosaminitol in the other. Products were fully characterized by 2D NMR spectroscopy. The azido moieties installed on these oligosaccharides can be further exploited in Cu(I) catalyzed azido-alkyne cycloaddition reactions.