Structural studies of the lipopolysaccharide from Haemophilus parainfluenzae strain 20

Haemophilus parainfluenzae is a Gram-negative bacterium that colonizes the upper respiratory tract of humans and is a part of normal flora. In this study, we investigated the lipopolysaccharide (LPS) expressed by H. parainfluenzae strain 20. Using NMR and MS techniques on LPS, oligosaccharide samples and lipid A, the structures for O-antigen, core oligosaccharide and lipid A could be established. It was found that the biological repeating unit of the O-antigen is →4)-α-d-GalpNAc-(1→P→6)-β-d-Glcp-(1→3)-α-d-FucpNAc4N-(1→, in which d-FucpNAc4N is 2-acetamido-4-amino-2,4,6-trideoxy-d-galactose. This sugar is in β-configuration when linked to O-4 of the glucose residue of β-d-Galp-(1→2)-l-α-d-Hepp-(1→2)-[PEtn→6]-l-α-d-Hepp-(1→3)-[β-d-Glcp-(1→4)]-l-α-d-Hepp-(1→5)-[PPEtn→4]-α-Kdo-(2→6)-lipid A. LPS from a wbaP mutant of H. parainfluenzae strain 20 did not contain an O-antigen, consistent with the wbaP gene product being required for expression of O-antigen in fully extended LPS.     

Mode of binding of the cytotoxic alkaloid berberine with the double helix oligonucleotide d(AAGAATTCTT)(2)

Berberine, an isoquinoline plant alkaloid, belongs to the structural class of protoberberines. Recently, the ability of these compounds to act as Topoisomerase I or II poisons, was related to the antitumor activity. The binding of protoberberins to DNA has been studied and the partial intercalation into the double helix has been considered responsible for their activity. We have studied the interaction of berberine with the double helix oligonucleotides d(AAGAATTCTT)(2), d(GCGATCGC)(2), d(CGTATACG)(2), d(CGTACG)(2), 5'-d(ACCTTTTTGATGT)-3'/5(ACATCAAAAAGGT)-3' and with the single strand 5'-d(ACATCAAAAAGGT)-3', by 1H, 31P NMR and UV spectroscopy. Phosphorus resonance experiments were performed to detect small conformational changes of the phosphoribose backbone, in the case that an intercalation process occurs. Our data reveal that berberine does not intercalate into the duplexes studied, and binds preferentially to AT rich sequences. The structure of the complex with d(AAGAATTCTT)(2) was determined by using proton 2D NOESY spectra, which allowed to obtain several NOE contacts between the drug and the nucleotide. Structural models were built up by Molecular Mechanics (MM) and Molecular Dynamics (MD) calculations, by using the inter-proton distances derived from the NOE values. Berberine results to be located in the minor groove, lying with the convex side on the helix groove and presenting the positively charged nitrogen atom close to the negative ionic surface of the oligomer. The large 1H chemical shifts variation, observed for the drug when it is added to the above duplexes, as well as to the single strand oligomer, was interpreted with non-specific ionic interactions. The binding constants were measured by UV and NMR spectroscopy. They are strongly affected by the ionic strength and by the self-association process, which commonly occurs with this type of drugs. A dimerisation constant was measured and the value was included in the calculations of the binding constants. The results obtained show that the non-specific ionic interactions represent the major contribution to the values of the binding constants. These parameters, as well as the protons chemical shift variation of the ligand, are thus not diagnostic for the identification of a drug/DNA complex.     

A Genome Wide Meta-Analysis Study for Identification of Common Variation Associated with Breast Cancer Prognosis

Objective

Genome wide association studies (GWAs) of breast cancer mortality have identified few potential associations. The concordance between these studies is unclear. In this study, we used a meta-analysis of two prognostic GWAs and a replication cohort to identify the strongest associations and to evaluate the loci suggested in previous studies. We attempt to identify those SNPs which could impact overall survival irrespective of the age of onset.

Methods

To facilitate the meta-analysis and to refine the association signals, SNPs were imputed using data from the 1000 genomes project. Cox-proportional hazard models were used to estimate hazard ratios (HR) in 536 patients from the POSH cohort (Prospective study of Outcomes in Sporadic versus Hereditary breast cancer) and 805 patients from the HEBCS cohort (Helsinki Breast Cancer Study). These hazard ratios were combined using a Mantel-Haenszel fixed effects meta-analysis and a p-value threshold of 5×10⁻⁸ was used to determine significance. Replication was performed in 1523 additional patients from the POSH study.

Results

Although no SNPs achieved genome wide significance, three SNPs have significant association in the replication cohort and combined p-values less than 5.6×10⁻⁶. These SNPs are; rs421379 which is 556 kb upstream of ARRDC3 (HR = 1.49, 95% confidence interval (CI) = 1.27–1.75, P = 1.1×10⁻⁶), rs12358475 which is between ECHDC3 and PROSER2 (HR = 0.75, CI = 0.67–0.85, P = 1.8×10⁻⁶), and rs1728400 which is between LINC00917 and FOXF1.

Conclusions

In a genome wide meta-analysis of two independent cohorts from UK and Finland, we identified potential associations at three distinct loci. Phenotypic heterogeneity and relatively small sample sizes may explain the lack of genome wide significant findings. However, the replication at three SNPs in the validation cohort shows promise for future studies in larger cohorts. We did not find strong evidence for concordance between the few associations highlighted by previous GWAs of breast cancer survival and this study.     

Type II Fatty Acid Synthesis Is Essential for the Replication of Chlamydia trachomatis

The major phospholipid classes of the obligate intracellular bacterial parasite Chlamydia trachomatis are the same as its eukaryotic host except that they also contain chlamydia-made branched-chain fatty acids in the 2-position. Genomic analysis predicts that C. trachomatis is capable of type II fatty acid synthesis (FASII). AFN-1252 was deployed as a chemical tool to specifically inhibit the enoyl-acyl carrier protein reductase (FabI) of C. trachomatis to determine whether chlamydial FASII is essential for replication within the host. The C. trachomatis FabI (CtFabI) is a homotetramer and exhibited typical FabI kinetics, and its expression complemented an Escherichia coli fabI(Ts) strain. AFN-1252 inhibited CtFabI by binding to the FabI·NADH complex with an IC₅₀ of 0.9 μm at saturating substrate concentration. The x-ray crystal structure of the CtFabI·NADH·AFN-1252 ternary complex revealed the specific interactions between the drug, protein, and cofactor within the substrate binding site. AFN-1252 treatment of C. trachomatis-infected HeLa cells at any point in the infectious cycle caused a decrease in infectious titers that correlated with a decrease in branched-chain fatty acid biosynthesis. AFN-1252 treatment at the time of infection prevented the first cell division of C. trachomatis, although the cell morphology suggested differentiation into a metabolically active reticulate body. These results demonstrate that FASII activity is essential for C. trachomatis proliferation within its eukaryotic host and validate CtFabI as a therapeutic target against C. trachomatis.     

Whole genome sequence analysis of Salmonella Typhi provides evidence of phylogenetic linkage between cases of typhoid fever in Santiago,Chile in the 1980s and 2010–2016

Typhoid fever epidemiology was investigated rigorously in Santiago, Chile during the 1980s, when Salmonella enterica serovar Typhi (S. Typhi) caused seasonal, hyperendemic disease. Targeted interventions reduced the annual typhoid incidence rates from 128–220 cases/10⁵ population occurring between 1977–1984 to <8 cases/10⁵ from 1992 onwards. As such, Santiago represents a contemporary example of the epidemiologic transition of an industrialized city from amplified hyperendemic typhoid fever to a period when typhoid is no longer endemic. We used whole genome sequencing (WGS) and phylogenetic analysis to compare the genotypes of S. Typhi cultured from acute cases of typhoid fever occurring in Santiago during the hyperendemic period of the 1980s (n = 74) versus the nonendemic 2010s (n = 80) when typhoid fever was rare. The genotype distribution between “historical” (1980s) isolates and “modern” (2011–2016) isolates was similar, with genotypes 3.5 and 2 comprising the majority of isolations, and 73/80 (91.3%) of modern isolates matching a genotype detected in the 1980s. Additionally, phylogenomically ‘ancient’ genotypes 1.1 and 1.2.1, uncommon in the global collections, were also detected in both eras, with a notable rise amongst the modern isolates. Thus, genotypes of S. Typhi causing acute illness in the modern nonendemic era match the genotypes circulating during the hyperendemic 1980s. The persistence of historical genotypes may be explained by chronic typhoid carriers originally infected during or before the 1980s.     

SNR analysis: molecular investigation of an anthrax epidemic

Background  

In Italy, anthrax is endemic but occurs sporadically. During the summer of 2004, in the Pollino National Park, Basilicata, Southern Italy, an anthrax epidemic consisting of 41 outbreaks occurred; it claimed the lives of 124 animals belonging to different mammal species. This study is a retrospective molecular epidemiological investigation carried out on 53 isolates collected during the epidemic. A 25-loci Multiple Locus VNTR Analysis (MLVA) MLVA was initially performed to define genetic relationships, followed by an investigation of genetic diversity between epidemic strains through Single Nucleotide Repeat (SNR) analysis.     

Monoamine oxidase-dependent histamine catabolism accounts for post-ischemic cardiac redox imbalance and injury

Monoamine oxidase (MAO), a mitochondrial enzyme that oxidizes biogenic amines generating hydrogen peroxide, is a major source of oxidative stress in cardiac injury. However, the molecular mechanisms underlying its overactivation in pathological conditions are still poorly characterized.Here, we investigated whether the enhanced MAO-dependent hydrogen peroxide production can be due to increased substrate availability using a metabolomic profiling method. We identified N¹-methylhistamine -the main catabolite of histamine- as an important substrate fueling MAO in Langendorff mouse hearts, directly perfused with a buffer containing hydrogen peroxide or subjected to ischemia/reperfusion protocol. Indeed, when these hearts were pretreated with the MAO inhibitor pargyline we observed N¹-methylhistamine accumulation along with reduced oxidative stress. Next, we showed that synaptic terminals are the major source of N¹-methylhistamine. Indeed, in vivo sympathectomy caused a decrease of N¹-methylhistamine levels, which was associated with a marked protection in post-ischemic reperfused hearts. As far as the mechanism is concerned, we demonstrate that exogenous histamine is transported into isolated cardiomyocytes and triggers a rise in the levels of reactive oxygen species (ROS). Once again, pargyline pretreatment induced intracellular accumulation of N¹-methylhistamine along with decrease in ROS levels. These findings uncover a receptor-independent mechanism for histamine in cardiomyocytes.In summary, our study reveals a novel and important pathophysiological causative link between MAO activation and histamine availability during pathophysiological conditions such as oxidative stress/cardiac injury.