Evaluation of Four Commercial Multiplex Molecular Tests for the Diagnosis of Acute Respiratory Infections

Acute Respiratory Infections (ARIs) are responsible for considerable morbidity and mortality worldwide. Documentation of respiratory specimens can help for an appropriate clinical management with a significant effect on the disease progress in patient, the antimicrobial therapy used and the risk of secondary spread of infection. Here, we compared the performances of four commercial multiplex kits used in French University Hospital diagnostic microbiology laboratories for the detection of ARI pathogens (i.e., the xTAG Respiratory Viral Panel Fast, RespiFinder SMART 22, CLART PneumoVir and Fast Track Diagnostics Respiratory Pathogen 33 kits). We used a standardised nucleic acids extraction protocol and a comprehensive comparative approach that mixed reference to well established real-time PCR detection techniques and analysis of convergent positive results. We tested 166 respiratory clinical samples and identified a global high degree of correlation for at least three of the techniques (xTAG, RespiFinder and FTD33). For these techniques, the highest Youden’s index (YI), positive predictive (PPV) and specificity (Sp) values were observed for Core tests (e.g., influenza A [YI:0.86–1.00; PPV:78.95–100.00; Sp:97.32–100.00] & B [YI:0.44–1.00; PPV:100.00; Sp:100.00], hRSV [YI:0.50–0.99; PPV:85.71–100.00; Sp:99.38–100.00], hMPV [YI:0.71–1.00; PPV:83.33–100.00; Sp:99.37–100.00], EV/hRV [YI:0.62–0.82; PPV:93.33–100.00; Sp:94.48–100.00], AdV [YI:1.00; PPV:100.00; Sp:100.00] and hBoV [YI:0.20–0.80; PPV:57.14–100.00; Sp:98.14–100.00]). The present study completed an overview of the multiplex techniques available for the diagnosis of acute respiratory infections.     

Biochemistry and Crystal Structure of Ectoine Synthase: A Metal-Containing Member of the Cupin Superfamily

Ectoine is a compatible solute and chemical chaperone widely used by members of the Bacteria and a few Archaea to fend-off the detrimental effects of high external osmolarity on cellular physiology and growth. Ectoine synthase (EctC) catalyzes the last step in ectoine production and mediates the ring closure of the substrate N-gamma-acetyl-L-2,4-diaminobutyric acid through a water elimination reaction. However, the crystal structure of ectoine synthase is not known and a clear understanding of how its fold contributes to enzyme activity is thus lacking. Using the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa), we report here both a detailed biochemical characterization of the EctC enzyme and the high-resolution crystal structure of its apo-form. Structural analysis classified the (Sa)EctC protein as a member of the cupin superfamily. EctC forms a dimer with a head-to-tail arrangement, both in solution and in the crystal structure. The interface of the dimer assembly is shaped through backbone-contacts and weak hydrophobic interactions mediated by two beta-sheets within each monomer. We show for the first time that ectoine synthase harbors a catalytically important metal co-factor; metal depletion and reconstitution experiments suggest that EctC is probably an iron-dependent enzyme. We found that EctC not only effectively converts its natural substrate N-gamma-acetyl-L-2,4-diaminobutyric acid into ectoine through a cyclocondensation reaction, but that it can also use the isomer N-alpha-acetyl-L-2,4-diaminobutyric acid as its substrate, albeit with substantially reduced catalytic efficiency. Structure-guided site-directed mutagenesis experiments targeting amino acid residues that are evolutionarily highly conserved among the extended EctC protein family, including those forming the presumptive iron-binding site, were conducted to functionally analyze the properties of the resulting EctC variants. An assessment of enzyme activity and iron content of these mutants give important clues for understanding the architecture of the active site positioned within the core of the EctC cupin barrel.     

The Type III Secretion System-Related CPn0809 from Chlamydia pneumoniae

Chlamydia pneumoniae is an intracellular Gram-negative bacterium that possesses a type III secretion system (T3SS), which enables the pathogen to deliver, in a single step, effector proteins for modulation of host-cell functions into the human host cell cytosol to establish a unique intracellular niche for replication. The translocon proteins located at the top of the T3SS needle filament are essential for its function, as they form pores in the host-cell membrane. Interestingly, unlike other Gram-negative bacteria, C. pneumoniae has two putative translocon operons, named LcrH_1 and LcrH_2. However, little is known about chlamydial translocon proteins. In this study, we analyzed CPn0809, one of the putative hydrophobic translocators encoded by the LcrH_1 operon, and identified an ‘SseC-like family’ domain characteristic of T3S translocators. Using bright-field and confocal microscopy, we found that CPn0809 is associated with EBs during early and very late phases of a C. pneumoniae infection. Furthermore, CPn0809 forms oligomers, and interacts with the T3SS chaperone LcrH_1, via its N-terminal segment. Moreover, expression of full-length CPn0809 in the heterologous host Escherichia coli causes a grave cytotoxic effect that leads to cell death. Taken together, our data indicate that CPn0809 likely represents one of the translocon proteins of the C. pneumoniae T3SS, and possibly plays a role in the translocation of effector proteins in the early stages of infection.     

Single Nucleotide Variant rs2232710 in the Protein Z-Dependent Protease Inhibitor (ZPI,SERPINA10) Gene Is Not Associated with Deep Vein Thrombosis

Rare mutations in PROC, PROS1 or SERPINC1 as well as common variants in F5, F2, F11 and SERPINC1 have been identified as risk factors for deep vein thrombosis (DVT). To identify novel genetic risk factors for DVT, we have developed and applied next-generation DNA sequencing (NGS) of the coding area of hemostatic and proinflammatory genes. Using this strategy, we previously identified a single nucleotide variant (SNV) rs6050 in the FGA gene and novel, rare SNVs in the ADAMTS13 gene associated with DVT. To identify novel coding variants in the genetic predisposition to DVT, we applied NGS analysis of the coding area of 186 hemostatic and proinflammatory genes in 94 DVT cases and 98 controls and we identified 18 variants with putative role in DVT. A group of 585 Italian idiopathic DVT patients and 550 healthy controls was used to genotype all the 18 risk-associated variants identified by NGS. Replication study in the Italian population identified the rs2232710 variant in the protein Z-dependent protease inhibitor (ZPI) gene to be associated with an increased risk of DVT (OR 2.74; 95% CI 1.33–5.65; P = 0.0045; Bonferroni P = 0.081). However, the rs2232710 SNV showed no association with DVT in two Dutch replication cohorts the LETS study (454 patients and 451 controls) and the MEGA study (3799 patients and 4399 controls), indicating that the rs2232710 variant is not a risk factor for DVT.     

The Effect of Optokinetic Stimulation on Perceptual and Postural Symptoms in Visual Vestibular Mismatch Patients

BackgroundVestibular patients occasionally report aggravation or triggering of their symptoms by visual stimuli, which is called visual vestibular mismatch (VVM). These patients therefore experience discomfort, disorientation, dizziness and postural unsteadiness.ObjectiveFirstly, we aimed to get a better insight in the underlying mechanism of VVM by examining perceptual and postural symptoms. Secondly, we wanted to investigate whether roll-motion is a necessary trait to evoke these symptoms or whether a complex but stationary visual pattern equally provokes them.MethodsNine VVM patients and healthy matched control group were examined by exposing both groups to a stationary stimulus as well as an optokinetic stimulus rotating around the naso-occipital axis for a prolonged period of time. Subjective visual vertical (SVV) measurements, posturography and relevant questionnaires were assessed.ResultsNo significant differences between both groups were found for SVV measurements. Patients always swayed more and reported more symptoms than healthy controls. Prolonged exposure to roll-motion caused in patients and controls an increase in postural sway and symptoms. However, only VVM patients reported significantly more symptoms after prolonged exposure to the optokinetic stimulus compared to scores after exposure to a stationary stimulus.ConclusionsVVM patients differ from healthy controls in postural and subjective symptoms and motion is a crucial factor in provoking these symptoms. A possible explanation could be a central visual-vestibular integration deficit, which has implications for diagnostics and clinical rehabilitation purposes. Future research should focus on the underlying central mechanism of VVM and the effectiveness of optokinetic stimulation in resolving it.     

Palaeoecological evidence from buried topsoils and colluvial layers at the Bronze Age fortification Corneşti-Iarcuri,SW Romania: results from palynological,sedimentological, chronostratigraphical and plant macrofossil analyses

Vegetation History and Archaeobotany - Located in the Romanian Banat region, the Late Bronze Age (LBA) fortification Corneşti-Iarcuri is the largest known prehistoric settlement in Europe....     

Relevance of the N-terminal NLS-like sequence of the prion protein for membrane perturbation effects

We investigated the nuclear localization-like sequence KKRPKP, corresponding to the residues 23-28 in the mouse prion protein (mPrP), for its membrane perturbation activity, by comparing effects of two mPrP-derived peptides, corresponding to residues 1-28 (mPrPp(1-28)) and 23-50 (mPrPp(23-50)), respectively. In erythrocytes, mPrPp(1-28) induced approximately 60% haemoglobin leakage after 30 min, whereas mPrPp(23-50) had negligible effects. In calcein-entrapping, large unilamellar vesicles (LUVs), similar results were obtained. Cytotoxicity estimated by lactate dehydrogenase leakage from HeLa cells, was found to be approximately 12% for 50 microM mPrPp(1-28), and approximately 1% for 50 microM mPrPp(23-50). Circular dichroism spectra showed structure induction of mPrPp(1-28) in the presence of POPC:POPG (4:1) and POPC LUVs, while mPrPp(23-50) remained a random coil. Membrane translocation studies on live HeLa cells showed mPrPp(1-28) co-localizing with dextran, suggesting fluid-phase endocytosis, whereas mPrPp(23-50) hardly translocated at all. We conclude that the KKRPKP-sequence is not sufficient to cause membrane perturbation or translocation but needs a hydrophobic counterpart.